| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Molecular & Cellular Proteomics 6:S21-S33, 2007.
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
Global Quantitative Analysis of the IR Response of p53K317R Knock-in Mouse Thymocytes
E. Appella1, L. M. Miller Jenkins1, S. J. Mazur1, M. Rossi1, and Y. Xu2
1National Cancer Institute, Bethesda, MD; 2University of California, San Diego, CA
The tumor suppressor protein p53 is a sequence-specific transcription factor that has crucial roles in apoptosis, cell cycle arrest, cellular senescence, and DNA repair. Following exposure to a variety of stresses, p53 becomes post-translationally modified, with concomitant increases in activity and stability. To elucidate the functions of specific post-translational modifications, knock-in mice were generated in which Lys317 (human Lys320) was mutated to Arg in both alleles of endogenous p53, maintaining a positive charge but removing the ability of the residue to be acetylated. In comparison with wild-type mice, apoptosis in thymocytes following exposure to ionizing radiation (IR) was increased two-fold. The effect on IR on the thymocytes of these mice was then studied at the global level to identify changes in the IR-response in the mutant mice. Using quantitative mass spectrometry, the effect of IR on protein levels in either the wild type or K317R thymocytes was first determined to find proteins that were up- and down-regulated in response to IR. A similar analysis of mRNA levels was performed using RNA microarrays. When the IR effect in the wild type and K317R samples was compared, several proteins were found to be differentially expressed in the two samples. The inability of p53 to be acetylated at Lys317 has widespread effects on mRNA and protein levels. Among the proteins that show differential regulation in the K317R sample are the products of known p53 targets and proteins whose genes contain p53 response elements in their promoter. Global pathway analysis further highlights genes and proteins whose IR response is differentially affected by the mutation. The genes and proteins that show differential expression have important bearing on the role that post-translational modifications play in modulating the functions of p53.
A.2
Withdrawn
A.3
Differential Recovery of Peptides from Sample Tubes and the Reproducibility of Quantitative Proteomic Data
S. Bark and V. Hook
Department of Pharmaceutical Chemistry, University of California at San Diego, La Jolla, CA
Differential recovery of peptides can seriously compromise reproducibility and quality of quantitative proteomic data obtained by liquid chromatography-mass spectrometry (LC-MS). It is likely that differences in non-specific adsorption of peptides to sample tubes contribute to the high differences observed in recovery of peptides.
This study demonstrates large variations in reproducibility and quantitation of LC-MS data for peptides derived from tryptic digests of BSA upon incubation in different sample tubes. This variability is manifested by increased standard deviations in both BPC and specific ion intensity profiles in replicate runs of peptides prepared from the same primary BSA trypsin digest. This BSA digest was diluted to 100 femtomole/µl and incubated for 15 minutes in either regular or low-retention Eppendorf sample tubes prior to LC-MS analysis. BPC and MS data show that the average relative standard deviation (rSD) of peak intensities in the regular Eppendorf data set was significantly higher than that in the low-retention Eppendorf data set. Some individual peaks showed increased SD values in regular tubes that were up to ten-fold greater than that in low-retention tubes. It is important to note that the absolute value of intensities for peptides in either BPC or for specific ions did not favor either regular or low-retention sample tubes in initial analysis. However, longer storage for 2 weeks at a higher 1 picomole/µl concentration showed strikingly deleterious effects on recovery for peptides stored in regular tubes, with nearly complete loss of detected peptides. Clearly, these results demonstrate sample tubes are a contributor to low reproducibility and losses of peptide signals observed from LC-MS data. Significantly, low-retention tubes reduced this variability and improved peptide recoveries.
While these experiments were designed to answer the simple question of reproducibility of proteomic data for peptide samples prepared in different tubes, there are profound implications from these results. Quantitation in proteomics, especially where comparison between data sets is anticipated, requires rigorous analysis of variances in data sets consisting of replicate experiments. Assignment of statistical significance cannot be reliably undertaken without defining this variation because the differences observed must be greater than the magnitude of the variances observed in each data set. Therefore, the improved reproducibility obtained by using low-retention sample tubes from the earliest steps in sample preparation will enhance observation of important differences between biological samples in quantitative proteomic studies.
A.4
Identification of Protein Complexes Associated to p8, a Protein Related to Tumor Progression
M. P. Valacco1,2, C. Varone1, J. L. Iovanna1, S. Moreno1, and A. L. Burlingame2
1Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, UBA, Buenos Aires, Argentina; 2Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
p8 is an 8 kDa protein which was first identified in rat due to its induction during the acute phase of pancreatitis. It was later characterized in mice and humans. Various functions related to cell growth control and stress have been attributed to p8 since its mRNA levels are increased in cell lines in response to stress and mitogenic factors. An important role in tumor progression was also assigned to p8 since it has been shown that, when fibroblasts obtained from p8+/+ mice are transformed with a retroviral vector expressing oncogene E1A, they are able to induce tumor formation when injected into nude mice; while transformed fibroblasts derived from p8–/– mice do not present these tumorigenic properties.
It is known that p8 binds DNA without preference for a specific sequence, it can be phosphorylated by PKA, and acetylated by p300 in vitro; it shares biochemical properties with the High Mobility Group Proteins.
Through sequence homology analysis in databases p8 was identified in many different species of higher eukaryotes. The sequence contains a highly conserved region, which corresponds to a putative bipartite NLS (Nuclear Localization Signal).
Immunocytochemistry experiments show that p8 presents nuclear localization in sub-confluent cells, but it localizes in the cytoplasm of confluent cells. It was also observed that nuclear import of p8 is energy dependent, and that its sub-cellular localization also changes with cell cycle stage and acetylation state of the cells.
It is remarkable that being small enough to diffuse between nucleus and cytoplasm passively, p8 should posses a NLS and a strictly controlled sub-cellular localization. This suggests that p8 is forming part of multiprotein complexes and that it could even be the mediator of the translocation of these complexes.
The aim of this study is to identify the members of these complexes. In order to do this, a HEK 293 cell line, that stably expresses p8 fused to N-terminal histidine-FLAG tags, was generated. Tandem affinity purification, using anti-flag antibodies and metal affinity chromatography (nickel and cobalt) was performed. The purified complexes were digested with either trypsin or chymotrypsin and analyzed by tandem mass spectrometry to identify the proteins that are associated to p8, and to detect any possible post translational modifications on this protein.
This approach has allowed us to identify a small group of putative partners of p8, the most interesting one being Ying Yang 1, a ubiquitous transcription factor which has no NLS but presents the same sub-cellular localization pattern as p8. Further studies are in progress in order to find p8's PTMs and to verify the specificity of the identified partners.
This work was supported by NIH Grant NCRR RR001614.
