Committee

This Symposium has Concluded

Speaker Abstracts

MS.1

New Technology for the Large-scale Proteomic Comparison of Human Embryonic Stem Cells, Induced Pluripotent Stem Cells, and Somatic Cells

Doug Phanstiel1, Justin Brumbaugh1, Graeme C. McAlister1, Craig D. Wenger1, Ron Stewart2, Shulan Tian2, James A Thomson2, and Joshua J. Coon1

1Department of Chemistry and 2Morgridge Institute for Research, University of Wisconsin, Madison, WI, USA

Induced puripotent stem cells (iPS) represent a breakthrough in stem cell research and pose remarkable therapeutic potential. They circumvent ethical issues surrounding the use of human embryonic stem cells (ES) and could eliminate immune rejection in transplantation therapies. In this work we investigate similarities and differences between human ES and iPS cells that may affect the use of iPS cells for research and therapeutic purposes. To perform these experiments we use novel mass spectrometry-based analyses in combination with isobaric tags for absolute and relative quantitation (iTRAQ). To date, we have identified 77,959 unique peptides and 6,534 unique proteins. The fold-differences observed between pluripotent lines and the somatic line showed a high correlation (R = 0.91) demonstrating remarkable similarity between ES and iPS cells. The set of proteins that was expressed at higher levels in pluripotent cells was mostly nuclear in nature and functionally enriched in the processes of transcriptional regulation and chromatin modification. Among these were numerous transcription factors and other proteins important to the maintenance of pluripotency including SOX2, OCT4, DPPA4, and LIN28. In agreement with current literature, comparison of protein expression changes to changes in mRNA expression revealed only a weak correlation (R = 0.68) highlighting the need for quantitative proteomic analysis.

MS.2

Electron Capture Dissociation in Radio-Frequency-Free Cell

Douglas F. Barofsky1, Valery G. Voinov1,3, Max L. Deinzer1, and Joseph S. Beckman2

Departments of 1Chemistry and 2Biochemistry and Biophysics, Oregon State University, Corvallis, OR, USA; 3Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia

A radio frequency-free (RFF), analyzer-independent cell has been devised for electron-capture dissociation (ECD) of ions. The device is based on interleaving a series of electrostatic lenses with the periodic structure of magnetostatic lenses commonly found in a traveling wave tube. A five-magnet version of the RFF electromagnetostatic ECD cell was installed in a Finnigan TSQ700 ESI triple quadrupole (QqQ) spectrometer, and its performance was evaluated by recording product-ion spectra of various peptides. These spectra were readily obtained without recourse to a buffering gas or synchronizing electron injection with a specific phase of an RF field. The mass spectra produced with the modified instrument appear in all respects (other than resolution and mass accuracy, which were limited by the mass spectrometer used) to be at least as good for purposes of peptide identification as those recorded with Fourier transform ion cyclotron resonance (FT ICR) instruments; however, the effort and time to produce the mass spectra were much less than required to produce their FT ICR counterparts. A two-magnet version of the electromagnetostatic ECD cell was installed in the same mass spectrometer and used to simultaneously obtain combined ECD/CID product-ion mass spectra that exhibit a-, b-, and c-type ion signals. Details of the cells design, construction, and operation will be presented and discussed.

MS.3

Decoding the Histone Code by Quantitative Proteomics

Gary LeRoy1, Mariana D. Plazas-Mayorca2, Nicolas L. Young1, and Benjamin A. Garcia1

Departments of 1Molecular Biology and 2Chemistry, Princeton University, Princeton, NJ, USAZ

Epigenetic refers to stable heritable changes in gene expression that are not due to changes in DNA sequence, such as DNA methylation, RNA interference and histone modifications. Histones are small basic proteins that function to package genomic DNA into repeating nucleosomal units (containing ~146 bp of DNA wrapped around two copies of each of histones H3, H4, H2A and H2B) forming the chromatin fiber and hence our chromosomes. In general, the packaging of DNA into chromatin is recognized to be a major mechanism by which the access of genomic DNA is restricted. A wide number of studies show that several covalent histone modifications such as methylation, acetylation, phosphorylation and ubiquitination located in the N-terminal tails correlate with both the regulation of chromatin structure during active gene expression, or heterochromatin formation during gene silencing. Here we are developing novel proteomic strategies to discover differentially expressed histone modifications, identify concurrent combinatorial histone modifications (Histone Codes), and characterize histone codes that are important various processes.

MS.4

Fragment Assignment by Visual Assistance (FAVA) Program for Analysis of Complex High Resolution MSMS Spectra

Shenheng Guan and A.L. Burlingame

Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA USA

Introduction: Fragmentation of intact proteins followed by high resolution detection in FTICRMS or OrbiTrap mass spectrometry in a typical “top-down’ experiment generates a complicated mass spectrum, in which spectral space is crowded by isotopic distributions from a large number of fragments in one or more charge states. Several programs, including THRASH can convert the isotopic distributions into monoisotopic mass values. However, they sometimes assign incorrect monoisotopic m/z peaks (or inaccurate charge states) and report erroneous deconvolved mass values. Large amounts of manual effort have been spent on verification of these assignments. We have improved our FAVA code to allow analysis of high resolution spectra recorded in either time domain (ion transient) or Fourier transformed spectral formats.

Methods: FAVA code was written in MatLab version 6 environment (MathWorks, Natick, MA).

Preliminary Data: A key feature of the FAVA code is that is allows for both manual and automatic modes of operation. In either mode, the program attempts to fit a theoretical isotopic pattern of a particular fragment in a particular charge state with the experimental spectrum. In the manual mode, the operator decides the correctness of an assignment by inspection of zoomed spectrum with theoretical isotopic patterns (visual assistance). In the automatic mode, mass accuracy, signal-to-noise ratio, peak width, and quality of fit (deviation of the fit) are used as criteria for assignment. The final output of the tool includes (1) a master spectrum containing annotations of peak assignments and (2) a table of parameters of ions being examined regardless of the assignment. Current improvements include (1) adaption of Unimod PTM annotation and (2) acceptance of standard RAW data format. .The result is the elimination of the data translation step and easier testing of sequence and PTM assignment candidates generated from a database search engine.

Support for this research was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A.L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614 and NIH NCRR RR019934.

MS.5

Use of Electron Transfer Dissociation to Analyze Combinations of Histone Post-translational Modifications on an LTQ-Orbitrap

Shannon Eliuk1, David Maltby1, Feixia Chu3, Barbara Panning2, and A.L. Burlingame1

1Mass Spectrometry Facility, Department of Pharmaceutical Chemistry and 2Department of Biochemistry & Biophysics, University of California, San Francisco, CA, USA; 3College of Life Sciences and Agriculture, University of New Hampshire, Durham, NH, USA

Post-translational modifications of histones are used to regulate DNA-chromatin interactions and ultimately gene expression. In many cases, the various histone PTMs do not regulate function individually. Instead, combinations of modifications are believed to act together to create a ‘histone code’. Elucidating these combinations through analyses of their proteolytic digests since is often not possible as much of this combinatorial information is lost following protein digestion. It is essential to be able to detect and assign the sites of these modifications in combination on particular protein isoforms to assess their significance.

Accordingly, we have developed approaches for both direct infusion and online reverse phase liquid chromatography separation for intact histone and large histone peptides (AspN or GluC digests) on an LTQ Orbitrap with electron transfer dissociation (ETD) fragmentation.

We have applied these methods for the analysis of post-translational modifications of histones in methyltransferase knockout mouse embryonic stem cells compared with wildtype cells. Combined with stable isotope labeling of amino acids in cell culture (SILAC), changes in stoichiometries of modification site occupancies are revealed by quantitative mass spectrometry. Through these techniques, we show that knock out of a specific methyltransferase leads to a variety of histone modification changes.

Support for this research was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A.L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614 and S10 RR019934.

MS

Electron Capture Dissociation for Structural Studies of Integral Membrane Proteins and Their Modifications

Julian Whitelegge

The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

Full structural analysis of the c-subunit of the Fo domain of ATP synthase demonstrated that the integral membrane proteins that constitute around one third of the proteome are amenable to top-down high-resolution mass spectrometry. Effective electron capture dissociation (ECD) was achieved through thermal excitation of ions using an infra-red laser for activated-ion, or aiECD, implying that it was necessary to break alpha-helix hydrogen bonds prior to electron capture. Collisionally activated dissociation (CAD) was also effective for top-down analysis of the c-subunit but aiECD clearly improved sequence coverage, allowing substantial sequence coverage within the two alpha-helical transmembrane domains. The need to use thermal excitation for effective ECD of this 8 kD protein suggested that integral membrane proteins may have a lower threshold for this requirement than soluble proteins. Subsequent studies of a variety of integral membrane proteins have not been consistent enough to answer this question. The most consistent feature of these experiments has been the frequent observation that while CAD yields useful product ion spectra ECD most often yields familiar charge reduction series even when IR laser is used to activate the ions. The origin of this problem seems to be the fact that integral membrane proteins typically carry fewer charges than soluble proteins after electrospray ionization giving them higher m/z. Strategies to achieve higher charging will be discussed.

O-GlcNAcylation: The Post-Translational Modification that Best Highlights the Value of ETD

Robert Chalkley

Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California San Francisco, CA, USA

O-GlcNAcylation is a widespread regulatory modification of nuclear and cytoplasmic proteins, analogous to phosphorylation. For a modification that was first discovered 25 years ago, surprisingly little is known about how it regulates protein functions, despite its clear role in problems such as diabetes and Alzheimer disease. Slow progress in characterizing this modification has largely been due to a lack of effective methods for detecting and locating O-GlcNAcylation sites. This is in a large part because the O-glycosidic link that attaches the single N-acetylglucosamine (GlcNAc) moiety to serines and threonines is highly labile under collision induced dissociation in a mass spectrometer (much more so than phosphorylation), so O-GlcNAc site assignment using this mass spectrometric approach has proven largely unsuccessful.

