Poster Session C

C.1 Epitope Tagging of Endogenous Proteins in Human Cells for Discovery of Novel Protein/Protein Interactions T. Waldman(1), J. Kim(1), W. Lane(2), and F. Bunz(3,) (1)Georgetown Medical School/Lombardi Cancer Center, Washington, DC; (2)Harvard University, Boston, MA; (3)Johns Hopkins School of Medicine, Baltimore, MD Epitope tagging is a powerful and commonly used approach for studying the physical properties of proteins and their functions and localization in eukaryotic cells. In the case of Saccharomyces cerevisiae, it has been possible to exploit the high efficiency of homologous recombination to tag proteins by modifying their endogenous genes, making it possible to tag virtually every endogenous gene and perform genome-wide proteomics experiments. However, due to the relative inefficiency of homologous recombination in cultured human cells, epitope-tagging approaches have been limited to ectopically expressed transgenes, with the attendant limitations of their nonphysiological transcriptional regulation and levels of expression. To overcome this limitation, a modification and extension of adeno-associated virus-mediated human somatic cell gene targeting technology is described that makes it possible to simply and easily create an endogenous epitope tag in the same way that it is possible to knock out a gene. Using this approach, we have created and validated human cell lines with FLAG-tagged alleles of the p53 and PTEN tumor suppressor genes in a variety of untransformed and transformed human cell lines. FLAG immunoprecipitation followed by mass spectrometry enabled the identififcation of both known and novel p53 and PTEN interacting proteins. This straightforward approach makes it possible to study the physical and biological properties of endogenous proteins in human cells without the need for specialized antibodies for individual proteins of interest. C.2 Quantitative Analysis of the Phosphoproteome of Resting and Activated Human Primary T Cells P. Ruperez(1), J. A. Oses-Prieto(1), A. Gago(2), and A. L. Burlingame(1) (1)Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA; (2)Department of Analytical and Food Chemistry, University of Vigo, Vigo, Spain Protein phosphorylation-dephosphorylation events play a primary role in regulation of almost all aspects of cell function including signal transduction, cell cycle or apoptosis. The activation of the immune response mediated by T lymphocytes is dependent on changes of the phosphorylated state of different proteins. Consequently, Indiana, USA order to understand the functionality of lymphocytes during the immune response we need to study the changes in phosphorylation profiles that occur in these cells in response to activation. Here we present a quantitative mass-spectrometry based analysis of the phosphoproteome of primary T cells, comparing resting cells with cells stimulated for 5 min using an anti-CD3 antibody. Relative phosphopeptide levels were quantified by stable isotope labeling methodology with i-TRAQ reagent, using 2 mg of total protein amounts in the combined extract. Titanium dioxide chromatography for phosphopeptide enrichment was performed followed by a fractionation by strong cation exchange chromatography. Fractions were analyzed by nano-LC-ESI-Qq-TOF mass spectrometry on a QStarElite instrument. A total of 139 phosphopeptides were identified from 101 proteins. Changes in relative levels were detected in 59 phosphopeptides; 43 of them increased in the activated T cells and 16 increased in the resting ones. All of the phosphorylation sites identified were on serine and threonine. Among all the proteins characterized we found several involved in the cytoskeleton reorganization, signal transduction, kinase activity, RNA binding. Some of these proteins are described to be part of the early events of T cell activation. Research supported 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 RR012961. C.3 ATAQS Computational Software Tool for High Throughput Transition Optimization and Validation for SRM Mi-Youn Brusniak(1), D. Campbell(1), J. Chen(1), M. Christiansen(1), E. Deutsch(1), C. Kwok(1), S. Letarte(1), H. Ramos(1), P. Picotti(2), L. Reiter(2), J. Watts(1), and R. Aebersold(2) (1)Institute for Systems Biology, Seattle, WA; (2)ETH - Swiss Federal Institute of Technology, Zurich, Switzerland SRM has recently emerged as a popular approach for targeted proteomics since it provides improved dynamic