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
Tyers M, Mann M. From genomics to proteomics. Nature. 2003 Mar 13;422(6928):193-7
Proteomics is the study of the function of all expressed proteins. Tremendous progress has been made in the past few years in generating large-scale data sets for protein–protein interactions, organelle composition, protein activity patterns and protein profiles in cancer patients. But further technological improvements, organization of international proteomics projects and open access to results are needed for proteomics to fulfil its potential.
Pandey A, Mann M. Proteomics to study genes and genomes. Nature. 2000 Jun 15;405(6788):837-46.
Proteomics, the large-scale analysis of proteins, will contribute greatly to our understanding of gene function in the post-genomic era. Proteomics can be divided into three main areas: (1) protein micro-characterization for large-scale identification of proteins and their post-translational modifications; (2) 'differential display' proteomics for comparison of protein levels with potential application in a wide range of diseases; and (3) studies of protein-protein interactions using techniques such as mass spectrometry or the yeast two-hybrid system. Because it is often difficult to predict the function of a protein based on homology to other proteins or even their three-dimensional structure, determination of components of a protein complex or of a cellular structure is central in functional analysis. This aspect of proteomic studies is perhaps the area of greatest promise. After the revolution in molecular biology exemplified by the ease of cloning by DNA methods, proteomics will add to our understanding of the biochemistry of proteins, processes and pathways for years to come.
Proteomics: An atlas of expression
Vivien Marx
The first draft of the complete human proteome has been more than a decade in the making. In the process, the effort has also delivered lessons about technology and biology.
http://www.nature.com/nature/journal/v509/n7502/full/509645a.html
Raphael I, Mahesula S1, Purkar A, Black D, Catala A, Gelfond JA, Forsthuber TG, Haskins WE Microwave & magnetic (M2) proteomics reveals CNS-specific protein expression waves that precede clinical symptoms of experimental autoimmune encephalomyelitis. Sci Rep. 2014 Sep 3;4:6210.
Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave &magnetic (M(2)) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M(2) proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M(2) proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M(2) proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE
http://www.nature.com/articles/srep06210
Müller C, Khabut A, Dudhia J, Reinholt FP, Aspberg A, Heinegård D, Önnerfjord P. Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution. Matrix Biol. 2014 Nov;40:34-45.
The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.
http://www.sciencedirect.com/science/article/pii/S0945053X14001668
Vivien Marx
The first draft of the complete human proteome has been more than a decade in the making. In the process, the effort has also delivered lessons about technology and biology.
http://www.nature.com/nature/journal/v509/n7502/full/509645a.html
Raphael I, Mahesula S1, Purkar A, Black D, Catala A, Gelfond JA, Forsthuber TG, Haskins WE Microwave & magnetic (M2) proteomics reveals CNS-specific protein expression waves that precede clinical symptoms of experimental autoimmune encephalomyelitis. Sci Rep. 2014 Sep 3;4:6210.
Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave &magnetic (M(2)) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M(2) proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M(2) proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M(2) proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE
http://www.nature.com/articles/srep06210
Müller C, Khabut A, Dudhia J, Reinholt FP, Aspberg A, Heinegård D, Önnerfjord P. Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution. Matrix Biol. 2014 Nov;40:34-45.
The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.
http://www.sciencedirect.com/science/article/pii/S0945053X14001668
Protein painting reveals solvent-excluded drug targets hidden within native protein–protein interfaces
Alessandra Luchini, Virginia Espina & Lance A. Liotta
Identifying the contact regions between a protein and its binding partners is essential for creating therapies that block the interaction. Unfortunately, such contact regions are extremely difficult to characterize because they are hidden inside the binding interface. Here we introduce protein painting as a new tool that employs small molecules as molecular paints to tightly coat the surface of protein–protein complexes. The molecular paints, which block trypsin cleavage sites, are excluded from the binding interface. Following mass spectrometry, only peptides hidden in the interface emerge as positive hits, revealing the functional contact regions that are drug targets. We use protein painting to discover contact regions between the three-way interaction of IL1β ligand, the receptor IL1RI and the accessory protein IL1RAcP. We then use this information to create peptides and monoclonal antibodies that block the interaction and abolish IL1β cell signalling. The technology is broadly applicable to discover protein interaction drug targets.
Analysing proteomic data
J. Barrett, , P.M. Brophy, J.V. Hamilton
The rapid growth of proteomics has been made possible by the development of reproducible 2D gels and biological mass spectrometry. However, despite technical improvements 2D gels are still less than perfectly reproducible and gels have to be aligned so spots for identical proteins appear in the same place. Gels can be warped by a variety of techniques to make them concordant. When gels are manipulated to improve registration, information is lost, so direct methods for gel registration which make use of all available data for spot matching are preferable to indirect ones. In order to identify proteins from gel spots a property or combination of properties that are unique to that protein are required. These can then be used to search databases for possible matches. Molecular mass, pI, amino acid composition and short sequence tags can all be used in database searches. Currently the method of choice for protein identification is mass spectrometry. Proteins are eluted from the gels and cleaved with specific endoproteases to produce a series of peptides of different molecular mass. In peptide mass fingerprinting, the peptide profile of the unknown protein is compared with theoretical peptide libraries generated from sequences in the different databases. Tandem mass spectroscopy (MS/MS) generates short amino acid sequence tags for the individual peptides. These partial sequences combined with the original peptide masses are then used for database searching, greatly improving specificity. Increasingly protein identification from MS/MS data is being fully or partially automated. When working with organisms, which do not have sequenced genomes (the case with most helminths), protein identification by database searching becomes problematical. A number of approaches to cross species protein identification have been suggested, but if the organism being studied is only distantly related to any organism with a sequenced genome then the likelihood of protein identification remains small. The dynamic nature of the proteome means that there really is no such thing as a single representative proteome and a complete set of metadata (data about the data) is going to be required if the full potential of database mining is to be realised in the future.