A.5
Quantification of Secreted Proteins from Mycobacterium tuberculosis Identifies Alternative Secretion Pathways and Elucidates Molecular Mechanisms of Secretion
M. Champion1, P. DiGiuseppe2, and J. Cox2
1Applied Biosystems, Foster City, CA; 2Department of Microbiology & Immunology, University of California, San Francisco, CA
In microbial pathogenesis, virulence originates from multiple sources, namely genetic content, changes in gene/protein expression, and protein localization. Mycobacterium tuberculosis is an acid-fast bacillus responsible for 1.75 million deaths annually. In addition to the general secretion pathway, M. tuberculosis utilizes an alternative secretion apparatus called ESX-1, which is essential for control of host-cell responses to infection. Despite its importance in virulence, this system is poorly characterized and has few known substrates. There are five near-duplicate loci of the ESX-1 system in the genome, which further increases the difficulty in defining the nature of substrate secretion. Previous efforts to map the secreted proteome of this bacterium have been less successful because of the inability to differentiate true secreted substrates from those that are present due to lysis or sample preparation. We established and quantified secreted substrates from M. tuberculosis using a novel application of isobaric mass tags and applied it to culture supernatants from wild-type M. tuberculosis and mutants deficient for components of ESX-1. Using this technique, we identified ESX-1 substrates and determined whether duplicate paralogs are secreted in an ESX-1-dependent manner. This has enabled us to definitively illustrate that most of the substrate paralogs are secreted in the absence of functional ESX-1 machinery, uncovering the presence of alternative secretion systems that have not been characterized. We paradoxically observed a quantitative increase in the secretion of substrates dependent upon Sec in ESX-1 mutants, suggesting cross-talk between general and alternative secretion systems. Further characterization of substrates by multiple-reaction-monitoring in dominant-negative mutants has helped elucidate mechanisms of ESX-1 substrate selection and secretion.
A.6
Methodological Refinements in iTRAQ Reagent-Based "Tagless" Strategy of Identification and Purification of Soluble Protein Complexes in Bacteria
M. Dong1,3, H. Liu2,3, S. Allen2,3, S. Hall2,3, S. Fisher1,2,3, T. Hazen1,3, J. Geller1,3, M. Singer1,3, L. Yang1,3, J. Jin1,3, M. Biggin1,3, and H. E. Witkowska2,3
1Lawrence Berkeley National Laboratory, Berkeley, CA; 2Department of Cell and Tissue Biology, University of California, San Francisco, CA; 3Virtual Institute for Microbial Stress and Survival, Berkeley, CA
Currently, two principle methods are used to identify physical interactions between proteins on a genome-wide basis: two-hybrid screens and tandem affinity purification (TAP). While each method has its undeniable strengths, neither offers an unbiased universal approach for isolating multiple protein complexes from native preparations without prior genetic manipulation. As part of the Genomics: GTL Protein Complex Analysis Project (PCAP) we are developing a high throughput pipeline to purify water soluble protein complexes from Desulfovibrio vulgaris (DvH), identify their polypeptide constituents by mass spectrometry, determine their stoichiometries, and provide samples suitable for single particle EM characterization. These methods will then be used as part of PCAP's effort to model stress responses relevant to the detoxification of metal and radionuclide contaminated sites.
Our strategy uses a novel "tagless" method (1) that fractionates the water soluble protein contents of a bacterium into a large number of fractions, and then identifies the polypeptide composition of a rational sampling of these fractions using a LC MALDI TOF/TOF mass spectrometry (MS) workflow. Protein fractionation employs four orthogonal separation steps optimized to preserve protein complex integrity. Protein elution through the final stage of separation is monitored with the aid of iTRAQ reagent labeling, i.e., iTRAQ reporter ions serve as beacons that signal the presence of the parent protein in each of the examined protein fractions. By definition, a putative complex consists of co-eluting proteins.
In preparation for the high-throughput phase of the project, we have examined and refined various aspects of our protocol to improve its robustness and reliability. We have adopted an efficient, highly reproducible MS sample preparation protocol that uses 96well PVDF multiscreen plates (2). We have introduced an internal protein standard to facilitate normalization of the iTRAQ-based quantification of protein levels throughout many protein fractions. We have also developed algorithms and graphical display tools for identifying protein complexes from MS data, including a method for cluster analysis of iTRAQ data to allow detection of co-migrating polypeptides and hence putative protein complexes. Using model systems (standard proteins and Escherichia coli) as well as the target organism (DvH), we have demonstrated that (i) iTRAQ methodology provides a faithful representation of the relative abundances of polypeptides in different chromatographic fractions; (ii) co-elution of components of known complexes can be discerned even under suboptimal conditions of high sample complexity; and (iii) heteromeric and homomeric complexes of DvH are preserved throughout a multistep separation process and detected with the aid of iTRAQ labeling.
References
1. M. Dong, et al., in Proceedings of the 54th ASMS Conference on Mass Spectrometry and Allied Topics, Seattle, Washington, May 28–June 1, 2006.
2. L. J. Basa et al., in Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, Texas, June 59, 2005.
A.7
Spectral Clustering for Increasing MSMS-based Peptide Identifications and Identifying Unknown Modifications
J. Falkner1, B. Searle2, and P. Andrews1
1Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI; 2Proteome Software, Portland, OR
Spectral clustering is an orthogonal approach to database-driven peptide identification using tandem mass spectrometry (MSMS) search engines. The approach relies on comparing two spectra and determining if they represent closely related structures. This process is different from database-dependent searches because the spectral comparison does not rely on first knowing the peptide sequence(s) represented by the spectra. Spectral comparison is helpful because it can be used as an orthogonal tool for quickly picking out known peptide identifications and for finding other related spectra that may not have been identified. This work summarizes use of a new spectral comparison algorithm, named Bonanza, with the existing Sequest, Mascot, and X!Tandem search engines. Bonanza is used to both refine the search parameters by the search engines and to intelligently pick out peptide modifications that the search engines may not have been configured to identify.
Initial results based on incorporating Bonanza in to a MSMS-based search strategy have provided significant gains in identified spectra. Bonanza identifies up to twice as many spectra, and using a control data set, it is shown that the new identifications maintain approximately the same ratio of false positives according to decoy database analysis. Additionally, Bonanza recognized many known peptide modifications and several unexpected modifications.