Electron transfer dissociation (ETD) is a recently developed radical-based fragmentation technique that fragments components at locations not defined by bond strength, so is able to maintain labile modifications on fragment ions. Hence, the modification is not cleaved during ETD mass spectrometric analysis, allowing O-GlcNAcylation site assignment.

In this talk I will present how ETD availability, along with strategies for enriching for modified peptides, is starting to transform the characterization of this modification.

This work was supported by NIH NCRR grant RR001614, SIG RR019934 and the Biotechnology and Biological Sciences Research Council of the UK.

MS.8

The Use of ECD for Proteomics-wide Identification and Quantification of iso-Asp Residues

Roman A. Zubarev

Division of Molecular Biometry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Protein deamidation is one of the major post-translational modifications (PTM) that lead to protein inactivation in vivo. It has been shown to relate with neurodegenerative diseases (e.g. Alzheimer’s disease) and the degradation of commercial protein products. Asparaginyl residue is a major source of deamidation in biological samples, the minor one being the glutaminyl residue. Asparagine deamidation is a non-enzymatic PTM that occurs spontaneously under physiological conditions, which results in a mixture of aspartyl (Asp) and isoaspartyl (isoAsp) residues. Asp isomerization also contributes to the increase in the isoAsp pool, albeit at a lower rate.

Electron capture dissociation (ECD) combined with Fourier transform mass spectrometry (FT MS) are able to distinguish the isoaspartyl peptides by unique ECD fragments of cn• + 58.0054 (C2H2O2) and zl-n – 56.9976 (C2HO2), where n is the position of the aspartyl residue and l is the peptide length.

In the present study, we tested the specificity of isoAsp detection using the accurate masses of these specific fragments. Totally, 466 isoAsp peptide candidates were identified from 32 whole and partial human proteome samples. Then additional criteria, like adjacent c/z fragments, specific losses from the reduced species, and the shape of the chromatographic peak, were applied to increase the specificity of the method. Upon detailed inspection, 219 isoAsp peptide candidates have been supported by at least one criterion other than the mass of the specific ECD fragments. Most stringent filtering of these candidates yielded several cases where the presence of isoAsp was beyond doubt. Among the identified proteins with isoAsp, actin, heat shock cognate 71 kDa protein and pyruvate kinase have previously been identified as substrates for L-isoaspartyl methyltransferase (PIMT), an important repair enzyme converting in vivo isoaspartyl to aspartyl. Quantification of relative isomerization degree was performed by the label-free approach using in-house developed software. This is the first attempt to analyze the human isoaspartome in a high-throughput manner. The developed workflow allows for further enhancement of the detection rate of isoaspartyl residues in biological samples.

1.1

Global Analysis of Small Molecule Interactions with Proteins

Xiyan Li and Michael Snyder

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA

Extensive effort has been made to characterize the interactions of proteins with other proteins, and for the case of transcription factors, their interaction with DNA. Natural small compounds comprise the majority of the cellular molecules, and have been shown to bind proteins as substrates, products, cofactors and regulatory ligands. Although they participate in such diverse processes, a large scale investigation of protein-small molecules has never been performed. We have developed a mass spectrometry assay for the global analysis protein-natural small molecule interactions in yeast, and applied this method to the analysis of molecules that bind lipid pathway biosynthetic proteins and protein kinases. We find that a large number of proteins bind small molecules. Some enzymes bind substrates, others bind their products, and many key regulatory proteins such as protein kinases bind small molecules. One common ligand bound by many proteins is ergosterol suggesting a general role for this compound in regulation. We further explore this by demonstration that the activity of the high conserved AKT protein kinase homolog of yeast depends upon the presence of ergosterol. Overall, our study helps define potential key regulatory steps in biosynthetic pathways and demonstrates that small molecules bind many proteins in a variety of biochemical and regulatory roles and suggest they can serve as regulators of protein activity.

1.2

Membrane-Assisted Sample Preparation for Online ESI-MS Analysis of Biomolecules

Juan Astorga-Wells1,2, Craig Whitehouse3, and Hans Jörnvall1

1Karolinska Institutet, Stockholm, Sweden; 2Biomotif AB, Täby, Sweden; 3Analytica of Branford, Connecticut, USA

In addition to further improvements in resolution, mass accuracy, sensitivity and throughput, advances in sample preparation are essential in order to fully unveil the potential of any mass spectrometer. A technology based on ion-selective membranes has been developed in order to perform online sample preparation before electrospray ionization. The technique permits to change the chemical composition of the solution and manipulate analytes a few microliters upstream from the electrospray process. Compounds can be removed or injected from an adjacent channel to the main flow stream through an ion-selective membrane that separates both channels. In this manner, protons can be injected into the flow stream to carry out rapid pH scans; deuterons can be delivered to perform deuterium/hydrogen exchange experiments in order to study protein structural changes; and metal ions can be delivered into the flow stream to study -for example- metal-binding peptides. Furthermore, a device with two membrane sections can be used to introduce an electric filed into the main channel in order to immobilize ions and perform pre-concentration, clean-up, multi-step micro reactions, solvent exchange and separation prior to mass analysis. Examples of each application will be shown as well as preliminary data of other applications.

1.3

Two-Dimensional Liquid Chromatography Coupled with ESI-MS for Protein Identification and Quantification

Jim Langridge1 ;Hans Vissers1; Scott Geromanos3; Martha Stapels3 ; Craig Dorschel3 Marc V Gorenstein3 ; Dan Golick3 and Hans Aerts2

1Waters Corporation, MS Technologies Center, Manchester, UK; 2AMC, University of Amsterdam, Department of Biochemistry, The Netherlands

Mass spectrometry is widely accepted as an essential tool to better understand protein function, facilitating both the identification and quantification of proteins in complex samples. Mass spectrometry based protein identification strategies have previously been described [1-3] that facilitate the simultaneous acquisition of qualitative and quantitative information, in a data independent fashion.

We have extended this approach to generate precise relative quantitation values for proteins contained in biological systems [4-5], and have constructed protein abundance curves for specific tissues, cell lysates and biofluids. This has been shown to be transferrable between different laboratories and independent of instrument type. An important aspect of this quantification approach is that it allows sample loading onto a given analytical column to be determined and optimized, to ensure that ideal chromatographic and mass spectrometric performance is obtained. This results in the maximum number of peptide and proteins being determined from the sample, whilst maintaining maximum accuracy for quantitative measurements. More recently this approach has been extended to cover a wider range of protein abundance by implementing a 2-dimenionsal reverse phase-reverse phase separation strategy, using differential pH. In this manner wide quantitative proteome coverage can be obtained.

Experimental information obtained from such studies will be compared to theoretical models of the given proteome; considering complexity, dynamic range and the inherent physiochemical properties of tryptic peptides in solution and the gas phase.

References

  1. Bateman et al. (2002) JASMS 13(7), 792-803.
  2. Purvine et al. (2003) Proteomics 3(6), 847-50.
  3. Silva et al. (2005) Anal Chem. 77(7), 2187-200.
  4. Silva et al. (2006) Mol Cell Proteomics 5(1),144-56.
  5. Hughes et al. (2006) J Proteome Res. 5(1), 54-63.

1.

Targeted Proteomic Approaches Provide Insights into Virion Assembly and Chromatin Remodeling during Viral Infection

Ileana M. Cristea

Department of Molecular Biology, Princeton University, Princeton, N.J., USA

Viruses have evolved finely tuned interactions with their hosts to manipulate and adapt complex cellular processes for their own use. The study of virus-host interactions has therefore emerged as a key driving force in the research of infectious disease during the post-genomic era. Despite these efforts, our understanding of the protein interactome remains, in large part, unknown. The development and incorporation of new approaches that can reveal the dynamics of virus-host protein interactions is a necessity. Modern proteomic techniques have the ability to provide access to such interactions, and the ever increasing sensitivity of mass spectrometry allows the identification and quantification of relatively low levels of proteins. This presentation will describe targeted proteomic approaches for studying virus-host macromolecular assemblies. Highlights will be shown from our studies on infections with human immunodeficiency virus (HIV) and human cytomegalovirus (HCMV).

We employed targeted genetic-proteomic approaches to study the virus-host interface either from the virus or host perspective. Using a library of tagged replication competent HCMV and HIV mutant viruses, we infected primary human fibroblasts (for HCMV) and CEM T cells (for HIV), and employed cryogenic cell lysis and rapid immunoaffinity purifications on magnetic beads to isolate virus-host assemblies. For studies on histone deacetylases (HDAC) during viral infections, we generated cell lines stably expressing green fluorescent protein tagged HDACs and probed their interactions and deacetylation activity. Isolated protein complexes were analyzed using a MALDI LTQ Orbitrap (Thermo Fisher Scientific) and the specificity of observed interactions was confirmed by immunofluorescence, reciprocal immunoprecipitation and metabolic labeling with stable isotopes (I-DIRT).

Two interesting findings will be highlighted: 1) studies on pUL32, pUL99, pUL83 and pTRS1 HCMV proteins demonstrated that parallel processes occur at distinct cellular sites during the assembly of HCMV virions, and 2) chromatin remodeling complexes, including histone deacetylases, are targeted by viruses, possibly in part to gain control over host gene expression and modulate the outcome of an infection.