range, sensitivity and reproducibility vs. shotgun based identification and quantification of peptides. SRM uses a tandem-in-space mass spectrometer, which provides two levels of mass screening (i.e. precursor and fragment ion) yielding better sensitivity and dynamic range. SRM experiments are thus hypothesis-driven, since they require a priori knowledge of the target peptides of interest, the mass of the precursors, and the most intense fragment ions for each target peptide. There is open source software like TIQAM-ProteinDigestor, for generating in silico digestion and fragmentation transitions and TIQAMViewer, for assisting users with validation of transitions by visualizing MS2-triggered SRM scans. However, these tools lack high throughput validation capability, and do not support heavy and isotopically normal peptide pair measurements for better identification and quantification. To the best of our knowledge, there is no open source software that provides for high throughput methods for validating measured transitions, based on sample type and complexity. SRM validation and quantification using current commercial software obtainable from MS vendors is typically a highly manual process, which is also subjective, and thus inherently variable. We introduce a new software pipeline: ATAQS (Automated and Targeted Analysis with Quantitative SRM). ATAQS provides an easy to use web browser interface to generate and filter candidate peptides of interest, and to generate, filter and validate peptide transitions. ATAQS also introduces a new proposed file format:TraML (Transition Markup Language) as a common data exchange format for validated transitions by downloading published validated transitions, and publishing your own validated transitions to a public SRM database such as MRMAtlas4. ATAQS provides algorithms to generate decoy SRM transitions for scoring and validation of transitions in automated manner. Finally, ATAQS provides a flexible pipeline for end users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists by easy extension of java algorithm classes for their own algorithm plug-in classes. C.4 Mass-Spectrometric Identification and Relative Quantification of N-linked Cell Surface Glycoproteins B. Wollscheid(1), D. Bausch-Fluck(1), C. Henderson(2), R. O'Brien(2), M. Bibel(3), R. Schiess(1), R. Aebersold(1,2), and J. D. Watts(2) (1)ETH - Swiss Federal Institute of Technology, Zurich, Switzerland; (2)Institute for Systems Biology, Seattle, Washington, USA; (3)Novartis Institutes for Biomedical Research, Basel, Switzerland Although the classification of cell types often relies on the identification of cell surface proteins as differentiation markers, flow cytometry requires suitable antibodies and currently permits detection of only up to a dozen differentiation markers in a single measurement. We use multiplexed mass-spectrometric identification of several hundred N-linked glycosylation sites specifically from cell surface-exposed glycoproteins to phenotype cells without antibodies in an unbiased fashion and without a priori knowledge. We apply our cell surface-capturing (CSC) technology, which covalently labels extracellular glycan moieties on live cells, to the detection and relative quantitative comparison of the cell surface N-glycoproteomes of T and B cells, as well as to monitor changes in the abundance of cell surface N-glycoprotein markers during T-cell activation and the controlled differentiation of embryonic stem cells into the neural lineage. A snapshot view of the cell surface N-glycoproteins will enable detection of panels of N-glycoproteins as potential differentiation markers that are currently not accessible by other means. References 1. Wollscheid et al. (2009) Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat. Biotechnol. 27(4), 37886. C.5 Chronic Ethanol Feeding Affects Proteasome Interacting Proteins M. P. Bousquet-Dubouch(1), S. Nguen(2), D. Bouyssié(1), O. Burlet-Schiltz(1), S. W. French(2), B. Monsarrat(1), and F. Bardag-Gorce(2) (1)CNRS IPBS, Toulouse University, France; (2)Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, CA Studies on alcoholic liver injury mechanisms show a significant inhibition of the proteasome activity. To investigate this phenomenon, we isolated proteasome complexes from the liver of rats fed ethanol chronically, and from the liver of their pairfed controls, using a nondenaturing multiple centrifugations procedure to preserve Proteasome Interacting Proteins (PIPs). Isotope-Coded Affinity Tagging (ICAT) and MS/MS spectral counting, further confirmed by Western blot, showed that the levels of several PIPs were significantly decreased in the isolated ethanol proteasome fractions. This was the case of PA28a/b proteasome activator subunits, and of three proteasome-associated deubiquitinases, Rpn11, ubiquitin C terminal hydrolase 14 (Usp14), and ubiquitin carboxyl-terminal hydrolase L5 (UCHL5). Interestingly, Rpn13 C-terminal end was missing in the ethanol proteasome fraction, which probably altered the linking of UCHL5 to the proteasome. 