A.8
Withdrawn
A.9
Distinct Assembly of Proteasome Complexes Contributes to Proteome Heterogeneity and Proteolytic Function in Murine Heart and Liver
A. V. Gomes, G. Young, Y. Wang, C. Zong, O. Drews, and P. Ping
Departments of Physiology and Medicine/Cardiology, University of California, Los Angeles, CA
The proteasome system plays a critical role in governing the intracellular protein degradation process in many mammalian cell types including heart and liver. However, it remains elusive whether populations of distinct proteasome complexes are assembled in a species- and cell-type-specific fashion and if so, whether proteome biology at the organelle level contributes to tissue heterogeneity and organelle function. We report that despite the same genetic makeup, the proteome biology of proteasomes is vastly different in the heart versus in the liver from the same mammals. Employing an organelle targeted proteomic approach we comprehensively characterized the molecular composition, subunits assembly, and proteolytic function of proteasomes in these two organs. Proteasome subunits exist in either free form with designated biological functions or they are assembled into complexes (e.g., 20S) by which they perform proteolytic functions. Immunoblotting analyses of cytosolic tissue (both free and assembled) showed that the liver possessed significantly more amount of the three inducible beta subunits; beta 1i, beta 2i, beta 5i, were expressed at 7 ± 1.5, 27 ± 3.8, and 14 ± 1.9 times more in the liver (n = 3 in all analyses), respectively. Subsequently, blue native electrophoresis combined with LC/MS/MS was applied to determine the ability of incorporating/assembling free subunits into fully functional cytosolic 20S complexes. The purified liver 20S complexes contained 95 ± 18% more of beta 1i, 40 ± 8% more of beta 2i, and 90 ± 12% more of beta 5i than that of the heart (n = 3 in all analyses), documenting that the liver 20S complexes were assembled with distinct organization of subunit composition different from that of the heart. Using an assembly index we established, our analysis confirmed that the liver 20S proteasomes were organized with relatively more inducible beta subunits; whereas in comparison, the heart as an organ, incorporates a higher percentage of its free inducible subunits into the 20S complexes. The differences in 20S complexes assembly were concomitant with diversity in proteasome function of these two organs observed. The chymotrypsin like proteolytic activity of the cardiac 20S was significantly greater than that of the liver 20S complexes; whereas the caspase like proteolytic activity of the liver 20S was greater than that of the cardiac 20S complexes; the cardiac 20S proteasomes were less sensitive to epoxomicin (a specific proteasome inhibitor) mediated inhibition of beta 5 activities than the liver 20S proteasomes. Taken together, these results demonstrated an organ specific heterogeneity of 20S proteasomes in the heart and in the liver. Our studies provide a unique prospective illustrating how the diversity in proteome biology of a specific organelle amongst different organs contributes to their respective functions at the systems level.
A.10
Characterization of Intact Prion Proteins
S. Guan1, F. Li1, A. Serban2, S. B. Prusiner2,3, and A. L. Burlingame1,2
1Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, 2Institute for Neurodegenerative Diseases, and 3Departments of Neurology and of Biochemistry and Biophysics, University of California, San Francisco, CA
Prions are infectious proteins that cause neurodegenerative diseases, including Creutzfeldt-Jakob disease and "mad cow" disease. We have performed intact protein analysis of synthetic prion proteins by use of electron capture dissociation (ECD). No cleavage is observed in the protein region held together by the disulfide bond, while the exposed regions yield extensive fragmentation. Reduced or alkylated forms of the same protein produce abundant fragments throughout the entire molecule –displaying excellent sequence coverage. ECD product ion charge distribution of reduced and alkylated forms shows marked difference from that observed in the original protein.
Financial support was provided by NIH NCRR Grant RR 01614.
A.11
De Novo Sequencing of Antibody Variable Regions Using Methods for the Capture or Detection of Peptides Containing Cysteine
L. Phu, V. Pham, J. Lill, and D. Arnott
Genentech, Inc., South San Francisco, CA
Over the past decade, murine, chimeric, and fully humanized monoclonal antibodies as therapeutics have proven to be successful in the treatments of infectious, malignant and autoimmune diseases. Production of therapeutic monoclonal antibodies involves full sequence characterization, which can typically be determined using genetic information. However, if the cDNA clone or the original hybridoma is not available for nucleotide sequencing then de novo sequencing of the monoclonal antibody at the protein level is necessary.
De novo sequencing of a monoclonal antibody is typically performed by producing complementary overlapping enzymatic and chemical proteolytic digestions which are analyzed by N-terminal sequencing and tandem mass spectrometry. The antibody is composed of both constant and variable regions and the parts of the antibody that allow antigen specificity are known as Complementarity Determining regions (CDRs). CDRs from the hypervariable regions of the antibody heavy and light chains are typically the most challenging part of the molecule to de novo sequence as the sequence is unpredictable, making selection of the appropriate reagent for proteolysis challenging. CDRs, however, are typically flanked by cysteine residues, hence capture or selective detection of cysteine-containing peptides could potentially increase throughput and success rates of full-length antibody de novo sequencing.
Here we explore four methods for cysteine capture and compare them for specificity, sensitivity, and ease of use.
All three techniques were initially optimized and applied to the analysis of a well-characterized standard antibody (Herceptin) prior to application to the de novo sequence analysis of the previously uncharacterized anti-BTLA monoclonal antibody.
Although these cysteine-containing peptide capture methodologies were investigated for the purpose of de novo sequencing bio-therapeutic monoclonal antibodies, they are also useful for a variety of other protein characterization studies including disulfide mapping and proteome simplification.
A.12
Following the Fate of the Radiolabel from [32P]PEP to Protein Targets Using a Combination of Chemical and Proteomics Approaches
M. Jeyasingham1, J. Crowley2, and G. Carlson3
1University of Kansas Medical Center, Kansas City, KS; 2Biomedical Mass Spectrometry Facility, Washington University, St. Louis, MO; 3Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, KS
Phosphoenolpyruvate (PEP) is a high-energy compound with a standard free energy of hydrolysis twice that of ATP and is a direct phosphoryl group donor in the formation of prokaryotic phosphoproteins. To determine whether PEP may act similarly in eukaryotes, [32P]PEP was synthesized, purified and incubated with extensively dialyzed mouse liver extracts. The formation of radiolabeled protein targets was observed by autoradiography of 2D-PAGE gels, and the proteins labeled were very different than observed with [
32P]ATP.
Several possible mechanisms for the incorporation of label from PEP include: (1) the direct transfer of the phosphoryl group from [32P]PEP to protein targets, as in formation of covalent enzyme intermediates; (2) an enzyme catalyzed reaction carried out by a novel protein kinase that utilizes PEP as phosphoryl donor; or (3) the conversion of PEP into an intermediate that labels proteins through one of the above two mechanisms.
In examining the last possibility, we followed the fate of [32P]PEP during its incubation with liver extracts. The formation of an unexpected new metabolite (X) was observed by ascending TLC and autoradiography. Metabolite X was isolated and shown by GC-MS to be 2-phospholactate. [2-32P]-Phospholactate was subsequently synthesized and used in radiometric assays with mouse liver extracts. Analysis of these samples by SDS-PAGE and autoradiography showed that several proteins were radiolabeled. The mechanism(s) leading to this labeling is under investigation.