1.5

Confident Assignment of Post-Translational Modifications Using Top-down Mass Spectrometry

Julian Whitelegge

The Pasarow Mass Spectrometry Laboratory, The NPI-Semel Institute, David Geffen School of Medicine, University of California, Los Angeles, CA, USA

Top-down proteomics uses high-resolution Fourier-transform mass spectrometry (FT-MS) to define proteins by their intact masses in combination with dissociation experiments for unambiguous primary structure determination. FT-MS has been used to characterize the integral and peripheral subunits of the integral membrane Photosystem II complex from the red alga Galdieria sulphuraria. The complex was analyzed by reverse-phase liquid chromatography with online electrospray-ionization mass spectrometry and concomitant fraction collection (LC-MS+). Selected fractions were transferred to static nanospray tips for nano-electrospray ionization on a hybrid linear ion-trap/Fourier-transform ion-cyclotron resonance mass spectrometer (7 Tesla LTQ-FT Ultra; Thermo Scientific) for extended averaging of transients. Collision-activated dissociation (CAD) was achieved in the ion-trap whereas electron-capture dissociation (ECD), IRMPD and activated-ion electron-capture dissociation (aiECD) were performed in the cyclotron cell using an infra-red laser and an electron source. Top-down datasets were deconvoluted using Xtract and matched to a Galdieria proteome database using Prosight PC (Thermo Scientific). The primary LC-MS+ separation yielded thirty-nine intact mass tags (IMTs) with 100 ppm mass accuracy on a low-resolution instrument. While several subunits could be identified based upon close coincidence of measured and calculated masses, the majority required use of sequence tag functionality of the software to yield candidate identifications for manual assessment. All of the subunits studied carried covalent modifications that required consideration in the analysis, including co-translational N-terminal formylation and post-translational removal of Met1 with or without N-acetylation, removal of signal peptides and attachment of cofactors. Approaches to identify and localize PTMs are considered. The precision afforded through use of FT-MS allows precursor and product ion matching at a 10 ppm tolerance with nearly all assignments achieving less than 5 ppm, such that false positives become highly unlikely. There are however limitations to the accurate assignment of PTMs and these will be illustrated with reference to labile modifications that can be displaced during dissociation. The lessons learned from top-down PTM analyses will be discussed in the context of current strategies for high-throughput bottom-up proteomics.

2.1

Protein Quantification Through Targeted Mass Spectrometry: The Way Out of Biomarker Purgatory?

Steven A. Carr

Broad Institute of MIT and Harvard, Cambridge, MA

The enormous potential of biomarkers to revolutionize clinical practice and improve patient care has been well documented. Given their high potential therapeutic and financial impact it is, on the surface, surprising that so few new protein biomarkers have been introduced into widespread clinical use recently. The reasons for the dearth of new protein biomarkers relate to the high false discovery rate of discovery “omics” methods (regardless of technology used), together with a lack of robust methods for biomarker verification in large clinical sample sets. It is now common for differential analysis of tissue or plasma by multidimensional LC-MS/MS (the workhorse tool for unbiased discovery) to provide confident identification of 1000’s of proteins, 100’s of which can vary 5-fold or more between case and control samples in discovery studies. Due to the extent of sample fractionation required to access proteins at lower abundance, it is not uncommon for the analysis of a single case/control sample pair to take up to two weeks of on-instrument time. This limits the numbers of samples that can be practically analyzed to typically 10 (or fewer) case vs control comparisons. These numbers are very small relative to the high dimensionality of the proteome (100,000’s or more possible components when posttranslational modifications and other variants are taken into account), and the scale of normal variation in the human population. Thus a very large fraction, possibly exceeding 95% of the protein biomarkers “discovered” in these experiments are false positives arising from biological or technical variability. Clearly discovery “omics” experiments do not lead to biomarkers of immediate clinical utility, but rather produce “candidates” that must be “qualified” and “verified” (1,2).

Lack of robust quantitative methods with sufficient sensitivity, reproducibility and throughput has significantly hampered our ability to credential candidates coming from unbiased proteomic discovery efforts since useful Ab reagents for the vast majority do not exist. Our laboratory has focused on addressing this serious barrier by developing robust targeted assay methods employing mass spectrometry to screen and quantify low abundance proteins in plasma. We have recently demonstrated that multiplexed assays for proteins at the low ng/mL level in plasma can be configured using Stable Isotope Dilution (SID) - Multiple Reaction Monitoring (MRM) Mass Spectrometry. Large-scale interlaboratory studies conducted under the auspices of the National Cancer Institute’s Clinical Proteomics Assessment for Cancer (CPTAC) program have demonstrated that these assays can be reproducibly configured, deployed and run in multiple laboratories with assay CV’s approaching clinical performance. Further improvements will come from the use of peptide immunoaffinity enrichment, referred to as SISCAPA, which holds particular promise for simplifying sample preparation and increasing both throughput and sensitivity of MRM-based assays. Using SISCAPA, assays can be readily configured that enable quantitation of proteins present at low ng/mL levels directly from plasma.

This presentation will focus on the further development and application of MRM-MS and SISCAPA technologies in the context of cancer and cardiovascular disease.

References

  1. Rifai, N., Gillette, M.A., and Carr, S.A. (2006) Protein biomarker discovery and validation: the long and uncertain path to clinical utility. Nat Biotechnol 24, 971-83.
  2. Paulovich, A.G., Whiteaker, J.R., Hoofnagle, A.N., and Wang, P. (2008) The Interface between Biomarker Discovery and Clinical Validation: The Tar Pit of the Protein Biomarker Pipeline. Proteom.- Clin. Appl. 2, 1386-1402.

2.2

Proteomics Targeted to Sub-cellular Compartments and Integration with Genomics for Candidate Biomarker Discovery in Colorectal Cancer

Connie R. Jimenez

OncoProteomics Laboratory, Department of Medical Oncology, VUmc-Cancer Center Amsterdam, VU University Medical Center, Amsterdam, The Netherlands.
Mass spectrometry-based proteomics applied to biomarker-rich sub-cellular compartments in (pre)-clinical samples is emerging as a powerful approach for discovery of tissue-derived biomarkers with close association to the disease. We have applied in-depth proteomics to sub-nuclear compartments, cell surface fractions and tumor secretomes for discovery of colorectal cancer-related proteins with potential use as imaging- and serum-based biomarkers. To this end, we analyzed preclinical (cell lines and genetic mouse model) as well as clinical samples. We have optimized the label-free GeLC-LTQ-FTMS/MS pipeline to allow for quantitation of individual proteins by spectral counting with good reproducibility. Integration of in-depth discovery proteomics with genomics data (microarray and arrayCHG) allows for a powerful approach to prioritize candidates for follow-up and validation by targeted strategies, including immunohistochemistry of tissue microarrays. Promising imageable biomarker candidates have been validated and validation of selected nuclear proteins and secretome differential proteins as serum and stool-based markers is underway.

2.3

Towards the Discovery of Biomarkers in Cerebrospinal Fluid by Combining Peptide Ligand Library Treatment and Label Free Protein Quantification on a LTQ-Orbitrap

Florence Roux-Dalvai1, Emmanuelle Mouton-Barbosa1, Anne Gonzalez de Peredo1, David Bouyssié1, Luc Guerrier2, Egisto Boschetti2, François Berger3, Odile Burlet-Schiltz1, and Bernard Monsarrat1

1CNRS IPBS , Toulouse University, France; 2BioRad, Gif-sur-Yvette, France; 3INSERM, Université Joseph Fourier, Grenoble, France;

Cerebrospinal Fluid (CSF) is the biological fluid in closest contact with the brain and thus contains some proteins and other products of neural cell origin, lending itself to proteomic analysis for potential biomarkers of neurological diseases. However, as in the case of other biological fluids, the main analytical challenge in proteomic characterization of the CSF is the very wide concentration range of proteins, largely exceeding the dynamic range of current analytical approaches. The most abundant protein, human serum albumin, constitutes alone around 45% of the total protein content in CSF, which renders extremely difficult the detection of lowabundant species.

Here, we used the peptide ligand library technology ProteominerTM to reduce the dynamic range of protein concentration in CSF and unmask previously undetected proteins by nanoLC-MS/MS analysis on an LTQ-Orbitrap mass spectrometer. This method was first applied on a large pool of CSF from different sources, with the aim to better characterize the protein content of this fluid, especially for the low abundance components. We were able to identify 1189 proteins in CSF, and among these, 755 were only detected after ProteominerTM treatment.

One drawback of this method is the large amount of biological fluid that has to be applied on conventional ProteominerTM columns, precluding its potential use for treatment of patient samples. The method has thus been optimized for clinical studies. First, the ProteominerTM treatment has been miniaturized to be compatible with the low CSF volume typically obtained after lumbar puncture. We could show that the treatment is still efficient with this miniaturized protocol, and that the dynamic range of protein concentration is actually reduced even with small amounts of beads, leading to an increase of more than 80% of the number of identified proteins in one LC-MS/MS run. Moreover, the reproducibility required for protein quantification has been checked for this new protocol in replicate experiments. Finally, a labelfree quantitative proteomic strategy dedicated to the analysis of large series of samples has been developed. For such studies, fractionation of the samples is generally not possible, but the analysis in one run of a complex sample, even after ProteominerTM treatment, limits the number of identified and quantified proteins. In order to increase this number, we implemented an approach based on the comparison of MS signals in individual runs with a previously generated MS/MS identification database containing m/z and retention time values associated with peptide sequences. The MFPaQ software was used to assign nonsequenced peptides MS signals to the identification database and to quantify them. The combination of ProteominerTM sample treatment with the bioinformatics workflow developed for data quantification allowed us to increase by a factor 3.4 the number of proteins identified and quantified in the same CSF sample. This work opens the way to future studies aiming at discovering biomarkers in CSF among the low abundance proteins.

