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Molecular cellular proteomics [journal]
- Identification of putative substrates for the periplasmic chaperone YfgM in Escherichia coli using quantitative proteomics. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 17.
How proteins are trafficked, folded and assembled into functional units in the cell envelope of Gram-negative bacteria is of significant interest. A number of chaperones have been identified, however the molecular roles of these chaperones are largely enigmatic because it has been challenging to assign substrates. Recently we discovered a novel periplasmic chaperone, called YfgM, which associates with the SecYEG translocon and operates in a network that contains Skp and SurA. The aim of the study presented here was to identify putative substrates of YfgM. We reasoned that substrates would be incorrectly folded or trafficked when YfgM was absent from the cell, and thus more prone to proteolysis (the loss-of-function rationale). We therefore used a comparative proteomic approach to identify cell envelope proteins that were lower in abundance in a strain lacking yfgM, and strains lacking yfgM together with either skp or surA. Sixteen putative substrates were identified. The list contained nine inner membrane proteins (CusS, EvgS, MalF, OsmC, TdcB, TdcC, WrbA, YfhB, YtfH) and seven periplasmic proteins (HdeA, HdeB, AnsB, Ggt, MalE, YcgK and YnjE), but it did not include any lipoproteins or outer membrane proteins. Significantly, AnsB (an asparaginase) and HdeB (a protein involved in the acid stress response), were lower in abundance in all three strains lacking yfgM. For both genes we ruled out the possibility that they were transcriptionally down-regulated, so it is highly likely that the corresponding proteins are misfolded / mistargeted and turned-over in the absence of YfgM. For HdeB we validated this conclusion in a pulse-chase experiment. The identification of HdeB and other cell envelope proteins as potential substrates of YfgM, will be a valuable resource for follow-up experiments that aim to delineate molecular function.
- Comprehensive and reproducible phosphopeptide enrichment using Fe-IMAC columns. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 13.
Advances in phosphopeptide enrichment methods enable the identification of thousands of phosphopeptides from complex samples. Current offline enrichment approaches using TiO2, Ti-IMAC and Fe-IMAC material in batch or microtip format are widely used, but suffer from irreproducibility and compromised selectivity. To address these shortcomings, we revisited the merits of performing phosphopeptide enrichments in an HPLC column format. We found that Fe-IMAC columns enable the selective, comprehensive and reproducible enrichment of phosphopeptides out of complex lysates. Column enrichment did not suffer from bead-to-sample ratio issues and scaled linearly from 100ug to 5mg digest. Direct measurements on an Orbitrap Velos mass spectrometer identified >7,500 unique phosphopeptides with 90% selectivity and good quantitative reproducibility (median CV of 15%). The number of unique phosphopeptides could be increased to over 14,000, when subjecting the IMAC eluate to a subsequent hydrophilic strong anion exchange (hSAX) separation. Fe-IMAC columns outperformed Ti-IMAC and TiO2 in batch or tip mode in terms of phosphopeptide identification and intensity. Permutation enrichments of flow troughs show that all materials largely bind the same phosphopeptide species, independent of physicochemical characteristics. However, binding capacity and elution efficiency did profoundly differ among the enrichment materials and formats. As a result, the often quoted orthogonality of the materials has to be called into question. Our results strongly suggest that insufficient capacity, inefficient elution and the stochastic nature of data dependent acquisition in mass spectrometry are the cause of the experimentally observed complementarity. The Fe-IMAC enrichment workflow using an HPLC format developed here enables rapid and comprehensive phosphoproteome analysis which can be applied to a wide range of biological systems.
- Hepatic proteome analysis of Atlantic salmon (Salmo salar) after exposure to environmental concentrations of human pharmaceuticals. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 13.