20S proteasome and most 19S subunits were however not changed but Ecm29, a protein known to stabilize the interactions between the 20S and its activators, was decreased in the isolated ethanol proteasome fractions. It is proposed that ethanol metabolism causes proteasome inhibition by several mechanisms, including by altering proteasome interacting proteins and proteasome regulatory complexes binding to the proteasome. C.6 Phosphorylation Dynamics at Synapses in the Central Nervous System J. C. Trinidad(1), A. Thalhammer(2), R. Schoepfer(2), and A. L. Burlingame(1) (1)Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA; (2)Department of Pharmacology, University College London, United Kingdom Glutamate is the main excitatory neurotransmitter in the central nervous system. This neurotransmitter is detected by ligand-gated ion channels in the membrane of postsynaptic neurons. These receptors are associated with a complex protein network known as the postsynaptic density (PSD). The PSD contains proteins of many functional classes including kinases/phosphatases, scaffolding molecules, and structural molecules. In addition to its primary role of mediating fast synaptic transmission, the PSD is a dynamic structure that alters its composition over longer time periods. Regulation of protein levels and post-translational modifications affects the efficacy of future synaptic transmission events and plays a role in such processes as learning and memory. In addition, many nervous system diseases affect synaptic protein levels and phosphorylation states. Using an established two-step sucrose density fractionation protocol, we have isolated postsynaptic density fractions. In previous work, we demonstrated that tryptic digestion of these PSD preparations, followed by a multidimensional LC-MS/MS approach, can allow for the identification of over 1200 proteins. In addition, we have also previously demonstrated the methodology to isolate and identify over 1400 phosphorylated peptides from these samples, by specifically enriching for such peptides using either immobilized metal affinity chromatography or titanium dioxide beads. Using stable isotope quantitative mass spectrometry, we profiled molecular changes following synaptic activity in cortical neurons. A mouse model of epilepsy was used, where seizures were induced by intraperitoneal injection of the muscarinic acetylcholine agonist, pilocarpine. Post-synaptic densities were isolated at time-points between zero and sixty minutes post-injection. The molecular composition of synapses was found to change in a complex fashion after seizure. Subsets of proteins displayed distinct patterns of localization at the synapse. Within a given subset of proteins, expression patterns were highly correlated, while different subsets showed distinct patterns. At the level of protein phosphorylation, we observed a greater than 2-fold increase in phosphorylation of serine 845 on the GluR1 receptor, consistent with published data. Overall, less than 5% of all phosphorylation sites had robust changes in response to seizure. This work has been supported by the Wellcome Trust and BBSRC (to RS) and 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. C.7 Linaclotide, a Novel Peptide Therapeutic Agent in Clinical Development for the Treatment of IBS-C and Chronic Constipation is Digested in the Mouse and Human Small Intestine to Small Peptides M. Kessler, R. W. Busby, J. D. Wakefield, W. P. Bartolini, P. Germano, A. P. Bryant, C. B. Kurtz, and M. G. Currie Ironwood Pharmaceuticals, Inc., Cambridge, MA Linaclotide (MD-1100) is an investigational peptide therapeutic agent in clinical development for the treatment of irritable bowel syndrome with constipation (IBS-C) and chronic constipation (CC). In Phase 2 clinical trials, linaclotide significantly reduced abdominal