3.

Quantitative Analysis of Proteome Localisation and Dynamics

Angus Lamond

Wellcome Trust Centre for Gene Regulation and Expression, MSI/WTB Complex, University of Dundee, Dundee, Scotland, UK

We are studying the functional organization of mammalian cell nuclei using a dual strategy that combines mass spectrometry (MS) based proteomics with live cell fluorescence imaging (see www.LamondLab.com). This applies two distinct but complementary quantitative techniques to analyse the same biological problem, providing a rigorous approach where potential artifacts or limitations of one method are avoided in the complementary approach and vice versa. The quantitative proteomic methods involve metabolic labeling of cellular proteins in cultured cell lines with the amino acids lysine and arginine containing heavy isotopes such as 13C and 15N. The quantitative imaging experiments, including time-lapse microscopy, FRAP, FLIP, FLIM and FLIM-FRET, are performed on mammalian cell lines stably expressing one or more fluorescent protein-tagged reporters. Both the proteomics and microscopy methods are used to study the same stable cell lines, allowing a direct comparison the resulting data from both techniques.

We have used this dual strategy to characterize in detail the molecular composition of nucleoli under different metabolic and growth conditions and at specific stages of cell cycle progression (see https://lamondlab.com/nopdb/). We have developed a MS-based proteomics strategy to perform quantitative analyses of subcellular protein localization – “spatial proteomics” - including the analysis of protein turnover rates in separate cell compartments. This provides a new approach for annotating the spatial organization of the proteome and for measuring how this changes in response to inhibitors and different cell growth conditions. We have also developed quantitative MS-based approaches for identifying specific protein-protein interactions. These strategies provide a general approach for characterizing the composition, dynamic properties and interactions of either cell organelles or multi-protein complexes.

3.2

Post-Translational Adenosine Monophosphate (AMP) Modification of Proteins

Carolyn A. Worby1,2,3,9, Seema Mattoo4,9, Robert P. Kruger5, Lynette B. Corbeil6,7, Antonius Koller8, Juan C. Mendez6, Bereket Zekarias6, Cheri Lazar1,2,3 and Jack E. Dixon1,2,3,4

Departments of 1Pharmacology, 2Cellular and Molecular Medicine, 3Chemistry and Biochemistry, and 4Howard Hughes Medical Institute, University of California, San Diego, La Jolla, CA, USA; 5Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA; 6Department of Pathology, University of California, San Diego Medical Center, San Diego, CA, USA; 7Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, CA, USA; 8Department of Pathology, Stony Brook University, Stony Brook, NY, USA

Eukaryotic cells have devised different strategies to regulate signaling pathways. The best known modification is phosphorylation, which attaches a phosphate group to serine, threonine or tyrosine residues in proteins, thereby regulating their activities. Here, we describe a new modification: the addition of adenosine monophosphate (AMP) on tyrosine residues. AMP addition to Rho GTPases by the Fic domain containing secreted surface antigen IbpA of the respiratory pathogen Histophilus somni leads to cytoskeletal collapse in host cells (1). Specifically, incubation of purified Rho GTPases (RhoA, Rac1 and Cdc42) with GST-tagged and purified Fic domain of IbpA in the presence of α32P-ATP, but not γ32P-ATP, allows transfer of the 32P-label to RhoA, Rac1 or Cdc42, thus indicating the addition of AMP versus a phosphorylation event. Unlike VopS, another Fic domain containing protein from V. parahemolyticus, that modifies AMP on threonine residues (2), mass spectrometric analysis of IbpA-treated Rho GTPases show that the IbpA Fic domain adds an AMP to a conserved tyrosine residue in the switch I region of Rho GTPases. In addition, we show that the only human protein containing a Fic domain, HYPE (Huntingtin Yeast-interacting Protein E), also has the ability to add AMP to tyrosine residues in Rho GTPases in vitro. Thus, we identify Fic domain containing proteins as a new class of enzymes that mediate not just bacterial pathogenesis, but also a previously unrecognized eukaryotic post-translational modification that may regulate key signaling events.

Interestingly, threonine and tyrosine AMP modified peptides behave similarly in the mass spectrometer as threonine and tyrosine phosphorylated peptides: whereas threonine modified peptides undergo neutral loss of AMP (plus 18Da) on fragments upon activation of the peptide, peptides fragments modified with AMP on tyrosine mostly stay intact, partially losing adenine as well as adenosine. In addition, AMP-Tyr modified peptides are only identifiable in an ion-trap CID fragmentation experiment, as fragmentation in an HCD cell (or CID in a QSTAR) will lead to a strong signal for adenine and only weak fragmentation peaks of the peptide backbone.

References

  1. Worby CA, Mattoo S, Kruger RP, Corbeil LB, Koller A, Mendez JC, Zekarias B, Lazar C, Dixon JE. (2009) The fic domain: regulation of cell signaling by adenylylation. Mol Cell 34(1), 93-103.
  2. Yarbrough ML, Li Y, Kinch LN, Grishin NV, Ball HL, Orth K. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. (2009) Science 323(5911), 269-272.

3.

Dissecting the Structure of the Human Spliceosome by Looking at its Pieces

Patricia Coltri1, Janine Ilagan1, Robert J. Chalkley2, A. L. Burlingame2, and Melissa S. Jurica1

1Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, Santa Cruz, CA, USA; 2Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA

Pre-mRNA splicing is the removal of the non-coding introns that interrupt most gene transcripts and serves an essential step in eukaryotic gene expression. The cellular machinery responsible for splicing, termed the spliceosome, is a large protein/RNA macromolecular complex comprised of five structural RNAs and over 100 individual polypeptides. The human complex assembles and functions via a progression of structural intermediates that are not yet fully characterized. The dynamics and complexity of the spliceosome have long posed challenges to detailed biochemical and structural studies that will provide insight into the spliceosome’s molecular mechanisms. In particular, isolating distinct conformations of this moving target in the amounts needed for standard biochemical and structural analyses is not simple. We made a key advance in this regard with our development of a substrate-based affinity method to isolate human spliceosomes arrested midway through splicing catalysis (C-complex). Initial mass spectrometry analysis of this complex identified over 200 proteins, ~100 of which were specific to splicing. Using cryo-electron microscopy (cryo-EM) and single particle reconstruction techniques, we solved the structure of C-complex spliceosomes to 30 Å resolution. This model represents an important first step in visualizing the structure of the spliceosome. However, before we can more fully interpret the model in functional terms we must answer questions regarding which components of the spliceosome are visualized/represented in our model and where they are located in the structure. Currently, we are finding ways to take the spliceosome apart and then examining the resultant pieces. Mass spectrometry analysis is critical for defining the protein composition of the pieces, enabling us to define interactions that underpin the spliceosomes architecture. We have examined the contribution of exon sequences in the composition and structure of C-complex and are now looking at the proteins that tightly associate with the intron vs. the region of the upstream exon poised for ligation. In addition to these studies, we have made progress in using chemical modification in conjunction with mass spectrometry to identify regions of proteins that are located at the surface of the spliceosome. This work will allow us to begin localizing these proteins’ positions in the complex. By combining the results of these studies with our structural investigation of the spliceosome, we are on the path to assembling a more detailed model of this critical cellular machine.

3.

Protein complexes and functional pathways in S. cerevisiae and E. coli

Mohan Babu1, Gareth Butland3, J. Javier Diaz-Mejia1,4, Pingzhao Hu1, Shuye Pu5, Gabriel Moreno-Hagelsieb4, Sarath Chandra Janga1, Shoshana Wodak2,5, Andrew Emili1,2, and Jack Greenblatt1,2

1Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, M5S 3E1, Canada, 2Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada, 3Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 4Department of Biology, Wilfrid Laurier University, Waterloo, ON, N2L 3C5 Canada, 5Hospital for Sick Children, Toronto, ON, Canada

We have used TAP-tagging and affinity-purification to sort the soluble proteins of S. cerevisiae into complexes. We combined this with systematic synthetic genetic interaction analysis for non-essential gene deletion mutants and essential gene hypomorphs, using the synthetic genetic array (SGA) approach, for genes related to nuclear processes. More recently, we have extended the yeast protein interaction network by focusing on the predicted yeast membrane proteins, purifying each protein three times in the presence of different detergents. We are testing the co-functionality of proteins in various membrane-associated protein complexes by comparing our protein complex data with synthetic genetic interaction data and assessments of the effects of the various proteins in a complex on the morphology of the intracellular compartment in which that complex is located.

We have also used dual affinity tagging followed by affinity purification and mass spectrometry to sort the soluble proteins of E. coli into protein complexes. Although our initial focus was on essential, evolutionarily conserved proteins, we have focused more recently on proteins of unknown function (functional orphans). We integrated our protein-protein interaction network with systematic genome context inferences to derive a probabilistic network of functional inferences encompassing almost all E. coli proteins (98%) and to assign about 57% of the orphans to discrete functional neighborhoods with high confidence. Many of these functional inferences were then confirmed by genome-scale phenotypic assessments. Functional pathways can be derived by systematically identifying genetic interactions, or epistasis, which tends to occur between genes involved in parallel pathways or interlinked biological processes. We have therefore developed a quantitative screening procedure, eSGA (E. coli synthetic genetic arrays), for monitoring bacterial genetic interactions based on conjugation of E. coli deletion or hypomorphic strains to create double mutants on a genome-wide scale. The patterns of synthetic lethality or sickness (aggravating genetic interactions) we observe for certain double mutant combinations provide information about functional relationships and redundancy between pathways and enable us to group E. coli genes into functional modules.

3.

