Molecular & Cellular Proteomics current issue

Protein Identification False Discovery Rates for Very Large Proteomics Data Sets Generated by Tandem Mass Spectrometry [Research]

Wed, 04 Nov 2009 11:52:05 PST

Comprehensive characterization of a proteome is a fundamental goal in proteomics. To achieve saturation coverage of a proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests, proteomics data sets are growing dramatically in size and heterogeneity. The trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide-spectrum matches. We present MAYU, a novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets. We validated and applied MAYU using various large proteomics data sets. The data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications. We particularly found that protein false discovery rates are significantly elevated compared with those of peptide-spectrum matches. The function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources. The MAYU software is available as standalone software and also integrated into the Trans-Proteomic Pipeline.

A New Approach for Quantitative Phosphoproteomic Dissection of Signaling Pathways Applied to T Cell Receptor Activation [Research]

Wed, 04 Nov 2009 11:52:05 PST

Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T cells. In our approach, label-free quantitation using normalization to copurified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T cell stimulation. Stable isotopic labeling of amino acids in cell culture (SILAC) ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy.

Heterochromatin Protein 1 Is Extensively Decorated with Histone Code-like Post-translational Modifications [Research]

Wed, 04 Nov 2009 11:52:05 PST

Heterochromatin protein 1 (HP1) family members (, β, and ) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation. Several previous reports have suggested that HP1s are post-translationally modified, yet sites of modification have not yet been exhaustively determined. Here we perform the first comprehensive proteomic analysis of all HP1 isoforms using tandem mass spectrometry. Our data reveal that all HP1 isoforms are highly modified in a manner analogous to histones including phosphorylation, acetylation, methylation, and formylation, including several sites having multiple different types of modifications. Additionally, many of these modifications are found in both the chromo- and chromoshadow domains, suggesting that they may have an important role in modulating HP1 interactions or functions. These studies are the first to systematically map the abundant sites of covalent modifications on HP1 isoforms and provide the foundation for future investigations to test whether these modifications are essential in heterochromatin maintenance or other nuclear processes.

Label-free Quantitative Proteomics Analysis of Etiolated Maize Seedling Leaves during Greening [Research]

Wed, 04 Nov 2009 11:52:05 PST

To better understand light regulation of C₄ plant maize development, we investigated dynamic proteomic differences between green seedlings (control), etiolated seedlings, and etiolated seedlings illuminated for 6 or 12 h using a label-free quantitative proteomics approach based on nanoscale ultraperformance liquid chromatography-ESI-MS^E. Among more than 400 proteins identified, 73 were significantly altered during etiolated maize seedling greening. Of these 73 proteins, 25 were identified as membrane proteins that seldom had been identified with two-dimensional electrophoresis methods, indicating the power of our label-free method for membrane protein identification; 31 were related to light reactions of chlorophyll biosynthesis, photosynthesis, and photosynthetic carbon assimilation. The expression of photosystem II subunits was highly sensitive to light; most of them were not identified in etiolated maize seedlings but drastically increased upon light exposure, indicating that the complex process of biogenesis of the photosynthetic apparatus correlates with the transition from a dark-grown to a light-grown morphology. However, transcriptional analysis indicated that most transcripts encoding these proteins were not regulated by light. In contrast, the levels of mRNAs and proteins for enzymes involved in carbon assimilation were tightly regulated by light. Additionally phosphoenolpyruvate carboxykinase, the key enzyme of the phosphoenolpyruvate carboxykinase C₄ pathway, was more tightly regulated by light than the key enzymes of the NADP-malic enzyme C₄ pathway. Furthermore phosphoenolpyruvate carboxylase 1C, which was originally reported to be specifically expressed in roots, was also identified in this study; expression of this enzyme was more sensitive to light than its isoforms. Taken together, these results represent a comprehensive dynamic protein profile and light-regulated network of C₄ plants for etiolated seedling greening and provide a basis for further study of the mechanism of gene function and regulation in light-induced development of C₄ plants.

Proline-rich Sequence Recognition: I. MARKING GYF AND WW DOMAIN ASSEMBLY SITES IN EARLY SPLICEOSOMAL COMPLEXES [Research]

Wed, 04 Nov 2009 11:52:05 PST

Proline-rich sequences (PRS) and their recognition domains have emerged as transposable protein interaction modules during eukaryotic evolution. They are especially abundant in proteins associated with pre-mRNA splicing and likely assist in the formation of the spliceosome by binding to GYF and WW domains. Here we profile PRS-mediated interactions of the CD2BP2/52K GYF domain by a site-specific peptide inhibitor and stable isotope labeling/mass spectrometry analysis. Several PRS hubs with multiple proline-rich motifs exist that can recruit GYF and/or WW domains. Saturating the PRS sites by an isolated GYF domain inhibited splicing at the level of A complex formation. The interactions mediated by PRS are therefore important to the early phases of spliceosomal assembly.