Nebija D, Noe CR, Urban E, Lachmann B. Quality control and stability studies with the monoclonal antibody, trastuzumab: application of 1D- vs. 2D-gel electrophoresis. Int J Mol Sci. 2014 Apr 15;15(4):6399-411.
Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.
Biron DG, Brun C, Lefevre T, Lebarbenchon C, Loxdale HD, Chevenet F, Brizard JP, Thomas F. The pitfalls of proteomics experiments without the correct use of bioinformatics tools. Proteomics. 2006 Oct;6(20):5577-96.
The elucidation of the entire genomic sequence of various organisms, from viruses to complex metazoans, most recently man, is undoubtedly the greatest triumph of molecular biology since the discovery of the DNA double helix. Over the past two decades, the focus of molecular biology has gradually moved from genomes to proteomes, the intention being to discover the functions of the genes themselves. The postgenomic era stimulated the development of new techniques (e.g. 2-DE and MS) and bioinformatics tools to identify the functions, reactions, interactions and location of the gene products in tissues and/or cells of living organisms. Both 2-DE and MS have been very successfully employed to identify proteins involved in biological phenomena (e.g. immunity, cancer, host-parasite interactions, etc.), although recently, several papers have emphasised the pitfalls of 2-DE experiments, especially in relation to experimental design, poor statistical treatment and the high rate of 'false positive' results with regard to protein identification. In the light of these perceived problems, we review the advantages and misuses of bioinformatics tools - from realisation of 2-DE gels to the identification of candidate protein spots - and suggest some useful avenues to improve the quality of 2-DE experiments. In addition, we present key steps which, in our view, need to be to taken into consideration during such analyses. Lastly, we present novel biological entities named 'interactomes', and the bioinformatics tools developed to analyse the large protein-protein interaction networks they form, along with several new perspectives of the field.
Rabilloud T. Paleoproteomics explained to youngsters: how did the wedding of two-dimensional electrophoresis and protein sequencing spark proteomics on: let there be light. J Proteomics. 2014 Jul 31;107:5-12.
Taking the opportunity of the 20th anniversary of the word "proteomics", this young adult age is a good time to remember how proteomics came from enormous progress in protein separation and protein microanalysis techniques, and from the conjugation of these advances into a high performance and streamlined working setup. However, in the history of the almost three decades that encompass the first attempts to perform large scale analysis of proteins to the current high throughput proteomics that we can enjoy now, it is also interesting to underline and to recall how difficult the first decade was. Indeed when the word was cast, the battle was already won. This recollection is mostly devoted to the almost forgotten period where proteomics was being conceived and put to birth, as this collective scientific work will never appear when searched through the keyword "proteomics".
BIBLIOGRAFÍA COMPLEMENTARIA
Steen H, Mann M. The ABC's (and XYZ's) of peptide sequencing. Nat Rev Mol Cell Biol. 2004 Sep;5(9):699-711.
Proteomics is an increasingly powerful and indispensable technology in molecular
cell biology. It can be used to identify the components of small protein
complexes and large organelles, to determine post-translational modifications and
in sophisticated functional screens. The key - but little understood - technology
in mass-spectrometry-based proteomics is peptide sequencing, which we describe
and review here in an easily accessible format.
http://www.nature.com/nrm/journal/v5/n9/full/nrm1468.html
Mass-spectrometry-based draft of the human proteome
Mathias Wilhelm, Judith Schlegl, Hannes Hahne, Amin Moghaddas Gholami, Marcus Lieberenz, Mikhail M. Savitski, Emanuel Ziegler, Lars Butzmann, Siegfried Gessulat, Harald Marx, Toby Mathieson, Simone Lemeer, Karsten Schnatbaum, Ulf Reimer, Holger Wenschuh, Martin Mollenhauer, Julia Slotta-Huspenina, Joos-Hendrik Boese, Marcus Bantscheff, Anja Gerstmair, Franz Faerber & Bernhard Kuster
Proteomes are characterized by large protein-abundance differences, cell-type- and time-dependent expression patterns and post-translational modifications, all of which carry biological information that is not accessible by genomics or transcriptomics. Here we present a mass-spectrometry-based draft of the human proteome and a public, high-performance, in-memory database for real-time analysis of terabytes of big data, called ProteomicsDB. The information assembled from human tissues, cell lines and body fluids enabled estimation of the size of the protein-coding genome, and identified organ-specific proteins and a large number of translated lincRNAs (long intergenic non-coding RNAs). Analysis of messenger RNA and protein-expression profiles of human tissues revealed conserved control of protein abundance, and integration of drug-sensitivity data enabled the identification of proteins predicting resistance or sensitivity. The proteome profiles also hold considerable promise for analysing the composition and stoichiometry of protein complexes. ProteomicsDB thus enables navigation of proteomes, provides biological insight and fosters the development of proteomic technology.
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