Researchers in many biological areas now routinely characterize proteins by mass spectrometry. Among the many formats for quantitative proteomics, stable-isotope labelling by amino acids in cell ...culture (SILAC) has emerged as a simple and powerful one. SILAC removes false positives in protein-interaction studies, reveals large-scale kinetics of proteomes and - as a quantitative phosphoproteomics technology - directly uncovers important points in the signalling pathways that control cellular decisions.
John Fenn’s electrospray mass spectrometry (ESMS) was awarded the chemistry Nobel Prize in 2002 and is now the basis of the entire field of MS-based proteomics. Technological progress continues ...unabated, enabling single cell sensitivity and clinical applications.
Numerous biological processes are concurrently and coordinately active in every living cell. Each of them encompasses synthetic, catalytic and regulatory functions that are, almost always, carried ...out by proteins organized further into higher-order structures and networks. For decades, the structures and functions of selected proteins have been studied using biochemical and biophysical methods. However, the properties and behaviour of the proteome as an integrated system have largely remained elusive. Powerful mass-spectrometry-based technologies now provide unprecedented insights into the composition, structure, function and control of the proteome, shedding light on complex biological processes and phenotypes.
Quantitative proteomics now provides abundance ratios for thousands of proteins upon perturbations. These need to be functionally interpreted and correlated to other types of quantitative genome-wide ...data such as the corresponding transcriptome changes. We describe a new method, 2D annotation enrichment, which compares quantitative data from any two 'omics' types in the context of categorical annotation of the proteins or genes. Suitable genome-wide categories are membership of proteins in biochemical pathways, their annotation with gene ontology terms, sub-cellular localization, presence of protein domains or membership in protein complexes. 2D annotation enrichment detects annotation terms whose members show consistent behavior in one or both of the data dimensions. This consistent behavior can be a correlation between the two data types, such as simultaneous up- or down-regulation in both data dimensions, or a lack thereof, such as regulation in one dimension but no change in the other. For the statistical formulation of the test we introduce a two-dimensional generalization of the nonparametric two-sample test. The false discovery rate is stringently controlled by correcting for multiple hypothesis testing. We also describe one-dimensional annotation enrichment, which can be applied to single omics data. The 1D and 2D annotation enrichment algorithms are freely available as part of the Perseus software.
Cellular function can be controlled through the gene expression program, but often protein post-translational modifications (PTMs) provide a more precise and elegant mechanism. Key functional roles ...of specific modification events—for instance, during the cell cycle—have been known for decades, but only in the past 10 years has mass-spectrometry-(MS)-based proteomics begun to reveal the true extent of the PTM universe. In this overview for the special PTM issue of Molecular and Cellular Proteomics, we take stock of where MS-based proteomics stands in the large-scale analysis of protein modifications. For many PTMs, including phosphorylation, ubiquitination, glycosylation, and acetylation, tens of thousands of sites can now be confidently identified and localized in the sequence of the protein. The quantification of PTM levels between different cellular states is likewise established, with label-free methods showing particular promise. It is also becoming possible to determine the absolute occupancy or stoichiometry of PTM sites on a large scale. Powerful software for the bioinformatic analysis of thousands of PTM sites has been developed. However, a complete inventory of sites has not been established for any PTM, and this situation will persist into the foreseeable future. Furthermore, although PTM coverage by MS-based methods is impressive, it still needs to be improved, especially in tissues and in clinically relevant systems. The central challenge for the field is to develop streamlined methods for determining biological functions for the myriad of modifications now known to exist.
The enormous complexity of the central nervous system has impeded its systemic exploration for decades but powerful “omic” technologies are now pushing forward the frontiers of neuroscience research ...at an increasing pace. This Primer reviews the most recent progress in mass spectrometry (MS)-based proteomics, focusing on the analysis of whole proteomes, protein-based interactions, and post-translational modifications. We also discuss how advanced workflows help to unravel spatial, regulatory, and temporal aspects of neuronal systems. These tools and approaches have already led to detailed and quantitative proteomic maps of the brain and its signaling architecture, generating new insights into health and disease. We predict that these new approaches will also accelerate biomarker discovery and contribute to novel therapeutics for neurodegenerative and other brain-related diseases.