Proline-rich Sequence Recognition: II. PROTEOMICS ANALYSIS OF Tsg101 UBIQUITIN-E2-LIKE VARIANT (UEV) INTERACTIONS [Research]

Wed, 04 Nov 2009 11:52:05 PST

The tumor maintenance protein Tsg101 has recently gained much attention because of its involvement in endosomal sorting, virus release, cytokinesis, and cancerogenesis. The ubiquitin-E2-like variant (UEV) domain of the protein interacts with proline-rich sequences of target proteins that contain P(S/T)AP amino acid motifs and weakly binds to the ubiquitin moiety of proteins committed to sorting or degradation. Here we performed peptide spot analysis and phage display to refine the peptide binding specificity of the Tsg101 UEV domain. A mass spectrometric proteomics approach that combines domain-based pulldown experiments, binding site inactivation, and stable isotope labeling by amino acids in cell culture (SILAC) was then used to delineate the relative importance of the peptide and ubiquitin binding sites. Clearly "PTAP" interactions dominate target recognition, and we identified several novel binders as for example the poly(A)-binding protein 1 (PABP1), Sec24b, NFB2, and eIF4b. For PABP1 and eIF4b the interactions were confirmed in the context of the corresponding full-length proteins in cellular lysates. Therefore, our results strongly suggest additional roles of Tsg101 in cellular regulation of mRNA translation. Regulation of Tsg101 itself by the ubiquitin ligase TAL (Tsg101-associated ligase) is most likely conferred by a single PSAP binding motif that enables the interaction with Tsg101 UEV. Together with the results from the accompanying article (Kofler, M., Schuemann, M., Merz, C., Kosslick, D., Schlundt, A., Tannert, A., Schaefer, M., Lührmann, R., Krause, E., and Freund, C. (2009) Proline-rich sequence recognition: I. Marking GYF and WW domain assembly sites in early spliceosomal complexes. Mol. Cell. Proteomics 8, 2461–2473) on GYF and WW domain pathways our work defines major proline-rich sequence-mediated interaction networks that contribute to the modular assembly of physiologically relevant protein complexes.

Differential 14-3-3 Affinity Capture Reveals New Downstream Targets of Phosphatidylinositol 3-Kinase Signaling [Research]

Wed, 04 Nov 2009 11:52:05 PST

We devised a strategy of 14-3-3 affinity capture and release, isotope differential (d₀/d₄) dimethyl labeling of tryptic digests, and phosphopeptide characterization to identify novel targets of insulin/IGF1/phosphatidylinositol 3-kinase signaling. Notably four known insulin-regulated proteins (PFK-2, PRAS40, AS160, and MYO1C) had high d₀/d₄ values meaning that they were more highly represented among 14-3-3-binding proteins from insulin-stimulated than unstimulated cells. Among novel candidates, insulin receptor substrate 2, the proapoptotic CCDC6, E3 ubiquitin ligase ZNRF2, and signaling adapter SASH1 were confirmed to bind to 14-3-3s in response to IGF1/phosphatidylinositol 3-kinase signaling. Insulin receptor substrate 2, ZNRF2, and SASH1 were also regulated by phorbol ester via p90RSK, whereas CCDC6 and PRAS40 were not. In contrast, the actin-associated protein vasodilator-stimulated phosphoprotein and lipolysis-stimulated lipoprotein receptor, which had low d₀/d₄ scores, bound 14-3-3s irrespective of IGF1 and phorbol ester. Phosphorylated Ser¹⁹ of ZNRF2 (RTRAYpS¹⁹GS), phospho-Ser⁹⁰ of SASH1 (RKRRVpS⁹⁰QD), and phospho- Ser⁴⁹³ of lipolysis-stimulated lipoprotein receptor (RPRARpS⁴⁹³LD) provide one of the 14-3-3-binding sites on each of these proteins. Differential 14-3-3 capture provides a powerful approach to defining downstream regulatory mechanisms for specific signaling pathways.

Proteomic Analysis of Microtubule-associated Proteins during Macrophage Activation [Research]

Patel, P. C., Fisher, K. H., Yang, E. C. C., Deane, C. M., Harrison, R. E. — Wed, 04 Nov 2009 11:52:06 PST

Classical activation of macrophages induces a wide range of signaling and vesicle trafficking events to produce a more aggressive cellular phenotype. The microtubule (MT) cytoskeleton is crucial for the regulation of immune responses. In the current study, we used a large scale proteomics approach to analyze the change in protein composition of the MT-associated protein (MAP) network by macrophage stimulation with the inflammatory cytokine interferon- and the endotoxin lipopolysaccharide. Overall the analysis identified 409 proteins that bound directly or indirectly to MTs. Of these, 52 were up-regulated 2-fold or greater and 42 were down-regulated 2-fold or greater after interferon-/lipopolysaccharide stimulation. Bioinformatics analysis based on publicly available binary protein interaction data produced a putative interaction network of MAPs in activated macrophages. We confirmed the up-regulation of several MAPs by immunoblotting and immunofluorescence analysis. More detailed analysis of one up-regulated protein revealed a role for HSP90β in stabilization of the MT cytoskeleton during macrophage activation.

Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum [Research]

Darula, Z., Medzihradszky, K. F. — Wed, 04 Nov 2009 11:52:06 PST

The lack of consensus sequence, common core structure, and universal endoglycosidase for the release of O-linked oligosaccharides makes O-glycosylation more difficult to tackle than N-glycosylation. Structural elucidation by mass spectrometry is usually inconclusive as the CID spectra of most glycopeptides are dominated by carbohydrate-related fragments, preventing peptide identification. In addition, O-linked structures also undergo a gas-phase rearrangement reaction, which eliminates the sugar without leaving a telltale sign at its former attachment site. In the present study we report the enrichment and mass spectrometric analysis of proteins from bovine serum bearing Galβ1–3GalNAc (mucin core-1 type) structures and the analysis of O-linked glycopeptides utilizing electron transfer dissociation and high resolution, high mass accuracy precursor ion measurements. Electron transfer dissociation (ETD) analysis of intact glycopeptides provided sufficient information for the identification of several glycosylation sites. However, glycopeptides frequently feature precursor ions of low charge density (m/z > ~850) that will not undergo efficient ETD fragmentation. Exoglycosidase digestion was utilized to reduce the mass of the molecules while retaining their charge. ETD analysis of species modified by a single GalNAc at each site was significantly more successful in the characterization of multiply modified molecules. We report the unambiguous identification of 21 novel glycosylation sites. We also detail the limitations of the enrichment method as well as the ETD analysis.

A Mixed Integer Linear Optimization Framework for the Identification and Quantification of Targeted Post-translational Modifications of Highly Modified Proteins Using Multiplexed Electron Transfer Dissociation Tandem Mass Spectrometry [Research]

DiMaggio, P. A., Young, N. L., Baliban, R. C., Garcia, B. A., Floudas, C. A. — Wed, 04 Nov 2009 11:52:06 PST

Here we present a novel methodology for the identification of the targeted post-translational modifications present in highly modified proteins using mixed integer linear optimization and electron transfer dissociation (ETD) tandem mass spectrometry. For a given ETD tandem mass spectrum, the rigorous set of modified forms that satisfy the mass of the precursor ion, within some tolerance error, are enumerated by solving a feasibility problem via mixed integer linear optimization. The enumeration of the entire superset of modified forms enables the method to normalize the relative contributions of the individual modification sites. Given the entire set of modified forms, a superposition problem is then formulated using mixed integer linear optimization to determine the relative fractions of the modified forms that are present in the multiplexed ETD tandem mass spectrum. Chromatographic information in the mass and time dimension is utilized to assess the likelihood of the assigned modification states, to average several tandem mass spectra for confident identification of lower level forms, and to infer modification states of partially assigned spectra. The utility of the proposed computational framework is demonstrated on an entire LC-MS/MS ETD experiment corresponding to a mixture of highly modified histone peptides. This new computational method will facilitate the unprecedented LC-MS/MS ETD analysis of many hypermodified proteins and offer novel biological insight into these previously understudied systems.

Recombinant Surface Proteomics as a Tool to Analyze Humoral Immune Responses in Bovines Infected by Mycoplasma mycoides Subsp. mycoides Small Colony Type [Research]

Hamsten, C., Neiman, M., Schwenk, J. M., Hamsten, M., March, J. B., Persson, A. — Wed, 04 Nov 2009 11:52:06 PST

A systematic approach to characterize the surface proteome of Mycoplasma mycoides subspecies mycoides small colony type (M. mycoides SC), the causative agent of contagious bovine pleuropneumonia (CBPP) in cattle, is presented. Humoral immune responses in 242 CBPP-affected cattle and controls were monitored against one-third of the surface proteins of M. mycoides SC in a high throughput magnetic bead-based assay. Initially, 64 surface proteins were selected from the genome sequence of M. mycoides SC and expressed as recombinant proteins in Escherichia coli. Binding of antibodies to each individual protein could then be analyzed simultaneously in minute sample volumes with the Luminex suspension array technology. The assay was optimized on Namibian CBPP-positive sera and Swedish negative controls to allow detection and 20-fold mean signal separation between CBPP-positive and -negative sera. Signals were proven to be protein-specific by inhibition experiments, and results agreed with Western blot experiments. The potential of the assay to monitor IgG, IgM, and IgA responses over time was shown in a proof-of-concept study with 116 sera from eight animals in a CBPP vaccine study. In conclusion, a toolbox with recombinant proteins and a flexible suspension array assay that allows multiplex analysis of humoral immune responses to M. mycoides SC has been created.