N-Terminomics: High Confidence, Broad Dynamic Range Coverage Utilizing Novel Polymers for Proteomics Reveals the Functional State of the Proteome

Christopher M. Overall

UBC Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada.

The nature of a protein’s N-terminus, its modifications and sequence has profound impacts on the function and localisation of proteins. Moreover, all proteomes are moulded by proteolysis and in all cases this changes the function of a protein, for example in enzyme and protein activation, inactivation, conversion to antagonists, as triggers for secretion, cell surface shedding and finally clearance. Therefore, to functionally annotate proteins, the N and C terminal peptides of proteins must be determined. To focus on these peptides, dedicated techniques are required. Hence, these semitryptic peptides are all too often overlooked in proteomics analyses.

We have developed novel polymers that target primary amine groups on peptides that are invaluable for such proteomics analyses because of their high derivatisation, excellent solubility, no non-specific binding properties, low cost and easy synthesis. Using these polymers in a new procedure termed TAILS (Terminal Amine Isotope Labelling of Substrates) we report a new proteomic and bioinformatics pipeline to rapidly identify natural N-termini and protease cleaved neo-termini of protein substrates after polymer enrichment. MS/MS both identifies the N-terminal peptide and both the substrate and sequence of protease cleavage sites in the same experiment. For most proteins multiple peptides are so identified enabling robust protein identification through multiple peptides. For proteins with single peptides identified, a new statistical analysis enables high confidence protein identification. The key to identifying specific protease substrates is the use of isotopic labelling of all primary amines in order to subtract background proteolysis that is always present. This can be achieved by dimethylation and iTRAQ labelling on primary amines in 8-plex analyses.

We applied TAILS for quantitative N-terminome analysis and for the global analysis of proteolysis in skin inflammation induced by TPA (12-O-tetradecanoyl-phorbol-13-acetate). First, we developed and successfully tested a mass spectrometry-compatible protein isolation and purification method for total skin lysates. Next, we combined this method with TAILS analysis to determine both the skin proteome and skin N-terminome and their perturbations in inflammation. Including wild-type and matrix metalloproteinase (MMP) 2 knockout mice in this multiplex approach allowed us to further identify novel bioactive substrates of this important family of inflammatory matrix metalloproteinase. Thereby, we identified 1,972 proteins with high confidence from murine skin samples with 84 being significantly up-regulated in TPA treated skin including known inflammatory markers such as acute phase proteins and components of the complement system. By TAILS we identified 1,677 N-terminal peptides for 1,032 proteins including 621 that had also been detected prior to N-terminal enrichment. Importantly, among the 411 proteins only identified after enrichment for N-termini were low abundance chemokines like the small inducible cytokines B5 (LIX) and macrophage inflammatory protein 2 (MIP2). As expected, the N-termini of these proteins were also included in a subset of 312 N-terminal peptides assigned to 184 proteins significantly induced by TPA treatment with a statistically significant enrichment of inflammation-related categories by Gene Ontology (GO) analysis. Notably, the analyses were neither skewed by proteins that are highly abundant in skin, such as keratin and filaggrin, nor by serum proteins (only 23 identified).

Hence, N-terminomics analyses using negative peptide selection enables broad proteome coverage with high dynamic range of complex proteomes.

4.1

Activity-based proteomics: applications for enzyme and inhibitor discovery

Benjamin F. Cravatt

Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA

Genome sequencing projects have revealed that eukaryotic and prokaryotic organisms universally possess a huge number of uncharacterized enzymes. The functional annotation of uncharacterized enzymatic pathways, thus, represents a grand challenge for researchers in the post-genomic era. To address this problem, global molecular profiling methods hold great promise, as they provide a relatively unbiased portrait of the biochemical composition of cells and tissues and can reveal unanticipated alterations in their metabolic and signaling networks. Nonetheless, the identification and functional characterization of enzymatic pathways that support human physiology and pathology have, to date, been hindered by a lack of “systems biology” techniques that can evaluate their activity in complex biological samples. To address this problem, we have introduced functional proteomic and metabolomic technologies that record dynamics in enzyme activity in directly in native biological systems. For example, the activity-based protein profiling (ABPP) technology utilizes active site-directed chemical probes to determine the functional state of large numbers of enzymes in proteomes. In this presentation, I will describe the integrated application of ABPP and complementary functional proteomic/metabolomic methods to discover and functionally annotate enzyme activities in mammalian (patho)physiological processes, including cancer and nervous system signaling. The long-term goal of these studies is to map new biochemical pathways that play important roles in human disease and develop selective chemical tools to perturb these pathways in living systems.

4.2

Global Profiling of Proteolytic Cleavage Sites in Apoptosis

Sami Mahrus1, Jonathan Trinidad6, David Barkan5, Huy Nguyen4, Andrej Sali1,2, A.L. Burlingame6, and James Wells1,3

Departments of 1Pharmaceutical Chemistry, 2Biopharmaceutical Sciences, 3Cellular and Molecular Pharmacology, 4Hematology/Oncology, and 5Graduate Group in Bioinformatics, University of California, San Francisco, CA, USA; 6Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA

The 600 or so proteases encoded in the human genome are involved in a diversity of biological processes. Some function as nonspecific degradative enzymes associated with protein catabolism, exhaustively cleaving many protein substrates at many sites. In contrast, several others function as selective post-translational modifiers, cleaving only a few protein substrates, usually at only one or a few sites. Apoptosis is an important example of a biological process regulated by widespread but specific intracellular proteolysis, predominantly carried out by the caspase family of proteases. This genetically programmed and non-inflammatory form of cell death is a central component of homeostasis, tissue turnover, and development. Since chemotherapeutics typically kill cells by induction of apoptosis, this process is also highly relevant from a therapeutic standpoint. We have developed a novel method for global profiling of proteolytic cleavage sites in complex biochemical mixtures based on use of an engineered peptide ligase, termed subtiligase, for selective biotinylation of free protein N-termini and positive enrichment of corresponding N-terminal peptides. Using this method to study apoptosis, we have sequenced 333 caspase-like cleavage sites distributed among 292 protein substrates in the acute T cell leukemia cell line Jurkat following treatment with the classic chemotherapeutic etoposide. Surprisingly, these sites are generally not predicted by in vitro caspase substrate specificity, but can be used to predict other physiological caspase cleavage sites. Structural bioinformatic studies show that caspase cleavage sites often appear in surface accessible loops and even occasionally in helical regions. We also find that a disproportionate number of caspase substrates physically interact, suggesting that these dimeric proteases target protein complexes and networks to elicit apoptosis, and that targeting of multiple components in each is required for a full commitment to apoptosis. Our current efforts are focused on quantitative analysis of how proteolysis in apoptosis varies as a function of time, target cell type, and apoptotic inducer.

Support for this research was provided by the Bio-Organic Biomedical Mass Spectrometry Resource at UCSF (A.L. Burlingame, Director) through the Biomedical Research Technology Program of the NIH National Center for Research Resources, NIH NCRR P41RR001614.

4.3

A Proteomics Approach to Overcoming Bacterial Drug Resistance: The Ribosomal QconCAT

Jill Barber1, Zubida Al-majdoub2, and Simon Gaskell2

1School of Pharmacy and Pharmaceutical Sciences and 2Manchester Interdisciplinary Biocentre, The University of Manchester, Manchester, UK

The ribosome is arguably the most important drug target in the bacterial cell, with seven distinct classes of drug inhibiting its action and assembly. These drugs include the macrolides, the safest known anti-bacterials, and the aminoglycosides, the most effective known anti-bacterials. Bacterial disease remains a global health problem not because we lack good drugs but because of the rise of bacterial resistance. The aminoglycosides also suffer from poor therapeutic indices, further limiting their use in the clinic.

We are therefore developing a proteomics approach to enhancing the lifetime and effectiveness of existing anti-bacterial drugs, especially the macrolides and aminoglycosides. We aim to identify targets whose inhibition will lead to synergy with aminoglycosides and macrolides, substantially reducing the likelihood of resistance and enhancing the effectiveness of the drugs. This requires that we can quantify the proteins of the translational machinery and determine the effect of sub-lethal antibiotic doses on the bacterial proteome.

To this end we have designed a flexible QconCAT for the quantification of the E. coli translational machinery by mass spectrometry. A QconCAT is an artificial protein, made up of signature peptides from each of the proteins under study. These peptides are concatenated together and expressed in labelled form using an artificial gene.1 Our initial QconCAT (the core) contains signature peptides from six central ribosomal proteins L2, L4 and L5 from the 50S subunit and S2, S7 and S8 from the 30S subunit. All these proteins are in place early in the ribosomal assembly process and their presence can be used as markers for ribosomes. The gene encoding the core contains restriction sites into which one or more cassettes encoding other peptides may be inserted. Thus the first cassette encodes signature peptides for the 30S ribosomal proteins.

Most of the ribosomal proteins are small and basic and a digestion protocol involving trypsin alone does not yield sufficient correctly-cleaved peptides for quantification. We therefore employ sequential digestion with endoproteinase Lys-C and trypsin to analyze ribosomal proteins, and the QconCATs are designed for this strategy.

Preliminary results on the effect of the aminoglycoside gentamycin on the bacterial proteome are described.

Reference

  1. Beynon RJ, Doherty MK, Pratt JM, Gaskell SJ. (2005) Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides. Nat Methods 2, 5879.

5.1

Characterization of the Velos, an Enhanced LTQ Orbitrap, for Proteomics

Jesper V. Olsen1,2, Michael L. Nielsen1,2, N. Eugen Damoc3, Jens Griep-Raming3, Thomas Moehring3, Alexander Makarov3 , Jay Schwartz4, Stevan Horning3, and Matthias Mann1

1Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried, Germany; 2NNF Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Denmark; 3Thermo Fisher Scientific, Bremen, Germany; 4Thermo Fisher Scientific, San Jose, USA.