This work was supported by NIH Grants DK072393, P41RR00954, P60DK20579, and P30DK56341.
A.13
Protein Dynamics and Assembly of dsRNA Bacteriophage by Hydrogen/Deuterium Exchange Detected by Mass Spectrometry
B. Suchanova1, V. Manole1, S. Kang2, L. Happonen1, A. Ora1, P. Prevelige2, S. Butcher1, and R. Tuma1
1Institute of Biotechnology, University of Helsinki, Finland; 2Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL
Viral capsids are dynamic structures which undergo series of structural transformations to form infectious viruses. Icosahedral dsRNA bacteriophages from Cystoviridae family represent excellent model systems for assembly of other dsRNA viruses such as members of Reoviridae family. Cystoviruses assemble in two stages, making first an empty polymerase complex (PC) into which ssRNA precursors are subsequently packaged by a viral packaging motor. The packaged RNA is then replicated inside PC by a virion-associated RNA polymerase. Upon RNA packaging and replication the capsid lattice expands and becomes the mature viral core. These processes are regulated by a sequence of conformational changes in the major capsid protein P1. Thus, PC is a relatively simple example of molecular machine and we are interested in delineating the mechanisms involved in its assembly and regulation.
This study focuses on the dynamics and interactions of the P1 protein within the PC assembly.
We monitored the local dynamics of the P1 protein by hydrogen/deuterium exchange combined with high-resolution mass spectrometry (1). This method allowed us to resolve kinetics of region-specific exchange and map them onto the primary structure. In the absence of high-resolution tertiary structure for P1 we are developing novel bioinformatics tools that exploit the exchange kinetics in the context of medium resolution electron density map obtained by cryo-microscopy (2). This will allow delineation of subunit interfaces and at the same time extend the interpretations of the cryo-microscopy results.
References
1. Lisal, J., Kainov, D. E., Lam, T. T., Emmett, M. R., Wei, H., Gottlieb, P., Marshall, A. G., and Tuma, R. (2006) Virology 351, 73–79.
2. Jaalinoja, H. T., Huiskonen, J. T., and Butcher, J. T. (2007) Structure 15, 157–167.
A.14
Microwave-assisted Nonspecific Proteolytic Digestion and Controlled Methylation for Glycomics Application
J. Li, X. Liu, and K. Chan
NRC Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario, Canada
Nonspecific proteolytic digestion of glycoproteins is an alternative approach in glycomics and glycoproteomics. Using nonspecific enzymes, i.e. Pronase E, glycoproteins could be digested into small glycopeptides/peptides with one to six amino acid residues, which were then analyzed using mass spectrometry. However, the time-consuming digestion procedure, typically one to three days, significantly limited its application due to the low sample throughputs. In this presentation, we report a microwave-assisted nonspecific proteolytic digestion for rapid glycoprotein characterization. The conditions for microwave-assisted enzymatic digestion were evaluated with power, digestion time, temperature and enzyme/protein ratio. Under the optimized conditions, three model glycoproteins, ribonuclease B, ovalbumin and transferrin, could be digested into small glycopeptides with one to three amino acid residues within 3 min. After desalting with porous carbon cartridge, the glycopeptides were analyzed by ESI-MS and MALDI-MS. It was found that the MALDI-MS spectra were dominated by the glycopeptides containing basic amino acids (K or R). To avoid the bias in ionization efficiency, a mild methylation procedure was then introduced, in which the glycopeptides were methylated with Methyl Iodide after converting to their corresponding sodiated forms. This methylation procedure resulted in the formation of a quaternary ammonium in amino group of N-terminus amino acid, but only for the glycopeptides containing no basic amino acid residues. On the other hand, no derivatized products were detected for the glycopeptides containing basic amino acid residues. In addition, it was demonstrated that the proposed strategy was also suitable for sialylated glycoprotein analysis due to the stabilization of sialic acids methyl-esterification. Interestingly, the stability study revealed that methylated products of glycopeptides with two or more amino acids were more stable than those glycopeptides with solo amino acid (Asn). For example, when the methylated samples were heated to 50 °C for 1.5 h, the glyco-Asn peaks in MALDI-TOF spectrum vanished and the spectrum was dominated by glyco-dipeptides or glyco-tripeptides. The stability feature thus offered a visualization approach to interpret MALDI-TOF spectra for determining glycan structures and glycosylation sites.
A.15
Characterization of GSK3-dependent Circadian Phosphoproteome
K. Kaasik1, J. Allen2, K. Shokat2, L. J. Ptá
ek1, and Y-H Fu1
1Department of Neurology and 2Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
Circadian rhythms are an adaptation to the daily solar cycle and are produced by transcriptional feedback loops. Defining features like period length and self sustained oscillations are regulated by protein kinases that phosphorylate clock associated transcription factors. Glycogen synthase kinases (GSK3
and GSK3ß) are among key regulators of circadian rhythms; however the functional significance of GSK3 catalyzed protein phosphorylation in the circadian proteome is poorly understood. GSK3 protein kinase activity itself has a circadian profile. Phosphorylated N-terminus of GSK3 prevents substrate bindings and blocks access to its catalytic center. Therefore we hypothesized that a subset of GSK3 substrates are regulated in a circadian manner. In order to identify direct GSK3 targets we are using chemical genetics approach and we have designed ATP analog-specific (as) kinases, which utilize bulky ATP analogs that are not utilized by wild-type kinases. We have further refined this method to allow antibody based detection and immunoaffinity isolation of kinase substrates. Immunoprecipitation with a thiophosphate-ester specific antibody allows isolation of direct kinase substrates from complex proteomes in the presence of hundreds of other kinases, which is particularly important for GSK3 which often requires a prior phosphorylation priming event to recognize its substrates. We have determined that a subset of GSK3 substrates in liver and brain tissues have a circadian phosphorylation pattern. Furthermore, we are in the process of identifying potentially novel GSK3 targets. Knowing the specificity of GSK3 and GSK3ß substrates and their circadian regulation will assist in design of novel therapies and more efficient use of current GSK3 inhibitors in medical treatment with fewer side effects
A.16
Fast Detection and Quantification of Legionella Spp. and Legionella pneumophila
X. Pennanec1,2, A. Dufour2, C. Charrêteur1, D. Haras2, and K. Réhel2
1Laboratoire CGI, Ploemeur, France; 2Laboratoire de Biotechnologie et Chimie Marines, Université de Bretagne Sud, Lorient, France
To reduce risks of legionellosis infection, a regular monitoring of water supplies is essential; for this reason the AFNOR (Association Française de NORmalisation) has published in April 2006 a standard project which holds a "Detection and Quantification of Legionella and/or Legionella pneumophila by concentration and genic amplification by polymerase chain reaction (PCR)." It calls into question the analyses of water such as they are carried out today. In accordance with the standard currently used relating to the detection and the quantification of Legionella by filtration and put in a culture medium, an incubation period of 10 days is necessary to have final results. This time can be reduced to a single day by combining two simple analytical methods: 1) MALDI ToF mass spectrometry allows a fast screening of bacteria in samples. 2) Realtime PCR, after DNA extraction, allow the quantification of Legionella spp. and more particularly Legionella pneumophila.