The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome: IDENTIFICATION, GLYCOSITE OCCUPANCY, AND MEMBRANE ORIENTATION [Research]

Wed, 04 Nov 2009 11:52:06 PST

Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.

Outside the Unusual Cell Wall of the Hyperthermophilic Archaeon Aeropyrum pernix K1 [Research]

Palmieri, G., Cannio, R., Fiume, I., Rossi, M., Pocsfalvi, G. — Wed, 04 Nov 2009 11:52:06 PST

In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.

Glucose-regulated Protein 78 Is an Intracellular Antiviral Factor against Hepatitis B Virus [Research]

Wed, 04 Nov 2009 11:52:06 PST

Hepatitis B virus (HBV) infection is a global public health problem that plays a crucial role in the pathogenesis of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. However, the pathogenesis of HBV infection and the mechanisms of host-virus interactions are still elusive. In this study, two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to analyze the host response to HBV using an inducible HBV-producing cell line, HepAD38. Twenty-three proteins were identified as differentially expressed with glucose-regulated protein 78 (GRP78) as one of the most significantly up-regulated proteins induced by HBV replication. This induction was further confirmed in both HepAD38 and HepG2 cells transfected with HBV-producing plasmids by real time RT-PCR and Western blotting as well as in HBV-infected human liver biopsies by immunohistochemistry. Knockdown of GRP78 expression by RNA interference resulted in a significant increase of both intracellular and extracellular HBV virions in the transient HBV-producing HepG2 cells concomitant with enhanced levels of hepatitis B surface antigen and e antigen in the culture medium. Conversely overexpression of GRP78 in HepG2 cells led to HBV suppression concomitant with induction of the positive regulatory circuit of GRP78 and interferon-β1 (IFN-β1). In this connection, the IFN-β1-mediated 2',5'-oligoadenylate synthetase and RNase L signaling pathway was noted to be activated in GRP78-overexpressing HepG2 cells. Moreover GRP78 was significantly down-regulated in the livers of chronic hepatitis B patients after effective anti-HBV treatment (p = 0.019) as compared with their counterpart pretreatment liver biopsies. In conclusion, the present study demonstrates for the first time that GRP78 functions as an endogenous anti-HBV factor via the IFN-β1-2',5'-oligoadenylate synthetase-RNase L pathway in hepatocytes. Induction of hepatic GRP78 may provide a novel therapeutic approach in treating HBV infection.

iTRAQ-based Proteomics Profiling Reveals Increased Metabolic Activity and Cellular Cross-talk in Angiogenic Compared with Invasive Glioblastoma Phenotype [Research]

Wed, 04 Nov 2009 11:52:06 PST

Malignant gliomas (glioblastoma multiforme) have a poor prognosis with an average patient survival under current treatment regimens ranging between 12 and 14 months. The tumors are characterized by rapid cell growth, extensive neovascularization, and diffuse cellular infiltration of normal brain structures. We have developed a human glioblastoma xenograft model in nude rats that is characterized by a highly infiltrative non-angiogenic phenotype. Upon serial transplantation this phenotype will develop into a highly angiogenic tumor. Thus, we have developed an animal model where we are able to establish two characteristic tumor phenotypes that define human glioblastoma (i.e. diffuse infiltration and high neovascularization). Here we aimed at identifying potential biomarkers expressed by the non-angiogenic and the angiogenic phenotypes and elucidating the molecular pathways involved in the switch from invasive to angiogenic growth. Focusing on membrane-associated proteins, we profiled protein expression during the progression from an invasive to an angiogenic phenotype by analyzing serially transplanted glioma xenografts in rats. Applying isobaric peptide tagging chemistry (iTRAQ) combined with two-dimensional LC and MALDI-TOF/TOF mass spectrometry, we were able to identify several thousand proteins in membrane-enriched fractions of which 1460 were extracted as quantifiable proteins (isoform- and species-specific and present in more than one sample). Known and novel candidate proteins were identified that characterize the switch from a non-angiogenic to a highly angiogenic phenotype. The robustness of the data was corroborated by extensive bioinformatics analysis and by validation of selected proteins on tissue microarrays from xenograft and clinical gliomas. The data point to enhanced intercellular cross-talk and metabolic activity adopted by tumor cells in the angiogenic compared with the non-angiogenic phenotype. In conclusion, we describe molecular profiles that reflect the change from an invasive to an angiogenic brain tumor phenotype. The identified proteins could be further exploited as biomarkers or therapeutic targets for malignant gliomas.