Fast and efficient peptide sequencing is a cornerstone of modern proteomics. We have evaluated and characterized a new and improved version of the LTQ Orbitrap for proteomics experiments. The new instrument is different in three key aspects from the current instrument. It’s source has a significantly higher ion transmission, it encompasses a significantly faster scanning linear ion trap and it has much more efficient Higher Energy C-trap Dissociation (HCD). These features – in combination with predictive automatic gain control – and parallel detector acquisition enable short cycle times (2.8 seconds) for Top20 MS/MS acquisition in the LTQ in combination with high-resolution full scan MS detection in the orbitrap. The new instrument also present a more than ten-fold improvement in its HCD capabilities in terms of speed and sensitivity, which in combination with the new brighter ion source allow for routine Top10 MS/MS cycles. The HCD spectra provide ppm-mass accuracy on all fragments spanning the full-mass-range (no low mass cut-off). High-resolution HCD in combination with online HPLC separation allow us to identify and quantify more than one thousand proteins in single runs of a complex peptide mixture. As a consequence of the spectacular HCD performance it is now possible to analyze both precursor and fragment ions with high-mass accuracy on a chromatographic scale without sacrificing acquisition speed or sensitivity. We have analyzed a whole yeast proteome lysate by peptide IEF fractionation in combination with HCD sequencing on the Velos and compared the haploid with the diploid state using SILAC. From three replicate analyses of 24 IEF fractions we are able to identify and quantify almost four thousand proteins with very low false discovery rate.

5.2

Analysis of the Yeast Kinase-Substrate Networks by Quantitative Phosphoproteomics

Bernd Bodenmiller1,2, Stefanie Wanka2,3, Claudine Kraft4, Jörg Urban5, David Campbell6, Patrick Pedrioli4, Bertran Gerrits7, Paola Picotti1, Henry Lam6, Olga Vitek8, Mi-Youn Brusniak6, Bernd Roschitzki7, Chao Zhang9, Ralph Schlapbach7, Kevan Shokat9, Alejandro Colman-Lerner10, Alexey Nesvizhskii11, Matthias Peter4, Robbie Loewith5, Christian von Mering3, and Ruedi Aebersold1,6,12

1Institute of Molecular Systems Biology, ETH Zurich, Switzerland; 2Zurich PhD Program in Molecular Life Sciences, Switzerland; 3Institute of Molecular Biology and Swiss Institute of Bioinformatics, University of Zurich, Switzerland; 4Institute of Biochemistry, ETH Zurich, 8093 Zurich, Switzerland; 5Department of Molecular Biology, University of Geneva, Switzerland, 6Institute for Systems Biology, Seattle, WA, USA; 7Functional Genomics Center Zurich, UZH | ETH Zurich, Switzerland; 8Purdue University, Departments of Statistics and Computer Science, West Lafayette, IN, USA; 9Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA; 10Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Argentina; 11 Department of Pathology, University of Michigan, Ann Arbor, MI, USA; 12Faculty of Science, University of Zurich, Switzerland

Reversible phosphorylation of proteins, carried out by kinases and phosphatases, constitutes one of the most important regulatory mechanisms in eukaryotic cells. The various kinases, phosphatases and their substrates form a network that controls and processes the flow of information, from sensors via signaling relays to effector molecules. This network is of fundamental importance for the function and robustness of essentially all biological systems.

Here, we describe a label-free, quantitative phosphoproteomics approach to determine the in vivo relationship between 97 kinases, 28 phosphatases and their cellular substrates in S. cerevisiae. We systematically detected and identified phosphopeptides that showed a significant and reproducible change in their abundance upon removal (or inactivation) of each of these kinases or phosphatases. In total, we identified over 7,000 regulated phosphorylation events on 1,213 substrate proteins, describing the first system-wide in vivo protein phosphorylation network of S. cerevisiae. Our data indicate the extent of biological activity of each kinase and phosphatase under the tested condition, associates many of them with specific cellular functions, and allows correlation of regulated phosphoproteins with yeast growth and morphological phenotypes. Overall, these results expand the systems-level understanding of phosphorylation-modulated signaling in yeast.

5.3

Quantitative phosphoproteomics to define kinase-substrate relationships in cell division

Judit Villén1, Liam J. Holt2, Brian B. Tuch2, Alexander D. Johnson2, David O. Morgan2 and Steven P. Gygi1

1Harvard Medical School, Boston, MA, USA; 2University of California, San Francisco, CA, USA

Protein phosphorylation is a main regulatory switch in the cell, controlling processes such as cell division, growth, proliferation, differentiation and survival. This control is performed by intricate signaling networks, which are capable of altering protein activities and rapidly communicating messages from different extracellular or internal cues to ultimately promote adequate cell readjustments. Numerous studies have addressed protein phosphorylation over the past decades, often on a single protein/pathway level. However, the global picture of signaling events can only be accomplished from comprehensive studies, which are becoming attainable by mass spectrometry (MS)-based proteomics.

The main difficulty in MS large-scale phosphorylation studies is the limitation in detecting phosphorylated species within complex sample mixtures due to their low abundance. However, the past five years have seen a steady improvement in phosphopeptide enrichment and MS data acquisition methods along with the development of computational tools for data analysis and validation, allowing us to routinely identify thousands of phosphorylation events from a single experiment. An overview of these efforts and the current status of technologies to profile the phosphoproteome will be given.

Furthermore, these strategies have been combined with stable isotope labeling for quantitative studies where two cell populations in different phosphorylation status are compared. One of the most challenging problems in signal transduction is establishing kinase-substrate relationships. We have combined chemical genetics and large-scale quantitative phosphoproteomics to identify in vivo substrates of the master mitotic kinase Cdk1 and meiotic kinase Ime2 in the budding yeast Saccharomyces cerevisiae. Using this approach, we expanded the number of known bona fide substrates to ~400 for each kinase, and pinpointed the precise sites of phosphorylation. We observed that substrates for Cdk1 and Ime2 vastly overlap; however the sites targeted and the linear motif preferred seem to differ, which provides an example on how different layers of regulation are assembled in complex systems.

5.4

,p>Global Analysis of Cdk1 Substrate Phosphorylation Sites in vivo

Liam J. Holt1, Judit Villén2, Brian B. Tuch3, Alexander D. Johnson3, Steven P. Gygi2, and David O. Morgan1

1Departments of Physiology and Biochemistry & Biophysics, and 3Microbiology and Immunology, University of California, San Francisco, CA, USA; 2Department of Cell Biology, Harvard Medical School, Boston, MA, USA

To explore the mechanisms and evolution of cell-cycle control, we analyzed the position and conservation of large numbers of phosphorylation sites for the cyclin-dependent kinase Cdk1 in the budding yeast Saccharomyces cerevisiae. We combined specific chemical inhibition of Cdk1 with quantitative mass spectrometry to identify the positions of 547 phosphorylation sites on 308 Cdk1 substrates in vivo. Comparisons of these substrates with orthologs throughout the ascomycete lineage revealed that the position of most phosphorylation sites is not conserved in evolution; instead, clusters of sites shift position in rapidly evolving disordered regions. We propose that regulation of protein function by phosphorylation often depends on simple nonspecific mechanisms that disrupt or enhance protein-protein interactions. The gain or loss of phosphorylation sites in rapidly evolving regions could facilitate the evolution of kinase signaling circuits.

6.1

Characterization and Quantification of Phosphosites in the Proteome of Human Primary T-Lymphocytes.

M.Carrascal, V.Casas, D.Ovelleiro, M.Gay, E.Gelpí and J.Abián

Laboratorio de Proteómica-CSIC/UAB, IIBB-CSIC, IDIBAPS, Barcelona, Spain.

T lymphocytes mediate cellular and humoral defense against foreign bodies or autoantigens. Protein phosphorylation-dephosphorylation is involved in many aspects of lymphocyte activation. In this context we are interested in the characterization of phosphoproteins and p-sites in human primary T-cells and we are studying the quantitative changes produced by different activators on the phosphoproteome of these cells. For qualitative analysis we used a strategy based on a multidimensional separation, involving preparative SDS-PAGE for prefractionation, in-gel digestion and sequential phosphopeptide enrichment using IMAC and TiO2 before LC-MSn analysis in an LTQ linear ion trap. Quantitative analyses are being performed by 2D-PAGE as well as by LC MS/MS using ITRAQ labeling.

Using these procedures we have identified more than 400 high confidence p-sites in resting cells, some of which had not been described previously, and quantified near 150 p-sites in resting lymphocytes vs lymphocytes treated for 4-hours with PMA-ionomicine. The complementarily of both enrichment techniques is also shown.1

The complete set of data obtained so far is stored in our LymPHOS database that is publicly available at https://lymphos.org.2 We have implemented an automatic workflow for the annotation of the database that includes tools for MS data filtering and accurate phosphorylation site assignation. The information stored in the database comprises most experimental and spectrometric data and data analysis information. All the spectra supporting each assignation are stored and presented in graphical form. Experimental data for each experiment is provided in a document that follows PSI MIAPE guidelines.

This data constitutes the only phosphorylation map available for human primary T-lymphocytes. Several novel lymphocyte specific p-sites are described and these could be a source of information for future studies on the role of phosphorylation in T-cell functions and the effect of pharmacological and immunological agonists and conditions in T-lymphocyte activation.

References

  1. Carrascal et al. (2008) Phosphorylation analysis of primary human t lymphocytes using sequential IMAC and titanium oxide enrichment. J. Proteom.Res.
  2. Ovelleiro and Carrascal et al. (2009) LymPHOS: Design of a phosphosite database of primary human T cells. Proteomics .