MALDI ToF mass spectrometry is a simple, efficient and rapid way to screen bacteria. Indeed, whole bacteria are analysed without any preliminary lysis step. Reproducibility of obtained spectra depends on conditions of culture (medium, time of growth, ... ) and analysis (matrix, solvent, deposition method,... ), a specific method has been developed. It allows to create a database of mass spectra which makes easier the identification of studied bacteria (single or within mixtures). For analysis by PCR, two probes are used, one is specific to Legionella pneumophila genus and the other one to Legionella spp. genus. This step allows to detect and quantify the presence of Legionella pneumophila responsible for 90% of cases of legionellosis. The mass spectrometry combined to realtime PCR is interesting in terms of cost in use as well for the duration of the analysis. Few minutes are enough to know if the quantification of legionellae step by PCR, lasting 4 hours, is necessary.
A.17
Proteomic Identification of the Cerebral Cavernous Malformation Signaling Complex
T. Hilder1, M. Malone1, S. Bencharit1, J. Colicelli3, T. Haystead4, G. Johnson1, and C. Wu5
1Department of Pharmacology and 2Department of Prosthodontics, University of North Carolina, Chapel Hill, NC; 3Department of Biological Chemistry, University of California, Los Angeles, CA; 4Department of Pharmacology and Cancer Biology, Duke University, Durham, NC; 5Department of Pharmacology, University of Colorado School of Medicine, Aurora, CO
Cerebral cavernous malformations (CCM) are sporadic or inherited vascular lesions of the central nervous system characterized by dilated, thin-walled, leaky vessels. Linkage studies have mapped autosomal dominant mutations to three loci: ccm1 (KRIT1), ccm2 (OSM), and ccm3 (PDCD10). All three proteins appear to be scaffolds or adaptor proteins, as no enzymatic function can be attributed to them. Our previous results demonstrated that OSM is a scaffold for the assembly of the GTPase Rac and the MAPK kinase kinase MEKK3, for the hyperosmotic stress-dependent activation of p38 MAPK. Herein, we show that the three CCM proteins are members of a larger signaling complex. To define this complex, epitope-tagged wild type OSM or OSM harboring the mutation of F217
A, which renders the OSM phosphotyrosine binding (PTB) domain unable to bind KRIT1, were stably introduced into RAW264.7 mouse macrophages. FLAG-OSM or FLAG-OSMF217A and the associated complex members were purified by immunoprecipitation using anti-FLAG antibody. OSM binding partners were identified by gel-based methods combined with electrospray ionization-MS or by multidimensional protein identification technology (MudPIT). Previously identified proteins that associate with OSM including KRIT1, MEKK3, Rac, and the KRIT1-binding protein ICAP-1 were found in the immunoprecipitates. In addition, we show for the first time that PDCD10 binds to OSM and is found in cellular CCM complexes. Other prominent proteins that bound the CCM complex include EF1A1, RIN2, and tubulin, with each interaction disrupted with the OSMF217A mutant protein. We further show that PDCD10 binds phosphatidylinositol di- and triphosphates and OSM binds phosphatidylinositol monophosphates. The findings define the targeting of the CCM complex to membranes and to proteins regulating trafficking and the cytoskeleton.
A.18
Analysis of the Dephosphorylation Kinetics of ß-Casein by Alkaline Phosphatase Using a Label-free Quantitation Method
J. Oses-Prieto, S. Guan, D. A. Maltby, and A. L. Burlingame
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, CA
Quantitative analysis of protein samples using mass spectrometry has been one of the main fields of high interest for the scientific community in the last years. Particularly, analysis of the dynamic population of posttranslational modifications in protein complexes during signaling events is considered to be essential for understanding of the cell physiology. Protein phosphorylation and dephosphorylation are main events during signal transduction and cell signaling processes, since they both can trigger activation or inactivation of different proteins involved in these pathways.
Isotopic labeling of samples from different systems in different phosphorylation states coupled with mass spectrometry has proven to be very useful. However, all isotopic labeling systems complicate experimental procedures and are prone to introduce artifacts to the samples. We have developed a label-free semiquantitation method taking advantage of the very high mass resolution and accuracy of an ion trap/ICR instrument. This method analyzes peptide digests after an LC/MSMS run relying on extraction of selected ion chromatograms, whose peptide identification is carried out by a database search of their MSMS spectra
As a control system, we have analyzed the dephosphorylation kinetics of the bovine phosphoprotein ß-casein by alkaline phosphatase (AP). Samples treated for different times with AP were digested with trypsin, either in solution or after running the proteins on an polyacrylamide gel, and analyzed by this label-free quantification method. Identical LC/MSMS runs were carried out to evaluate the errors due to sample manipulation and instrumental variability. A close correlation was observed between logarithms of the intensities of the ions for most peptides observed in all related samples. However, some peptides deviated from this statistical linear trend. Differential miscleavage frequency and oxidation in the samples during digestion were partially responsible for this finding. Also phosphate containing peptides were observed to disappear, and the intensities of the corresponding dephosphorylated peptides rise, during AP treatment. Our preliminary results show how this label-free quantitation approach can be useful for the analysis of dynamic changes of PTM, at least on systems of limited complexity.
This work was supported by NIH Grant NCRR RR001614.
A.19
Sample Preparation for Structure Characterization of Novel Antibiotics Produced by Bacillus Strains
J.-M. Schmitter, P. Bressollier, B. Verneuil, A. Ballade, and M. Urdaci
European Institute of Chemistry and Biology (IECB), Pessac, France
Biologically active compounds produced by Bacillus strains include already well known antibiotics. Small lipopeptides having structures built on the basis of a lipid chain grafted to a cyclic peptidic part account for a large number of these active compounds, belonging to the surfactin, iturin, bacillomycin, mycosubtilin and fengycin series, among others. However, the occurrence of compounds in the 2–4 kDa mass range such as lantibiotics produced by ribosomal synthesis is also reported.
Although MALDI ionization has found many applications for fast screening of bacterial strains, the series of compounds revealed by this type of analysis rarely account for the whole set of biological activities displayed by a given strain. The loss of information related to absence of detection of small molecules is partly responsible for this situation. However, the main cause of information loss is the occurrence of strong spectral suppression effects within the complex mixtures of lipopeptides, urging the search for adequate sample preparation.
