Bischoff R, Schlüter H. Aminoacids: Chemistry, funcionality and selected non-enzymatic post-translational modifications.J Proteomics:. 2012 Apr 18;75(8):2275-96.
The ultimate goal of proteomics is determination of the exact chemical composition of protein species, including their complete amino acid sequence and the identification of each modified side chain, in every protein in a biological sample and their quantification.We are still far from achieving this goal due to limitations in analytical methodology and data analysis but also due to the fact that we surely have not discovered all amino acid modifications that occur in nature. To detect modified side chains and to discover new, still unknown amino acid derivatives, an understanding of the chemistry of the reactive groups of amino acids is mandatory. This tutorial focuses on the chemistry of the aminoacid side chains and addresses non-enzymatic modifications. By highlighting some exemplary reactions a glimpse of the huge diversity of modified amino acids provides the reader with sufficient insight into amino acid chemistry to raise the awareness for unexpected side chain modifications. We further introduce the reader to a terminology, which enables the comprehensive description of the exact chemical composition of a protein species, including its full amino acid sequence and all modifications of its amino acid side chains. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP number 10).
http://www.sciencedirect.com/science/article/pii/S1874391912000838
Structural Analysis of PTM Hotspots (SAPH-ire) – A Quantitative Informatics Method
Enabling the Discovery of Novel Regulatory Elements in Protein Families
Henry M. Dewhurst‡, Shilpa Choudhury, and Matthew P. Torres
Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)-a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits-conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.
http://www.mcponline.org/content/14/8/2285.long
Integrated microfluidics for protein modification discovery.
Noach-Hirsh M, Nevenzal H, Glick Y, Chorni E1, Avrahami D1, Barbiro-Michaely E, Gerber D, Tzur A.
Protein post-translational modifications (PTMs) mediate dynamic cellular processes with broad implications in human disease pathogenesis. There is a large demand for high-throughput technologies supporting PTM research, and both mass spectrometry (MS) and protein arrays have been successfully utilized for this purpose. Protein arrays override the major limitation of target protein abundance inherently associated with MS analysis. This technology, however, is typically restricted to pre-purified proteins spotted in a fixed composition on chips with limited life-time and functionality. In addition, the chips are expensive and designed for a single use, making complex experiments cost-prohibitive. Combining microfluidics with in situ protein expression from a cDNA microarray addressed these limitations. Based on this approach, we introduce a modular integrated microfluidic platform for multiple PTM analysis of freshly synthesized protein arrays (IMPA). The systems potency, specificity and flexibility are demonstrated for tyrosine phosphorylation and ubiquitination in quasicellular environments. Unlimited by design and protein composition, and relying on minute amounts of biological material and cost-effective technology, this unique approach is applicable for a broad range of basic, biomedical and biomarker research.
http://www.mcponline.org/content/early/2015/08/14/mcp.M115.053512.long
Nature Milestones: Mass Spectrometry
10 September 2015 | doi:10.1038/nmeth.3558
Nature Milestones are special supplements that aim to highlight the outstanding technological developments and scientific discoveries that have helped to define a particular field. Nature Milestones in Mass Spectrometry, a collaboration between five Nature Publishing Group journals, presents a historical look back at the key technical developments in mass spectrometry and the chemical and biological applications that stemmed from these advances. Each short Milestone article, written by a Nature Publishing Group editor, covers one breakthrough, highlighting the main papers that contributed to the advance and discussing both their value at the time and their lasting influence on mass spectrometry today.
http://www.nature.com/milestones/milemassspec/pdf/milemassspec_all.pdf
PALEONTOPROTEOMICA
Pathogens and host immunity in the ancient human oral cavity.
Warinner C, Rodrigues JF, Vyas R, Trachsel C, Shved N, Grossmann J, Radini A, Hancock Y, Tito RY, Fiddyment S, Speller C, Hendy J, Charlton S, Luder HU, Salazar-García DC, Eppler E, Seiler R, Hansen LH, Castruita JA, Barkow-Oesterreicher S, Teoh KY, Kelstrup CD, Olsen JV, Nanni P, Kawai T, Willerslev E, von Mering C, Lewis CM Jr, Collins MJ, Gilbert MT, Rühli F, Cappellini E.
Nat Genet. 2014 Apr;46(4):336-44. doi: 10.1038/ng.2906.
Calcified dental plaque (dental calculus) preserves for millennia and entraps biomolecules from all domains of life and viruses. We report the first, to our knowledge, high-resolution taxonomic and protein functional characterization of the ancient oral microbiome and demonstrate that the oral cavity has long served as a reservoir for bacteria implicated in both local and systemic disease. We characterize (i) the ancient oral microbiome in a diseased state, (ii) 40 opportunistic pathogens, (iii) ancient human-associated putative antibiotic resistance genes, (iv) a genome reconstruction of the periodontal pathogen Tannerella forsythia, (v) 239 bacterial and 43 human proteins, allowing confirmation of a long-term association between host immune factors, 'red complex' pathogens and periodontal disease, and (vi) DNA sequences matching dietary sources. Directly datable and nearly ubiquitous, dental calculus permits the simultaneous investigation of pathogen activity, host immunity and diet, thereby extending direct investigation of common diseases into the human evolutionary past.
http://www.nature.com/ng/journal/v46/n4/full/ng.2906.html
Detecting the immune system response of a 500 year-old Inca mummy.