pain, abdominal discomfort, and bloating and improved bowel function. The most common adverse event in linaclotide-treated patients was diarrhea. This 14 amino-acid peptide contains three disulfide bonds and is a potent agonist of the receptor guanylate cyclase C (GC-C) located in the intestinal lumen. Linaclotide is locally acting, has low oral bioavailability in animals, and no detectable systemic exposure in humans at therapeutic doses. Linaclotide has an active metabolite, MM-419447, which lacks the C-terminal tyrosine. Both of these peptides are stable in simulated gastric fluid and highly resistant to proteolysis under oxidative conditions. Previous studies have shown that linaclotide incubated in the presence of fluid taken from rat small intestinal ligated loops (RIF) is metabolized to MM-419447 and then the disulfide bonds in both peptides are enzymatically reduced. The reduced peptides are proteolytically degraded to small peptide fragments and ultimately to L-amino acids. The present studies were conducted to determine the metabolic fate of linaclotide in mouse intestinal fluid (MIF), obtained from ligated intestinal loops, and in human intestinal fluid (HIF), obtained from the luminal contents of cadavers. Incubation of linaclotide in MIF resulted in rapid degradation with a t1/2 of 3 min and immediate formation of MM-419447; however, this metabolite was also degraded and was not detectable after 60 min. Linaclotide is degraded in HIF with the immediate formation of MM-419447, followed by degradation to small peptide fragments. These peptide fragments were also observed when linaclotide is metabolized in RIF. HIF contains free thiols in millimolar concentrations and the components of the glutaredoxin/glutathione reductase system, as is the case with RIF. Even with rapid metabolic degradation in mice, orally administered linaclotide significantly increased intestinal fluid secretion and intestinal transit, and it decreased visceral hypersensitivity. This indicates sufficient intestinal residence time for the parent and/or active metabolite to bind to GC-C receptors and mediate the effects described on GI function. In conclusion, these studies support a linaclotide metabolism/digestion pathway in mice and human similar to the pathway observed in rats that involves formation of the active metabolite MM-419447 and reduction of the disulfide bonds in both linaclotide and MM-419447, followed by subsequent proteolytic degradation. Linaclotide is present long enough to have an effect, but its low permeability and rapid degradation result in no detectible exposure as demonstrated in clinical studies. C.8 Large Scale Multiplex Stable Isotope Dimethyl Labeling Applied to the Quantitative Analysis of Tyrosine Phosphorylation P. Boersema(1), L. Y. Foong(2), V. Ding(2), S. Lemeer(1), S. Mohammed(1), R. Raijmakers(1), B. van Breukelen(1), J. Boekhorst(1), A. B. H. Choo(2), and Albert J. R. Heck(1) (1)Utrecht University, Utrecht, The Netherlands; (2)A*STAR (Agency for Science, Technology and Research), Singapore Introduction: Accurate quantification of protein expression in biological systems is an increasingly part of proteomics research. Incorporation of differential stable isotopes in samples for relative protein quantification has been widely used. Stable isotope incorporation at the peptide level using dimethyl labeling is a reliable, cost-effective and undemanding procedure that can be easily automated and applied in high-throughput proteomics experiments and it uses inexpensive reagents and is applicable to virtually any sample(1). Here, we developed several labeling approaches and apply the method to investigate phospho-tyrosine signaling in human cells. Methods: Cell lysate digestion derived peptides are dimethyl labeled by reductive amination with isotopomers of formaldehyde (CH2O, light; CD2O, intermediate; 13CD2O, heavy) followed by reduction with cyanoborohydride (light and intermediate) or cyanoborodeuteride (heavy). Small sample amounts (< 1 μg) are labeled while trapped on a nanoLC reversed phase (RP) column that is connected online with a mass spectrometer. Larger protein amounts are labeled in-solution or on-column using RP SPE columns. Peptides derived from digestion of HeLa cells that were mock or pervanadate treated or stimulated with EGF for 0, 10 or 30 min were differentially labeled and combined. Immunoprecipitation of tyrosine phosphorylated peptides was achieved