This work describes sample preparation prior to ESI/MS/MS and MALDI/MS/MS of lipopeptides and peptidic antibiotics, allowing detection of new compounds and their complete characterization. About 50 Bacillus strains grown in Petri dishes were screened for anti-microbial activity. These strains were then first characterized by MALDI-MS/MS. Owing to the high salt content of culture media (Na+ and K+ ions), sample preparation included an ion exchange process with Li+ ions. The combination of accurate mass measurement and MS/MS spectra obtained from (M+Li)+ ions with the MALDI-Q-ToF instrument provided the basis for unambiguous identification of compounds belonging to already known families of lipopeptides. Selected strains displaying strong activities and producing unknown compounds were grown in liquid medium, in order to isolate enough material for structure analysis. Solvent extracts were characterized by combined LC-ESI-MS/MS. This analysis revealed the occurrence of low molecular weight compounds and confirmed that MALDI mass spectra of lipopeptides were strongly affected by spectral suppression.
Among many unknown compounds, a new 2 kDa peptidic antibiotic displaying a strong anti-Staphylococcus aureus activity was isolated by liquid chromatography. This compound was characterized by a combination of techniques including total hydrolysis, ring opening by chemical derivatization and MS/MS analysis, prior to complete structure determination by NMR. This new compound belongs to the family of lantibiotics and its structure is related to that of mutacin. More biologically active compounds are currently analyzed with a similar protocol, and their structure determination is underway.
A.20
Quantitative Analysis of Rapamycin Effects in Yeast
M. Fournier, N. Pavelka, M. Sardiu, K. Zueckert-Gaudenz, C. Seidel, S. Swanson, L. Florens, and M. Washburn
Stowers Institute for Medical Research, Kansas City, MO
Quantitative proteomics, monitoring dynamic changes of a proteome in response to different stimuli, has emerged as a powerful approach to analyze protein function.
In this study, Multidimensional Protein Identification Technology (MudPIT) combined with a metabolic labeling strategy and Normalized Spectral Abundance Factor (NSAF) is used to elucidate the molecular mechanisms of rapamycin, an immunosuppressive and anticancer drug. Rapamycin inhibits the protein kinase TOR (Target of Rapamycin), a central controller of cell growth in eukaryotic organisms. The targets of TOR have not yet been identified and gaps in the signalling pathway remain to be filled. In order to gain insights into TOR function and a global understanding of rapamycin effects, we performed a time course analysis of protein and mRNA changes in Saccharomyces cerevisiae in response to rapamycin. Proteomic and transcriptomic profiles have been compared between Saccharomyces cerevisiae grown in N15 media (none treated) versus N14 media (treated) during 6 hours of rapamycin treatment. We have demonstrated that NSAF-based MudPIT data can be modelled by a power law global error model (PLGEM) emerging as a new statistical tool to identify protein abundance changes in large scale shotgun proteomics data. Using PLGEM, we observed a decrease of ribosomal proteins abundance which could be correlated with the inhibition of translation caused by rapamycin. We also observed a significant increasing amount of the tricarboxylic acid cycle enzymes and the proteins Pdr5 (membrane ABC transporter) and Dld3 (D-lactate dehydrogenase). This metabolic reconfiguration, known to be apart of the retrograde pathway (RTG), indicates a mitochondrial dysfunction induced by rapamycin treatment. The overall correlation between mRNA and protein abundance changes was low at early time point. Interestingly, this correlation increased in a delayed fashion when changes at the mRNA level at early time point were correlated with changes at the protein level at later time point. The most apparent example concerned the ribosomal proteins for which the highest correlation is observed between the 20 minutes time point at the mRNA level and the 6 hours time point at the protein level. This result suggests that protein synthesis is still effective during this range of time explaining the accumulation of the RTG effectors. The observation that transcriptomic and proteomic profiles were highly correlated in a delayed fashion, demonstrates the significance of performing such dynamic analyses and integrating data over time. This leads to a better understanding of the physiological state of an organism, particularly in this study focused on drug molecular mechanisms.
A.21
Candidate Biomarker Identification in the Time-dependant Progression of a Mouse Model of Lung Fibrosis
K. Williams1, C. Lombardo2, K. Blese2, L. Wong2, G. Hampton2, and C. Hunter1
1Applied Biosystems, Foster City, CA; 2Celgene Corporation, Summit, NJ
Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix in the interstitium resulting in respiratory failure. The mechanisms underlying the development and progression of the disease are not fully understood. Intratracheal instillation of bleomycin into rodents serves as a model for human lung fibrosis. The goal of this study is to identify biomarkers with clear linkage to the biology associated with the progression of fibrosis. We employed an unbiased proteomic analysis using multiplex isotope labeling to identify and quantify changes in the proteome during bleomycin induced progression of fibrosis.
Lung tissue was collected at days 0, 1, 7, and 14 from six bleomycin treated mice per time point. Relative protein expression levels were measured using 8plex iTRAQ reagent labeling. The combined labeled samples were separated by 2D HPLC MS/MS and analyzed on a MALDI TOF/TOF platform. Protein identification and quantitation of relative protein ratios were performed using ProteinPilot software and clustering heat map data analysis.
A number of proteins were identified which show differential expression during the progression of fibrosis, and fall into several functional classes consistent with the pathology of the disease. In concordance with this, the appearance of specific markers for the endothelial-mesenchymal transition has also been observed. The quantitative isotopic labeling data was validated for a subset of proteins using immunoblotting techniques.
The multiplex nature of the 8plex iTRAQ reagent strategy for biomarker discovery allowed simultaneous experiments to be performed in one LCMS/MS run. In this study, a 4 point time course with 6 mice at each time point was used to measure both protein abundance trends and inter-animal variability. Although inter-animal variability exists in disease model studies and is often a confounding factor, several proteins were observed that exhibited >1.5 fold changes with a S.D. of <25%.
A.22
Targeted Quantitative MRM Analysis for Validation of Changes in hESCs During Noggin- and BMP4-Induced Differentiation
A. Yocum1, C. Hunter3, K. S. O'Shea2, and P. Andrews1
1Department of Biological Chemistry and 2Deparment of Cell and Developmental Biology, University of Michigan, Ann Arbor, MI; 3Applied Biosystems, Foster City, CA
Since their derivation, human embryonic stem cells (hESCs) have become the most promising source for in vitro production of cells for regenerative medicine and treatment of diseases. Despite extensive research aimed at elucidating and controlling the cellular processes of self-renewal and lineage-specific differentiation, much needs to be learned before their clinical potential can be realized. Exogenous treatment of hESCs to various growth factors can inhibit or induce lineage specific differentiation. Quantitative analysis of treated hESCs using non-isobaric chemical labeling reagents and multiple reaction monitoring (MRM) on a hybrid triple quadrupole linear ion trap 4000 Q TRAP mass spectrometer led to verification of quantification of several lineage-dependent marker proteins in hESCs discovered from both global quantitative proteomics and other molecular biology technologies. After analysis of the quantitative discovery phase, we proposed that we could design and provide a high throughput methodology for measuring specific biomarkers of stem cell differentiation and self-renewal, validate the global proteomics findings (both the proteins’ existence and quantity), and develop a specific assay to distinguish between different isoforms utilizing the MRM technology.