Pharmaceuticals are pseudo persistent aquatic pollutants with unknown effects at environmentally relevant concentrations. Atlantic salmon (Salmo salar) were exposed to environmentally relevant concentrations of Acetaminophen; Atenolol and Carbamazepine for 5 days. After Acetaminophen treatment, 19 proteins were differently expressed, of which 11 were significant with respect to the control group (eight up-regulated and three down-regulated). After Atenolol treatment, 7 differently expressed proteins were obtained in comparison with the control, of which 6 could be identified (four up-regulated and 2 down-regulated). Carbamazepine exposure resulted in 15 differently expressed proteins compared with the control, with 10 of them identified (seven up-regulated and three down-regulated). Out of these, 3 features were common between Acetaminophen and Carbamazepine and one between Carbamazepine and Atenolol. One feature was common across all treatments. Principal component analysis and heat map clustering showed a clear grouping of the variability due to the applied treatments. The obtained data suggest (1) that exposure to environmentally relevant concentrations of the pharmaceuticals alters the hepatic protein expression profile of the Atlantic salmon; and (2) the existence of treatment specific processes that may be useful for biomarker development. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD001354.
- Glycoproteomic analysis of seven major allergenic proteins reveals novel post-translational modifications. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 11.
Allergenic proteins such as grass pollen and house dust mite (HDM) proteins are known to trigger hypersensitivity reactions of the immune system, leading to what is commonly known as allergy. Key allergenic proteins including sequence variants have been identified but characterization of their post-translational modifications (PTMs) is still limited. Here, we present a detailed PTM characterization of a series of the main and clinically relevant allergens used in allergy tests and vaccines. We employ Orbitrap-based mass spectrometry with complementary fragmentation techniques (HCD/ETD) for site-specific PTM characterization by bottom-up analysis. In addition, top-down mass spectrometry is utilized for targeted analysis of individual proteins, revealing hitherto unknown PTMs of HDM allergens. Moreover, we identified more complex glycan structures than previously reported on the major grass pollen group 1 and 5 allergens, implicating important roles for carbohydrates in allergen recognition and response by the immune system. The new findings are important for understanding basic disease-causing mechanisms at the cellular level, which ultimately may pave the way for instigating novel approaches for targeted de-sensitization strategies and improved allergy vaccines.
- PIMAX approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 10.
Many biomedically critical proteins are underrepresented in proteomics and biochemical studies due to difficulties of their production in E. coli. These proteins may possess a posttranslational modification vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator", such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and applications of a PIMAX system (protein interaction module-assisted function X) that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of hyperphosphorylated tau protein intimately linked to Alzheimer's disease, and an enzymatically active cyclin-dependent kinase complex. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer's disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad impacts on proteomics and biomedical research.
- C-terminomics screen for natural substrates of cytosolic carboxypeptidase 1 reveals processing of acidic protein C-termini. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 7.
Cytosolic carboxypeptidases (CCPs) constitute a new subfamily of M14 metallocarboxypeptidases associated to axonal regeneration and neuronal degeneration, amongst others. CCPs are deglutamylating enzymes, able to catalyze the shortening of polyglutamate side-chains and the gene-encoded C-termini of tubulin, telokin and myosin light chain kinase. The functions of these enzymes are not entirely understood, in part due to lack of information about C-terminal protein processing in the cell and its functional implications. By means of C-terminal COFRADIC, a positional proteomics approach, we searched for cellular substrates targets of CCP1, the most relevant member of this family. We here identified 7 new putative CCP1 protein substrates, including ribosomal proteins, translation factors and high mobility group proteins. Furthermore, we showed for the first time that CCP1 processes both glutamates as well as C-terminal aspartates. The implication of these C-termini in molecular interactions furthermore suggests that CCP1-mediated shortening of acidic protein tails might regulate protein-protein and protein-DNA interactions.
- Acetylome profiling reveals overlap in the regulation of diverse processes by sirtuins, Gcn5 and Esa1. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 7.