Corthals A, Koller A, Martin DW, Rieger R, Chen EI, Bernaski M, Recagno G, Dávalos LM.
PLoS One. 2012;7(7):e41244. doi: 10.1371/journal.pone.0041244.
Disease detection in historical samples currently relies on DNA extraction and amplification, or immunoassays. These techniques only establish pathogen presence rather than active disease. We report the first use of shotgun proteomics to detect the protein expression profile of buccal swabs and cloth samples from two 500-year-old Andean mummies. The profile of one of the mummies is consistent with immune system response to severe pulmonary bacterial infection at the time of death. Presence of a probably pathogenic Mycobacterium sp. in one buccal swab was confirmed by DNA amplification, sequencing, and phylogenetic analyses. Our study provides positive evidence of active pathogenic infection in an ancient sample for the first time. The protocol introduced here is less susceptible to contamination than DNA-based or immunoassay-based studies. In scarce forensic samples, shotgun proteomics narrows the range of pathogens to detect using DNA assays, reducing cost. This analytical technique can be broadly applied for detecting infection in ancient samples to answer questions on the historical ecology of specific pathogens, as well as in medico-legal cases when active pathogenic infection is suspected.
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0041244
Proteomic analysis of a pleistocene mammoth femur reveals more than one hundred ancient bone proteins.
Cappellini E, Jensen LJ, Szklarczyk D, Ginolhac A, da Fonseca RA, Stafford TW, Holen SR, Collins MJ, Orlando L, Willerslev E, Gilbert MT, Olsen JV.
J Proteome Res. 2012 Feb 3;11(2):917-26. doi: 10.1021/pr200721u.
We used high-sensitivity, high-resolution tandem mass spectrometry to shotgun sequence ancient protein remains extracted from a 43 000 year old woolly mammoth ( Mammuthus primigenius ) bone preserved in the Siberian permafrost. For the first time, 126 unique protein accessions, mostly low-abundance extracellular matrix and plasma proteins, were confidently identified by solid molecular evidence. Among the best characterized was the carrier protein serum albumin, presenting two single amino acid substitutions compared to extant African ( Loxodonta africana ) and Indian ( Elephas maximus ) elephants. Strong evidence was observed of amino acid modifications due to post-mortem hydrolytic and oxidative damage. A consistent subset of this permafrost bone proteome was also identified in more recent Columbian mammoth ( Mammuthus columbi ) samples from temperate latitudes, extending the potential of the approach described beyond subpolar environments. Mass spectrometry-based ancient protein sequencing offers new perspectives for future molecular phylogenetic inference and physiological studies on samples not amenable to ancient DNA investigation. This approach therefore represents a further step into the ongoing integration of different high-throughput technologies for identification of ancient biomolecules, unleashing the field of paleoproteomics.
http://pubs.acs.org/doi/abs/10.1021/pr200721u
Direct evidence of milk consumption from ancient human dental calculus.
Warinner C, Hendy J, Speller C, Cappellini E, Fischer R, Trachsel C, Arneborg J, Lynnerup N, Craig OE, Swallow DM, Fotakis A, Christensen RJ, Olsen JV, Liebert A, Montalva N, Fiddyment S, Charlton S, Mackie M, Canci A, Bouwman A, Rühli F, Gilbert MT, Collins MJ.
Sci Rep. 2014 Nov 27;4:7104. doi: 10.1038/srep07104.
Milk is a major food of global economic importance, and its consumption is regarded as a classic example of gene-culture evolution. Humans have exploited animal milk as a food resource for at least 8500 years, but the origins, spread, and scale of dairying remain poorly understood. Indirect lines of evidence, such as lipid isotopic ratios of pottery residues, faunal mortality profiles, and lactase persistence allele frequencies, provide a partial picture of this process; however, in order to understand how, where, and when humans consumed milk products, it is necessary to link evidence of consumption directly to individuals and their dairy livestock. Here we report the first direct evidence of milk consumption, the whey protein β-lactoglobulin (BLG), preserved in human dental calculus from the Bronze Age (ca. 3000 BCE) to the present day. Using protein tandem mass spectrometry, we demonstrate that BLG is a species-specific biomarker of dairy consumption, and we identify individuals consuming cattle, sheep, and goat milk products in the archaeological record. We then apply this method to human dental calculus from Greenland's medieval Norse colonies, and report a decline of this biomarker leading up to the abandonment of the Norse Greenland colonies in the 15(th) century CE.
http://www.nature.com/articles/srep07104
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