This work represents a successful transition from quantitative discovery proteomics to target verification using MRM and allowed the elucidation and confirmation of five proteins which can be further utilized as biomarkers for stem cell biology in a high throughput manner that is highly selective and sensitive. Two of these markers, tubulin ß-III and cytokeratin 8 were previously recognized as markers for neuronal and epi/endoderm differentiation, respectively. During development of the MRM assay using MRM triggered MS/MS strategies, we specifically targeted unique peptides that differentiated among the many isoforms. These markers not only demonstrate the validity of our experimental conditions and design but offer substantiating evidence regarding the differences between mouse and human ESCs. Further, we propose additional biomarkers: nuclear autoantigenic sperm protein (NASP) and the isoforms of collaspin-response mediator protein (CRMP2) and (CRMP4), for self-renewal and neuronal differentiation, respectively. These proteins and others targeted in this approach were used for development of a highly effective MRM methodology using non-isobaric chemical labeling. This combination of technologies has the potential for very high sensitivity and throughput while maintaining specificity. With development and measurement of specific MRMs, we successfully discriminated among highly homologous protein targets, and provided additional verification for the relative quantification. These results demonstrate the power of MRM applied to verification of discovery proteomics experiments and contributes to our understanding of hESC differentiation.
A.23
Associating Partners Modulate 20S Proteasome Functions in Mammalian Tissues
G. Young, A. Gomes, C. Zong, and P. Ping
Departments of Physiology and Medicine, Division of Cardiology, School of Medicine, University of California, Los Angeles, CA
Investigations into the sub-proteome of the proteasome offer a view into the regulatory and functional components of this family of large multi-protein complexes. Previous studies in our laboratory suggest that an additional level of complexity and functional capacity is gained through a growing list of associating partners. These associating partners co-reside with the endogenous 20S proteasome complexes and regulate the proteolytic function. Here we report a proteomic-based study that identified associating partners of the 20S proteasomes from both the murine heart and liver using multidimensional chromatography combined with mass spectrometry analyses. Characterization of the purified proteasomes by LC/MS/MS (LTQ, Thermo) identified both known [heat shock protein 60 (HSP 60)] and novel [serine hreonine protein phosphatase 1 (PP1), microtubule associated serine hreonine kinase (MAST2) and a zinc finger containing protein (ZFHX4)] associating partners of the 20S proteasomes in liver and heart. All proteins identified through mass spectrometry were confirmed as associating partners of the native 20S complexes by non-denaturing gel electrophoresis and subsequent immunoblotting. PP1 alpha and HSP60 were detected with at least five matching peptides in both heart and liver. While MAST2 and ZFHX4 were both identified with two peptides in the liver, no peptides were detected in the heart. This is consistent with the lower apparent stoichiometry of these partners in heart as assessed by immunoblotting of non-denaturing gels. Significant heterogeneity exists between associating partners of the liver and the heart 20S proteasomes. Interestingly, PP1 activity did not impact trypsin-like and caspase-like proteolytic activities of the liver and cardiac proteasomes, while PP1 had a selective effect on the chymotrypsin-like activity in an organ specific manner. Addition of PP1 to purified cardiac 20S proteasomes enhanced the chymotryptic activity (50% ± 2% increase when compared to heat-inactivated PP1), whereas it had no detectable effect on the chymotryptic activity in liver 20S proteasomes. Endogenous PP1 inhibition by calyculin A (10 nM) also inhibited only the chymotryptic activity in heart when compared to liver. Although similar amounts of PP1 were found in purified 20S complexes from both the liver and heart, the amounts of HSP60, ZFHX4 and MAST2 exhibited varying ratios between the tissue types. These data show that associating partners participate in the regulation of proteasome function in the liver and in the heart via distinct cellular mechanisms. The sensitivity by which these molecules modulate organ proteolytic function is cell type specific. Our investigation underscores the importance of differential regulatory mechanisms of proteasome function in different organs and affords potential therapeutic opportunities whereby organ-selective regulation of proteolytic function may be achieved.
A.24
The Mammalian 20S Proteasomes Are Methylated on Arginine and Lysine Residues
G. Young, A. Gomes, and P. Ping
Departments of Physiology and Medicine, Division of Cardiology, School of Medicine, University of California, Los Angeles, CA
The proteasome complexes represent the largest proteolytic system in the mammalian cell and is primarily responsible for protein degradation in these cells. Previous investigations from our laboratory implicate that these proteasome subunits exhibit heavy posttranslational modifications, which in turn, may contribute to the regulation of their biological function. In this study, we report that, in addition to the commonly observed posttranslational modifications (e.g., phosphorylation), we observed protein methylation of proteasome subunits in a reproducible fashion. Protein methylation is a form of posttranslational modification that is not well understood; recent studies suggest that protein methylation is involved in the regulation of gene expression, protein function, RNA metabolism, and oncogenesis. Protein methylation most commonly takes place on arginine or lysine residues. Arginine can be methylated once (monomethylated arginine) or twice (dimethylated arginine) whereas lysine can be methylated once, twice or three times (trimethylated lysine). To characterize the methylation of proteasome subunits, we isolated endogenous 20S proteasome complexes from both murine liver and heart; these proteasome samples were then digested by trypsin and then subjected to either one- and/or two-dimensional gel electrophoresis combined with LC/MS/MS (LTQ). Data analysis was carried out with the Sequest program using the following stringent search criteria: X Corr values 1+, 3 0.2; 2+, 3 0.25; 3+, 3 0.38; Rsp = 1, 
CN > 0.1, and a minimum peptide probability of 0.001. Using this approach, we identified the following proteasome sites of methylation in the murine heart: alpha 1 and alpha 5 are monomethylated at arginines while alpha 7 and alpha 2 are mono and dimethylated respectively at lysine residues. In the liver, alpha 4 and alpha 7 had mono and dimethylated lysine resides respectively, while beta 6 was methylated at arginine and beta 5i was mono and dimethylated at two separate arginine residues. The methylation patterns of the heart and liver show considerable heterogeneity as neither organ displayed any modified sites in common. To confirm these data by mass spectrometry, we performed immunoblotting using antibodies specific for either mono and dimethylated arginine or mono and dimethylated lysine. Data from the immunoblotting analysis confirmed the presence of methylated residues on proteasome subunits. This is the very first report to show methylation of 20S proteasome subunits in any cell type or species.
A.25
Erythromycin B—An Old Drug for the New Millennium?