6.2

Analysis of Ubiquitin Chain Editing by Quantitative Mass Spectrometry

Donald S. Kirkpatrick1, Lilian Phu1, Daisy J. Bustos1, Jennie R. Lill1, Ivan Bosanac2, Sarah G. Hymowitz2, Vishva M. Dixit3, Michael H. Glickman4

Departments of 1Protein Chemistry, 2Protein Engineering, and 3Physiological Chemistry, Genentech Inc, S. San Francisco, CA, USA; 4Technion Institute, Haifa, Israel

The assembly of a ubiquitin signal on a protein substrate requires the coordinated actions of E1-activating, E2-conjugating and E3-ligase enzymes. Ubiquitin signals are recognized by ubiquitin receptor proteins that recruit modified substrates to the proteasome for degradation or into a multi-protein signaling/trafficking complexes. Both of these processes are opposed by ubiquitin isopeptides which disassemble ubiquitin signals and prevent ubiquitin dependent processes. Recent evidence suggests that fine tuning of cellular processes by the ubiquitin system can occur through the process of ubiquitin editing or chain remodeling- conversion of one ubiquitin signal into another by concerted disassembly and reassembly efforts. To further investigate this process and evaluate its role in cells, we have implemented the Ubiquitin-AQUA method. Ubiquitin-AQUA measures the amount of each polyubiquitin linkage relative to isotopically labeled internal standard peptides designed toward the tryptic branched signature peptides. Isotopically labeled unbranched peptides from ubiquitin are used to quantify the total amount of ubiquitin. Digested peptides and isotopically labeled standards are analyzed either by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap or by multiple reaction monitoring on a QTrap mass spectrometer. To improve analysis of the N-terminus of ubiquitin and as well as signature peptides toward K6-linked and linear polyubiquitin chains, we have defined conditions for oxidizing these Met containing peptides to the sulfoxide and sulfone states. Quantification of total ubiquitin based upon multiple loci is performed to minimize possible interference from complex ubiquitin signals. Using these methods we provide evidence that the majority of substrate bound ubiquitin in cells at the steady state is conjugated to substrates as mono- rather than poly-ubiquitin. Furthermore, by combining quantitative mass spectrometry with ubiquitin linkage specific antibodies, we can show that polyubiquitinated substrates purified from cells may be modified by more than one chain linkage. These observations provide a cellular context to our growing understanding of ubiquitin editing.

6.3

Age Determination in the Adult Human Brain and Body Using Bomb-Carbon

K.L. Spalding1, O. Bergmann1, S. Bernard2, H. Druid3, B. Buchholz4, P.A. Arner5, J. Frisen1

Departments of 1Cell and Molecular Biology, 3Forensic Medicine, and 5Medicine, Karolinska Institute, Stockholm, Sweden; 2Institut Camille Jordan, University of Lyon, France; 4Lawrence Livermore National Laboratories, Livermore, CA, USA

Much of the impetus in regenerative medicine is fuelled by the prospect of promoting cell replacement, or blocking unwanted cell production. Without knowing, however, if a specific cell type is renewed in the healthy or pathological situation, it remains uncertain whether it may be realistic and rational to modulate this process. Despite the importance of this information, remarkably little is known about the age of cells in many regions of the adult human brain and body. This is largely due to difficulties in studying this process in humans. Using recently established methodology, which integrates biomedical approaches with recent developments in nuclear physics, it is possible to establish the turnover of cells in human tissues. By measuring 14C derived from nuclear bomb tests in DNA, it is possible to retrospectively establish the birth date of cells. Findings of neuronal turnover in the adult human brain, fat cell turnover in humans (lean and obese), and age-determination in humans will be discussed.

7.1

Signaling to Transcription Networks in Nerve Injury Response

Izhak Michaelevski1, Yael Segal-Ruder1, Ophir Shalem2, Katalin F. Medzihradszky3, Meir Rozenbaum1, Giovanni Coppola4, Daniel Geschwind4, Yitzhak Pilpel2, A.L. Burlingame3, and Mike Fainzilber1

Departments of 1Biological Chemistry and 2Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel; 3Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA, USA; 4Center for Neurobehavioral Genetics, University of California, Los Angeles, CA, USA

Investigations of the molecular mechanisms underlying responses to peripheral nerve injury have highlighted the importance of the retrograde transport system in axons, showing that a protein complex containing importin b1, vimentin and p-ERK 1/2 associates with dynein motors to signal retrogradely from an axonal lesion site to the neuronal cell body. In order to obtain a comprehensive view of the signaling and transcriptional networks involved in retrograde injury signaling, we have now applied proteomics approaches to characterize axonal signaling ensembles, and transcriptomics to characterize the cell body response. These efforts yielded large data-sets, with over 879 proteins and 2465 phosphorylation sites implicated in axonal retrograde injury signaling in rat sciatic nerve, and ~4500 transcripts regulated in the cell body response in dorsal root ganglia. Computational tools were then employed to link these data-sets, thus identifying combined signaling to transcription networks underlying the response to nerve injury. Network redundancies suggest a high level of robustness in the injury response system.

7.2

Regulation of Neuronal Protein Levels at Sub-cellular Sites Distant from the Cell Body

J. Coleman, S. Alda, D. Vuppalanchi, S. Yoo, D.E. Willis, and J.L. Twiss

Nemours Biomedical Research, A.I. duPont Hospital for Children, Wilmington, DE, USA; Department of Biology, University of Delaware, Newark, DE, USA

Localized synthesis of new proteins in subcellular regions provides a means to rapidly respond to extracellular stimuli and cellular events. In the nervous system, protein synthesis in dendrites plays a role in synaptic plasticity and proteins generated locally in axons are used for growth and injury responses. With geographically separated processes, neurons are a very attractive model system to study protein dynamics in subcellular compartments. We have used several approaches to determine what proteins are generated locally in axonal process and how transport and localized translation of the mRNAs is regulated. mRNAs are transported into axons as ribonucleoprotein complexes. Cues that regulate directionality of axonal growth modify both the populations of mRNAs sent into axons and the localized translation of mRNAs. Response to some stimuli, including axotomy, also requires localized proteolysis. The majority of studies of these mechanisms have been limited to analyses of functional responses or single protein species. Analyses of mRNAs have shown that their transport into axonal processes is regulated with a surprising degree of specificity. It is highly likely that mRNA translation will show similar complexity with specific changes in protein production and protein degradation being linked to extracellular events. Understanding the specificity of these events will require unbiased approaches to quantify proteins dynamics in axons and dendrites. Knowledge of the molecular composition of the transported ribonucleoprotein complexes will also be needed to dissect regulatory mechanisms underlying specificity of mRNA transport into subcellular regions. However, the limited quantities of materials derived from cultured neurons where these cellular processes can be isolated to purity have thus far restricted proteomics approaches.

7.3

Organelle Proteomics: Linking Axonal Transport to Nerve Regeneration

Namiko Abe1, Angels Almenar-Queralt2, Concepcion Lillo2, Zhouxin Shen2, Jean Lozach2, Steven P. Briggs2, David S. Williams2,3, Lawrence S. B. Goldstein2, and Valeria Cavalli1

1Washington Unviersity in St Louis, MO, USA; 2University of California, San Diego, CA, USA; 3University of California, Los Angeles, CA, USA

The extreme polarized morphology of neurons poses a challenging problem for intracellular trafficking pathways. The distant synaptic terminals must communicate via axonal transport with the cell soma for neuronal survival, function and repair. Multiple classes of organelles transported along axons may establish and maintain the polarized morphology of neurons, as well as control signaling and neuronal responses to extracellular cues such as neurotrophic or stress factors. We reported previously that the motorbinding protein Sunday Driver (syd), also known as JIP3 or JSAP1, links vesicular axonal transport to injury signaling. To better understand syd function in axonal transport and in the response of neurons to injury, we developed a purification strategy based on antisyd antibodies conjugated to magnetic beads to identify sydassociated axonal vesicles. Electron microscopy analyses revealed two classes of sydassociated vesicles of distinct morphology. To identify the molecular anatomy of syd vesicles, we determined their protein composition by mass spectrometry. Gene ontology analyses of each vesicle protein content revealed their unique identity and indicated that one class of syd vesicles belong to the endocytic pathway, while another may belong to an anterogradely transported vesicle pool. To validate these findings, we examined the transport and localization of components of syd vesicles within axons of mouse sciatic nerve. Together, our results lead us to propose that endocytic syd vesicles function in part to carry injury signals back to the cell body, while anterograde syd vesicles may play a role in axonal outgrowth and guidance.