using an immobilized antibody against tyrosine phosphorylation. The eluate was analyzed by LC-MS without further affinity enrichment. Results: We developed in-solution, online and on-column protocols for stable isotope dimethyl labeling of sample amounts ranging from sub-micrograms to milligrams in a very quick and undemanding manner (1). The online and on-column methods combine the sample clean-up and dimethyl labeling steps thereby minimizing sample loss and speeding up the sample handling process. The online approach is particularly suited for minute amounts of protein since it is performed on a nano-LC system that is online with a mass spectrometer. On-column dimethyl labeling is suited for larger (milligrams) protein amounts and allows consecutive 2D-LC separation. The in-solution approach can be used when several samples are to be labeled in parallel. We applied our methods to the quantification of tyrosine phosphorylation upon pervanadate treatment and EGF stimulation. Immunoprecipitation of differentially labeled tyrosine phosphorylation allowed the quantification of differences in tyrosine phosphorylation of respectively 128 and 73 unique phosphotyrosine peptides. Most of the upregulated phosphotyrosine peptides are involved in the EGF signaling pathway, validating our approach. However, one new site and several others that have not been firmly established earlier to be involved in EGF receptor signaling were also identified. In general, we show that the combination of immuno-affinity purification of tyrosine phosphorylated peptides with large scale stable isotope dimethyl labeling and only a single LC-MS run provides a cost-effective approach to obtain a rather complete qualitative and quantitative picture of a signaling event. References 1. P. J. Boersema et al. (2009) Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics. Nat. Protoc. 4, 484–494. C.9 Structural Characterization of Novel Components from the Venom of the Mexican Scorpion Vaejovis mexicanus smithi by Electron Capture Dissociation and Electron Transfer Dissociation S. P. Salas-Castillo, K. F. Medzihradszky, S. Guan, and A. L. Burlingame Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA Polypeptides from animal venoms, such as of snakes, spiders, conus snails, scorpions or insects display remarkable biological activities. For example, antimicrobial, enzymatic and ion channels blocking properties. For this reason these polypeptide toxins may be excellent drug candidates and have great potential in agricultural applications. They could have use as tools for biological research as well. Amino acid sequences and secondary structures (if any) of these toxins have to be deciphered in order to understand the structure-biological activity relationship. In most cases, we have to rely on de novo sequencing for a majority of species because genomic information is not available. Mass spectrometry has been proven a powerful tool for such purposes. However, sequencing of these toxic polypeptides presents a special challenge. They are relatively large peptides (∼3–8 kDa), may not contain specific cleavage sites evenly spread, and may feature unexpected and unusual post-translational modifications. At the same time the very same properties may make them excellent candidates for intact peptide de novo sequencing. This work presents the characterization of some components of the crude venom from the Mexican scorpion Vaejovis mexicanus smithi. ECD and ETD analyses were utilized for sequence determination from the intact polypeptides. We compare the utility of the two dissociation techniques. We illustrate some of the difficulties in peak-picking and fragment assignment. Last but not least, we present that once tentative sequence had been determined, an in-house developed software, FAVA will help to decipher which possibility corresponds to the correct structural assignment. Research support 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, by the grants NIH NCRR RROO1614, NIH NCRR RR019934, NIH NCRR P41RR001614 and NIH NCRR RR015804. C.10 Proteomics Analysis Reveals a New Player Involved in DNA Repair and/or Replication J. Gilmore, M. E. Sardiu, S. Venkatesh, B. Stutzman, and M. P. Washburn Stowers Institute for Medical Research, Kansas City, MO DNA processes including replication, repair and transcription are tightly regulated by chromatin structure. During DNA replication in eukaryotes, the cellular machinery performing