While histone acetylation and deacetylation machineries (HATs and HDACs) regulate important aspects of cell function by targeting histone tails, recent work highlights that non-histone protein acetylation is also pervasive in eukaryotes. Here, we use quantitative mass-spectrometry to define acetylations targeted by the sirtuin family, previously implicated in the regulation of non-histone protein acetylation. To identify HATs that promote acetylation of these sites, we also performed this analysis in gcn5 (SAGA) and esa1 (NuA4) mutants. We observed strong sequence specificity for the sirtuins and for each of these HATs. While the Gcn5 and Esa1 consensus sequences are entirely distinct, the sirtuin consensus overlaps almost entirely with that of Gcn5, suggesting a strong coordination between these two regulatory enzymes. Furthermore, by examining global acetylation in an ada2 mutant, which dissociates Gcn5 from the SAGA complex, we found that a subset of Gcn5 targets did not depend on an intact SAGA complex for targeting. Our work provides a framework for understanding how HAT and HDAC enzymes collaborate to regulate critical cellular processes related to growth and division.
- Investigating the interaction between the neonatal Fc receptor and monoclonal antibody variants by hydrogen/deuterium exchange mass spectrometry. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 6.
The recycling of immunoglobulins by the neonatal Fc receptor (FcRn) is of crucial importance in the maintenance of IgG antibody levels in plasma and is responsible for the long half-lives of endogenous and recombinant monoclonal antibodies. From a therapeutic point of view there is great interest in understanding and modulating the IgG-FcRn interaction to optimize antibody pharmacokinetics and ultimately improve efficacy and safety. Here we study the interaction between a full length human IgG1 and human FcRn by hydrogen/deuterium exchange mass spectrometry (HDX-MS) and targeted electron transfer dissociation to map sites perturbed by binding on both partners of the IgG-FcRn complex. Several regions in the antibody Fc region and the FcRn were protected from exchange upon complex formation in good agreement with previous crystallographic studies of FcRn in complex with the Fc fragment. Interestingly, we demonstrate that several regions in the IgG Fab region also show reduced deuterium uptake. Our findings indicate the presence of hitherto unknown FcRn interaction sites in the Fab region or a possible conformational link between the IgG Fc-Fab regions upon FcRn binding. Further, we have investigated the role of IgG glycosylation on the conformational response of the IgG-FcRn interaction. Removal of antibody glycans increases the flexibility of the FcRn binding site in the Fc region. Consequently, FcRn binding does not induce a similar conformational stabilization of deglycosylated IgG as observed for the wild type glycosylated IgG. Our results provide new molecular insight into the IgG-FcRn interaction and illustrate the capability of HDX-MS to advance structural proteomics by providing detailed information on the conformation and dynamics of large protein complexes in solution.
- Epitope identification from fixed-complexity random-sequence peptide microarrays. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 3.
Antibodies play an important role in modern science and medicine. They are essential in many biological assays, and have emerged as an important class of therapeutics. Unfortunately, current methods for mapping antibody epitopes require costly synthesis or enrichment steps, and no low cost universal platform exists. In order to address this, we tested a random sequence peptide microarray consisting of over 330,000 unique peptides sequences sampling 83% of all possible tetramers and 27% of pentamers. It is a single, unbiased platform capable of performing many different types of tests, it does not rely on informatic selection of peptides for a particular proteome(s), and it does not require iterative rounds of selection. In order to optimize the platform, we developed an algorithm that considers the significance of k-length peptide subsequences (k-mers) within selected peptides that come from the microarray. We tested eight monoclonal antibodies and seven infectious disease cohorts. The method correctly identified 5/8 monoclonal epitopes, and identified both reported and unreported epitope candidates in the infectious disease cohorts. This algorithm may greatly enhance the utility of random-sequence peptide microarrays, by enabling rapid epitope mapping and antigen identification.
- Nic1 Inactivation Enables Stable Isotope Labeling with 13C6 15N4-Arginine in Schizosaccharomyces pombe. [JOURNAL ARTICLE]
- Mol Cell Proteomics 2014 Nov 3.
Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Due to compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We showed that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1 in ammonium-supplemented medium, blocks this re-cycling to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M and G2 cell cycle phases in S. pombe.