7.4

Quantitative Phosphoproteomics Identifies Sites in K-Cl Co-transporters that Regulate Cell Volume and Neuronal Excitation

Jesse Rinehart1,2, Yelena D. Maksimova4, Jessica E. Tanis5, Kathryn L. Stone2,3, Caleb A. Hodson1, Junhui Zhang1, Mary Risinger6, Weijun Pan7, Dianqing Wu7, Christopher M. Colangelo2,3, Biff Forbush5, Clinton H. Joiner6, Erol E. Gulcicek2,3, Patrick G. Gallagher4, and Richard P. Lifton1,2

Departments of 1Genetics, Howard Hughes Medical Institute, 4Pediatrics, 5Cellular and Molecular Physiology, and 7Pharmacology, Yale University School of Medicine, New Haven, CT, USA; 2Yale/NHLBI Proteomics Center, Yale University, New Haven, CT, USA; 3Keck Biotechnology Resource Laboratory, New Haven, CT, USA; 6Cincinnati Comprehensive Sickle Cell Center, Division of Hematology/Oncology, University of Cincinnati College of Medicine & Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA

Modulation of intracellular chloride concentration ([Cl-]i) plays a fundamental role in cell volume regulation, response to osmotic stress, and neuronal excitation. Cl- exit via K-Cl co-transporters (KCCs) is a major determinant of [Cl-]i, however the mechanisms governing their activities are poorly understood. Using a SILAC approach, we identified two highly conserved phosphorylation sites in human KCC3 that are de-phosphorylated in response to cell swelling. Alanine substitutions at these sites result in constitutively active cotransport. Quantitative studies utilizing multiple reaction monitoring (MRM) confirm that these same sites are modulated during neuronal maturation in vivo. Parallel MRM studies also showed that these sites are highly phosphorylated in plasma membrane KCC3 in isotonic conditions, suggesting that de-phosphorylation increases KCC3’s intrinsic transport activity. Studies with phospho antibodies demonstrate how human red blood cells control cell volume via a similar mechanism. Inhibition of the kinase WNK1 via RNAi reduces phosphorylation at these sites. Phosphopeptide mapping via mass spectrometry and antibody based validation studies showed that homologous sites are phosphorylated in all human KCCs. These findings provide new insights into regulation of cell volume and neuronal function and highlight the power of emerging proteomic techniques.

8.1

N-terminal & ‘Genome Free’ Proteomics; de novo Sequence Analysis by a Combination of LysN Protein Digestion and Electron Transfer Dissociation

Albert J.R. Heck

Biomolecular Mass Spectrometry and Proteomics Group, Utrecht University, and Netherlands Proteomics Centre, Utrecht, The Netherlands

In this talk targeted novel targeted proteomics technologies will be discussed used to analyze I) protein N-termini and II) proteomes of species of uncharacterized genomes.

Although N-terminal processing of proteins is an essential process, not many large inventories are available, in particular not for human proteins. Using modern day mass spectrometry based proteomics techniques it is now possible to unravel N-terminal processing in a semi-comprehensive way. Strong cation exchange chromatography with improved separation of singly charged peptides was exploited for the targeted analysis of N-acetylated protein termini from human HEK293 cells. Taking advantage of the complementarity between Lys-N, Lys-C, and trypsin for protein digestion, a total of 1391 non-redundant acetylated protein N-termini could be identified in a multi-protease approach, representing the largest dataset of human acetylated protein N-termini to date. Sequence analysis and comparison of the dataset with related datasets from D. melanogaster, S. cerevisiae and H. salinarum provides new insights into N-terminal processing across these species.

For species with un-sequenced or poorly characterized genomes de novo sequencing of MS/MS fragmentation spectra is essential. However, de novo sequencing is challenging due to the complexity of common CID fragmentation spectra. Lys-N enzymatic cleavage in combination with ETD analysis results in fragmentation spectra almost exclusively containing N-terminal fragment ions. These, easy to interpret, ladder sequences open up a completely new window for de novo sequencing. As a proof of concept we analyzed the proteomes of ostrich muscle and hibernating bear heart. We performed a proteomics study of ostrich through Lys-N proteolytic cleavage followed by low-pH SCX fractionation, RP-nanoLC separation and ETD dissociation. The SCX fractionation is used for isolation of the ‘de novo sequence-able’ peptides. These peptides produce fragmentation spectra, after ETD, dominated by c-type ions, which are relatively easy to interpret. De novo analyses of the ETD spectra is performed by an in-house developed algorithm, called LysNDeNovo, which utilizes the presence of a single fragment ion series to assign the peptide sequence. Our de novo sequencing approach results in a significant higher number of peptides identified than searching the ETD Ostrich proteomics dataset using the Mascot search engine. Moreover, the de novo results allow the determination of point mutations as well as conserved regions between proteins of different species.

References

  1. N. Taouatas , A.F. M. Altelaar, M. M. Drugan, A. O. Helbig, S. Mohammed and A. J.R. Heck. (2009) SCX-based fractionation of Lys-N generated peptides facilitates the targeted analysis of post-translational modifications. Mol. Cell. Proteomics 8, 190-200.
  2. S. Gauci, A. O. Helbig, M. Slijper, J. Krijgsveld, A. J.R. Heck, S. Mohammed. (2009) Lys-N and Trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem 81, 4493-4501.

8.2

Quantitative Proteomics Analysis of C/EBPα Transcription Factor Complexes in Leukemia

Jarrod A. Marto

Department of Biological Chemistry and Molecular Pharmacology, Havard Medical School, Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, MA, USA

Acute myeloid leukemia (AML) remains a highly lethal malignancy with limited therapeutic options. Full transformation in AML requires coupling of aberrant molecular events associated with multiple cellular processes. For example, recent work has linked oncogenic FLT-3 kinase, the most common molecular abnormality in acute myeloid leukemia (AML), with functional disruption of otherwise normally expressed C/EBPα, a key transcription factor in hematopoiesis. Moreover, there is growing evidence that combined FLT-3 and C/EBPα karyotypes provide useful prognostic indicators for AML patients. Despite these strong molecular and clinical links, we know surprisingly little about the mechanisms that underlie normal C/EBPα gene activation, and how FLT-3 signaling may interfere with C/EBPα function in the context of AML. We are testing the hypothesis that FLT-3 mediated phosphorylation of C/EBPα governs the assembly of transcriptionally active or repressed protein complexes. Towards this end we have established inducible expression of dual-affinity tagged C/EBPα in myeloid cells that also exhibit constitutive FLT-3 activity. We observe that inhibition of FLT-3 signaling modulates C/EBPα phosphorylation in a dose-dependent manner. Next, quantitative proteomics methodology, including iTRAQ labeling and nanoflow LC coupled with online multidimensional RP/RP fractionation was used to monitor remodeling of C/EBPα protein complexes as a function of FLT-3 mediated phosphorylation. Our data significantly expand upon the known repertoire of C/EBPα interactors, including more than 100 proteins involved in chromatin organization, transcriptional modulation, and cell cycle regulation. Furthermore, our quantitative proteomics data demonstrate that (i.) C/EBPα interacts with proteins genetically linked to leukemia; and (ii.) many of these interact with C/EBPα in a phosphorylation-dependent manner. Genetic depletion of newly-identified, leukemia-associated protein interactors reduced the ability of C/EBPα to drive expression of granulocytic target genes. In addition we have confirmed co-localization of C/EBPα and proteins identified in our proteomics analysis at the promoters of early, myeloid-specific genes. Collectively our data demonstrate direct physical and functional links between the tumor suppressor C/EBPα and other leukemia-associated, putative oncogenes. Our ability to quantitatively monitor multiple leukemia-related gene products in the context of C/EBPα protein complexes provides valuable insight into the mechanisms by which oncogenic kinase activity disrupts transcription and leads to leukemogenesis.

8.3

Rapid, Near Proteome-wide, Quantitative Analysis of Aneuploid Budding Yeast

Noah Dephoure1, Eduardo M. Torres2, Judit Villen1, Angelika Amon2, and Steven P. Gygi1

1Harvard Medical School, Boston , MA, USA; 2Howard Hughes Medical Institute, MIT, Cambridge, MA, USA

Mass spectrometry based proteomics holds the promise to extend the global analysis of gene expression afforded by DNA microarrays to the measurement of cellular proteins. Recent advances in methodology and instrumentation have brought the elusive goal of facile global protein measurement closer. However, the extensive analysis time required to achieve high protein coverage renders routine experimentation impractical for most researchers. Using stable isotope labeling with amino acids in cell culture (SILAC) and strong cation exchange chromatography we are able to routinely and reproducibly quantify ~3,000 budding yeast proteins in less than two days of instrument analysis time. We have used this method to characterize protein level changes in a collection of aneuploid yeast strains, harboring two copies of a single chromosome (disomic). In all strains examined to date, proteins coded on the disomic chromosome were enriched ~2-fold, while levels of other proteins remained constant. Such small changes can be extremely challenging to detect by traditional methods such as immunoblotting, but were easily discernible by our method. We also observed a subset of disomic gene products whose levels were unchanged and are trying to understand the mechanisms that allow these proteins to escape the increase in gene dosage.

8.4

Advancing Epigenetics Research by Proteomics: Technologies, Applications and Perspectives

Ole N. Jensen

Centre for Epigenetics and Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark

“An epigenetic trait is a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” (Berger et al, 2009).

Histone proteins plays a major role in maintaining chromatin structure, regulating gene activity and DNA integrity. Post-translational modifications of histone proteins that constitute the nucleosome modulate the interactions of these proteins with DNA, transcription factors and chromatin modifying enzymes. There is emerging evidence that combinations of post-translational modifications are key regulators of cellular development and differentiation programs, including epigenetic mechanisms, and that errors in these systems lead to a variety of diseases.

Mass spectrometry plays a prominent role in mapping and quantifying histone modifications, including multi-site, cooperative modifications of histone tails. In this lecture I will describe some of our analytical strategies that are aimed at detailed characterization of histone proteins in the context of pathogenic microorganisms (Salcedo-Amaya et al, 2009; Trelle et al , 2009), drug development (Beck et al, 2006), stem cell research and cancer (Jung et al, submitted). I will also discuss some of the current bottlenecks in functional proteomics and emerging areas of research where proteomics is likely to play a major role.

References:

  1. Beck et al (2006) Mol Cell Proteomics. 5(7):1314-25.
  2. Berger et al (2009) Genes Dev. 23, 781-783.
  3. 3. Salcedo-Amaya et al (2009) Proc Natl Acad Sci U S A. 106(24):9655-60.
  4. 4. Trelle et al (2009) J Proteome Res. 8(7):3439-3450.

National Institute of General Medical SciencesAdelson Medical Research Foundation