these tasks needs to gain access to the DNA. Likewise, chromatin structure provides an obstacle to the DNA repair machinery attempting to gain access the DNA lesion. Experimental evidence has led to the hypothesis that the histone octomer is removed from the DNA by histone chaperones and chromatin remodeling complexes during theses processes (Chromatin disassembly), and placed back on the DNA after the process is complete (Chromatin assembly). MudPIT analyses of all four purified histones were conducted and many proteins involved in DNA repair/replication were identified. In addition, a protein of unknown function designated YDL156w (Cmr1) was also identified in the analyses. Next, MudPIT analyses were performed on purified Cmr1 and on other proteins that were relatively abundant in the Cmr1 pull down. Hierarchical clustering analysis was performed on the relative protein abundances expressed as dNSAFs. Results indicate that Cmr1 is in close proximity to proteins involved in DNA repair and replication. GOstat analysis was performed on the gene products identified from MudPIT analysis of purified Cmr1. The collection of proteins pulled down by Cmr1 was enriched for several biological processes including DNA damage response and chromatin assembly/disassembly. Cmr1 associated proteins were confirmed using size exclusion chromatography in conjunction with MudPIT analyses, and suggests that Cmr1 does associate with proteins involved in DNA replication and/or repair. Furthermore, proteinprotein interactions with Cmr1 are being validated using Co-IPs. C.11 Quantitative Comparison of the Shigella dysenteriae Proteome Assessed by Two Label-free Global Profiling Methods, APEX and 2D gels S. Kuntumalla(1), J. C. Braisted(1), S.-T. Huang(1), P. P. Parmar(1), D. J. Clark(1), H. Alami(1), Q. Zhang(2), A. Donohue-Rolfe(2), S. Tzipori(2), R. D. Fleischmann(1), S. N. Peterson(1), and R. Pieper(1) (1)Pathogen Functional Genomics Resource Center, J. Craig Venter Institute, Rockville, MD; (2)Cummings School of Veterinary Medicine, Tufts University, North Grafton, MA The human pathogen Shigella dysenteriae serotype 1 (SD1) is the most virulent of the four Shigella serotypes and is a causative agent of shigellosis. In this study, the stationary phase proteome of SD1 was quantitatively analyzed in Coomassie Brilliant Blue (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method for Absolute Protein Expression Measurements (APEX). Of the 4502 genome-predicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A Spearman rank correlation analysis revealed a reasonably good correlation (Rs = 0.81) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. To assess APEX abundance measurements in the context of subunit stoichiometries for characterized multi-subunit protein complexes, we compared protein abundance ratios from the SD1 APEX dataset with stoichiometric ratios designated for orthologous E. coli protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, including chaperones and proteins involved in energy metabolism. The observed APEX stoichiometry was also indicative of the biological function and dynamics of protein complexes such as peroxidases, where the examined stationary phase of SD1 cells appeared to favor the reduced, active state linked to substrate reduction, thus demonstrating versatile applications of the APEX methodology in quantitative proteomics. This work was supported by the National Institute of Allergy and Infectious Disease, National Institutes of Health, Department of Health and Human Services (NIAID/NIH/DHHS) under contract number N01-AI-15447 to the Pathogen Functional Genomics Resource Center at JCVI. At Tufts University, this project has been funded in whole or in part with Federal funds from NIAID/NIH/DHHS under contract number N01-AI-30050. C.12 Characterization of Protein Kinase C-catalyzed CYP3A4 Phosphorylation by LC Tandem Mass Spectrometry Y. Q. Wang(1), S. Guan(2), A. L. Burlingame(2), and M. A. Correia(2) (1)Department of Cellular and Molecular Pharmacology and (2)Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA CYP3A4, a major human liver P450, is degraded via the ubiquitin-dependent 26S-proteasomal pathway. In the course of this degradation, the protein is phosphorylated and this phosphorylation is enhanced after its structural inactivation by cumene hydroperoxide (CuOOH). We have previously identified three sites: S478 phosphorylated by undefined liver cytosolic