Hosp and Mann describe the value of proteomics for neurobiology by reviewing recent advances in whole proteome analyses, protein-based interactions, and post-translational modifications, offering thoughtful perspectives on how to unravel spatial, regulatory, and temporal aspects of neuronal systems by proteomics.
Efficient analysis of very large amounts of raw data for peptide identification and protein quantification is a principal challenge in mass spectrometry (MS)-based proteomics. Here we describe ...MaxQuant, an integrated suite of algorithms specifically developed for high-resolution, quantitative MS data. Using correlation analysis and graph theory, MaxQuant detects peaks, isotope clusters and stable amino acid isotope-labeled (SILAC) peptide pairs as three-dimensional objects in m/z, elution time and signal intensity space. By integrating multiple mass measurements and correcting for linear and nonlinear mass offsets, we achieve mass accuracy in the p.p.b. range, a sixfold increase over standard techniques. We increase the proportion of identified fragmentation spectra to 73% for SILAC peptide pairs via unambiguous assignment of isotope and missed-cleavage state and individual mass precision. MaxQuant automatically quantifies several hundred thousand peptides per SILAC-proteome experiment and allows statistically robust identification and quantification of >4,000 proteins in mammalian cell lysates.
Aging promotes lung function decline and susceptibility to chronic lung diseases, which are the third leading cause of death worldwide. Here, we use single cell transcriptomics and mass ...spectrometry-based proteomics to quantify changes in cellular activity states across 30 cell types and chart the lung proteome of young and old mice. We show that aging leads to increased transcriptional noise, indicating deregulated epigenetic control. We observe cell type-specific effects of aging, uncovering increased cholesterol biosynthesis in type-2 pneumocytes and lipofibroblasts and altered relative frequency of airway epithelial cells as hallmarks of lung aging. Proteomic profiling reveals extracellular matrix remodeling in old mice, including increased collagen IV and XVI and decreased Fraser syndrome complex proteins and collagen XIV. Computational integration of the aging proteome with the single cell transcriptomes predicts the cellular source of regulated proteins and creates an unbiased reference map of the aging lung.
Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and blood biomarkers are used to categorize patients and to support treatment decisions. However, existing ...biomarkers are far from comprehensive and often lack specificity and new ones are being developed at a very slow rate. As described in this review, mass spectrometry (MS)‐based proteomics has become a powerful technology in biological research and it is now poised to allow the characterization of the plasma proteome in great depth. Previous “triangular strategies” aimed at discovering single biomarker candidates in small cohorts, followed by classical immunoassays in much larger validation cohorts. We propose a “rectangular” plasma proteome profiling strategy, in which the proteome patterns of large cohorts are correlated with their phenotypes in health and disease. Translating such concepts into clinical practice will require restructuring several aspects of diagnostic decision‐making, and we discuss some first steps in this direction.
The performance of mass spectrometry (MS)‐based proteomics has reached a sensitivity and dynamic range that makes it suitable for biomarker studies. This Review discusses plasma proteome profiling strategies and how they can be translated into clinical practice.
A main bottleneck in proteomics is the downstream biological analysis of highly multivariate quantitative protein abundance data generated using mass-spectrometry-based analysis. We developed the ...Perseus software platform (http://www.perseus-framework.org) to support biological and biomedical researchers in interpreting protein quantification, interaction and post-translational modification data. Perseus contains a comprehensive portfolio of statistical tools for high-dimensional omics data analysis covering normalization, pattern recognition, time-series analysis, cross-omics comparisons and multiple-hypothesis testing. A machine learning module supports the classification and validation of patient groups for diagnosis and prognosis, and it also detects predictive protein signatures. Central to Perseus is a user-friendly, interactive workflow environment that provides complete documentation of computational methods used in a publication. All activities in Perseus are realized as plugins, and users can extend the software by programming their own, which can be shared through a plugin store. We anticipate that Perseus's arsenal of algorithms and its intuitive usability will empower interdisciplinary analysis of complex large data sets.
PDF