M. Mordi, V. Boote, P. Bhadra, G. Morris, and J. Barber
Schools of Pharmacy and Chemistry, University of Manchester, United Kingdom
The antibiotic erythromycin A was discovered in 1952 and has been of huge clinical importance ever since, especially in the treatment of deep-seated Gram positive infections, such as tuberculosis and Legionnaire's disease. Erythromycin B was isolated at the same time, as a minor contaminant of erythromycin A-producing cultures, but has never been exploited clinically.
In even mildly acidic conditions (pH < 7), erythromycin A undergoes cyclisation reactions between the 12-hydroxyl and 9-keto groups, leading to inactivation of the drug. Erythromycin B has no 12-hydroxyl group and cannot be inactivated in this way; an investigation of its properties seemed long overdue.
We have investigated the aqueous solution chemistry of erythromycin B compared with erythromycin A and clarithromycin (a second generation analogue) by NMR spectroscopy, supported by electrospray ionisation mass spectrometry. Erythromycin B (t1/2 >200 min at pH 2.5) was shown to be much more stable than erythromycin A (t1/2 < 10 min at pH 2.5). Microbiological studies showed that the two drugs had comparable antibacterial activity in vitro.
Finally we studied the metabolism of erythromycins A and B and clarithromycin in a small number of rats, monitoring the concentrations of drug in the bloodstream following an oral dose (50 mg kg–1), using liquid chromatography–electrospray ionisation mass spectrometry. All three drugs were well-absorbed, giving peak blood concentrations after about 2 hours. The peak erythromycin A concentration was about 150 ng ml–1, both the other drugs achieved double this blood concentration. Three metabolites of erythromycin A were observed and identified, whereas clarithromycin and erythromycin B gave a single metabolite. Clarithromycin, however, was cleared from the blood more slowly than erythromycin B.
Although clarithromycin out-performs erythromycin B in pharmacokinetic terms, it has proved very difficult to prepare pediatric formulations of this drug. By contrast, we have been able adapt the solution chemistry of erythromycin B to the preparation of taste-free prodrugs suitable for pediatric use.
A.26
Phosphoproteome Profiling Approaches for Comprehensive Monitoring of Cell Signaling Events in Stimulated Macrophages
M. Marcantonio1,2, M. Trost1,3, M. Courcelles1,2, M. Desjardins3, and P. Thibault1,2
1IRIC, Institut of Research in Immunology and Cancer, Université de Montréal, Montréal, Quebec, Canada; 2Department of Biochemistry, Université de Montréal, Montréal, Quebec, Canada; 3Department of Pathology and Cell Biology, Université de Montréal, Montréal, Quebec, Canada
The cascade of signalling pathways leading to host immune response in macrophage cells is primarily guided through phosphorylation and dephosphorylation events. In addition, studies have shown that secretion of interferon- by T-cells activates the JAK/STAT signalling pathway in macrophages and induces phosphorylation of many additional substrates. Hence, phosphoproteome analysis in macrophages is essential for a better understanding of the immune response. In the present study, we conducted kinetic profiles (0, 5, 10, 30, 60 and 180min) of differential phosphoproteome following interferon- administration to mouse J774 macrophages. Phosphopeptides from tryptic digests of cytosolic proteins were isolated using TiO2 affinity media and analyzed by liquid chromatography-mass spectrometry (LC-MS). Amongst the different trends observed, a higher proportion of phosphopeptides showed a sudden increase in phosphorylation 5 min after interferon- induction. The comparison of the macrophage phosphoproteome with and without interferon- using in-house bioinformatic tools indicated that 23% and 18% ion clusters showed more than 3-fold over- and under-expression, respectively. A total of 1607 phosphopeptides were identified including 1314 novel phosphorylation sites. The kinetic profiles of phosphopeptide abundances lead to the identification of early signalling and regulatory events including the enhanced phosphorylation of members of ROS complex (p40 and p67 phox) and vesicle trafficking (VAMP-4 and -taxilin). Furthermore, significant increase of putative phosphopeptide identification was obtained using 2D-LC-MS and alkaline phosphatase treatment. Among new potential phosphoproteins showing statistically meaningful change in abundance were CAD protein, cytosolic Phospholipase-A2, p47 phox and AP-3 complex. Overall, analytical tools enabled the identification of important phosphorylated candidates upon interferon- induction of macrophages which leads to a better understanding of the signalling pathways of the immune response.
A.27
Affinity Tagged Reductive Amination; a Novel Chemical Cross-linking Strategy
M. J. Trnka and A. L. Burlingame
Department of Pharmaceutical Chemistry. University of California, San Francisco, CA
Chemical cross-linking with analysis by high resolution/mass accuracy mass spectrometry is a means to stabilize transient protein interactions, map interacting domains of proteins, and provide distance constraints from which low-resolution structures of protein complexes can be inferred. While the structural insights derived by cross-linking are necessarily of lower resolution than other methods, the advantage of this technique is that it is able to probe conformational dynamics and/or subunit rearrangement in response to a variety of biochemical stimuli. Furthermore, it is applicable to studying proteins in the solution state, in cell culture, or even in vivo. Thus, a robust cross-linking methodology complements other structural techniques, such as crystallography or electron microscopy that have higher resolution, but are slow and limited to non-physiological conditions.
Cross-linking analysis of binary protein complexes has flourished with the proliferation of modern mass spectrometers and proteomics analysis. However, existing cross-linking reagents are not suitable for the analysis of the large multi-megaDalton protein machines that perform the most critical cellular functions, due to their propensity to hydrolyze at either one or both reactive sites of the reagent. This leads to an overwhelming quantity of hydrolyzed reagent and so called "dead-end" modified peptides. Typically, the yield of cross-linked peptide pairs containing useful structural information is extremely low. The underlying chemistry and yield of cross-linking reactions must be improved in order to expand the utility of cross-linking analysis to the scale of large, dynamic protein complexes such as the spliceosome or the histone deacetylase complex.
Many of the problems concerning poor yield and hydrolysis can be overcome by changing the underlying reactivity of the cross-linking reagent from activated NHS-ester chemistry, which is prone to hydrolysis, to reductive amination of lysine side chains by electrophilic aldehyde moieties. Aldehydes form Schiff bases with primary amines that can be stabilized in aqueous conditions by a mild reducing agent such as sodium cyanoborohydride. In addition to not being hydrolyzable, this strategy also leaves a chemical "handle" on molecules of cross-linker that have not reacted fully. This handle can be used to separate truly cross-linked peptide pairs from unmodified and "dead-end" modified peptides by selective application of affinity tags. This presentation describes the affinity tagged, reductive amination cross-linking strategy as well as the design, synthesis, and preliminary evaluation of the first generation of these reagents.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Journal of Biological Chemistry |
| Journal of Lipid Research | ASBMB Today |