kinases, T264 and S420 by protein kinases (PKA and PKC). To determine the physiological relevance of this CYP3A4 phosphorylation we heterologously expressed wild type and CYP3A4 with single, double or triple Ala-mutations of these residues in S. cerevisiae. Our findings indicated that the CYP3A4 S478A mutant was significantly stabilized in yeast strains and this stabilization was further synergized by coupled T264A and S420A mutations. However, these mutations greatly reduced, but did not totally abolish CYP3A4 phosphorylation. To further obtain a more complete characterization of CYP3A4 phosphorylation, we applied mass spectrometric analyses to identify the CuOOH-inactivated CYP3A4 phosphorylation sites after in vitro incubation with several kinases including PKC. Six new sites phosphorylation signals of S116, S119, S131, T92, T284, and S259 together with two known sites of T264 and S420 have been identified thus far. A label free quantification method is currently applied to estimate stoichiometries of phosphorylation at those sites and these findings will aid in further investigation of functional significance of protein phosphorylation for CYP3A4. Research was supported by NIH grant GM44037, NIH NCRR P41RR001614, NIH NCRR P41RR001614 and NIH NCRR RR019934. C.13 Analysis of H5N1 Influenza Hemagglutinin Glycosylation by LC/MS/MS Utilizing Hydrazide Capture SPE and HILIC Separation of Intact Glycopeptides T. A. Blake, T. L. Williams, J. L. Pirkle, and J. R. Barr Centers for Disease Control and Prevention, Atlanta, GA Influenza hemagglutinin (HA) is the antigenic glycoprotein that binds and fuses to the cell during infection and is the primary component of seasonal vaccines. Glycosylation of HA is thought to affect the virulence of an influenza strain by interfering with the cell recognition binding site or by masking antigenic regions of the protein. Identification of differences among strains in the number/conservation of glycosylation sites as well as the size/complexity of the glycans themselves is necessary for characterizing HA from emerging strains and for determining the efficacy of alternative virus propagation systems for vaccine production. This work represents a first step towards a platform for investigating differences in HA glycosylation due to virus propagation conditions. Whole virus reagents from three strains of H5N1 influenza (rgA/Vietnam/1203/2004; A/Indonesia/05/2005; A/Bar-headed goose/Qinghai Lake/1A/05) and a reassortant virus (Ind05/PRB-RG2) grown in embryonated chicken eggs were analyzed. Solid-phase extraction (SPE) via hydrazide capture was utilized to specifically isolate tryptic glycopeptides in order to determine glycosylation site occupation via deglycosylation and subsequent reverse-phase liquid chromatography-tandem mass spectrometry (LCMS/MS). Hydrophilic interaction liquid chromatography (HILIC) was also utilized to improve LC-MS/MS analysis of intact glycopeptides. De novo peptide sequencing was used for peptide confirmation. ExPASy's GlycoMod Tool was utilized to aid in the interpretation of intact glycopeptide MS/MS data. All six predicted N-linked glycosylation sites within the N-terminal ectodomain of HA were found to be occupied for the reagent strains examined. We identified the presence of glycosylation site 3 for the bar-headed goose strain, even though the exact protein sequence for the selected bar-headed goose strain was not in the database and all other strains within this subset did not predict the presence of this site. This methodology also determined occupied glycosylation sites when the predicted site was ambiguous (i.e. NNST). This approach was then used to propose compositions for multiple glycoforms at the occupied glycosylation sites on HA from the reassortant strain (Ind05/PRB-RG2). We have applied an approach for determining N-linked glycosylation site occupation and investigating the glycans attached to those sites for tryptic digests of influenza virus samples via selective sample preconcentration and LC/MS/MS analysis. We have used this data on glycosylation site occupation/conservation to assist in the examination of intact glycopeptides generated from a tryptic digest of a reassortant H5N1/H1N1 influenza virus grown in eggs. We have proposed potential sugar compositions for the multiple glycoforms associated with the occupied glycosylation sites on HA. This approach can also be utilized to determine changes in glycosylatio