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.
Selected reaction monitoring (SRM) is a targeted mass spectrometry technique that is emerging in the field of proteomics as a complement to untargeted shotgun methods. SRM is particularly useful when ...predetermined sets of proteins, such as those constituting cellular networks or sets of candidate biomarkers, need to be measured across multiple samples in a consistent, reproducible and quantitatively precise manner. Here we describe how SRM is applied in proteomics, review recent advances, present selected applications and provide a perspective on the future of this powerful technology.
The question of how genomic information is expressed to determine phenotypes is of central importance for basic and translational life science research and has been studied by transcriptomic and ...proteomic profiling. Here, we review the relationship between protein and mRNA levels under various scenarios, such as steady state, long-term state changes, and short-term adaptation, demonstrating the complexity of gene expression regulation, especially during dynamic transitions. The spatial and temporal variations of mRNAs, as well as the local availability of resources for protein biosynthesis, strongly influence the relationship between protein levels and their coding transcripts. We further discuss the buffering of mRNA fluctuations at the level of protein concentrations. We conclude that transcript levels by themselves are not sufficient to predict protein levels in many scenarios and to thus explain genotype-phenotype relationships and that high-quality data quantifying different levels of gene expression are indispensable for the complete understanding of biological processes.
Genomic information becomes translated into phenotypes via the complex and highly regulated process of gene expression and translation. Here, we review the relationship between mRNA and protein levels under various scenarios and conclude that transcript abundance by itself is not always sufficient to predict protein levels, emphasizing the complexity of the genotype-phenotype relationships.
Chemical cross-linking in combination with LC-MS/MS (XL-MS) is an emerging technology to obtain low-resolution structural (distance) restraints of proteins and protein complexes. These restraints can ...also be used to characterize protein complexes by integrative modeling of the XL-MS data, either in combination with other types of structural information or by themselves, to establish spatial relationships of subunits in protein complexes. Here we present a protocol that has been successfully used to generate XL-MS data from a multitude of native proteins and protein complexes. It includes the experimental steps for performing the cross-linking reaction using disuccinimidyl suberate (a homobifunctional, lysine-reactive cross-linking reagent), the enrichment of cross-linked peptides by peptide size-exclusion chromatography (SEC; to remove smaller, non-cross-linked peptides), instructions for tandem MS analysis and the analysis of MS data via the open-source computational software pipeline xQuest and xProphet (available from http://proteomics.ethz.ch). Once established, this robust protocol should take ∼4 d to complete, and it is generally applicable to purified proteins and protein complexes.
The vast majority of proteomic studies to date have relied on mass spectrometric techniques to identify, and in some cases quantify, peptides that have been generated by proteolysis. Current ...approaches differ in the types of instrument used, their performance profiles, the manner in which they interface with biological research strategies, and their reliance on and use of prior information. Here, we consider the three main mass spectrometry (MS)-based proteomic approaches used today: shotgun (or discovery), directed and targeted strategies. We discuss the principles of each technique, their strengths and weaknesses and the dependence of their performance profiles on the composition of the biological sample. Our goal is to provide a rational framework for selecting strategies optimally suited to address the specific research issue under consideration.
In recent years, chemical crosslinking of protein complexes and the identification of crosslinked residues by mass spectrometry (XL-MS; sometimes abbreviated as CX-MS) has become an important ...technique bridging mass spectrometry (MS) and structural biology. By now, XL-MS is well established and supported by publicly available resources as a convenient and versatile part of the structural biologist's toolbox. The combination of XL-MS with cryo-electron microscopy (cryo-EM) and/or integrative modeling is particularly promising to study the topology and structure of large protein assemblies. Among the targets studied so far are proteasomes, ribosomes, polymerases, chromatin remodelers, and photosystem complexes. Here we provide an overview of recent advances in XL-MS, the current state of the field, and a cursory outlook on future challenges.
Chemical crosslinking followed by the mass spectrometric analysis of cross-linked peptides (XL-MS) identifies contact sites between residues within a single or between multiple proteins.
The application of XL-MS to many biologically relevant molecular machines has been shown, with a rapidly growing number of successful studies reported in the past 2–3 years.
Crosslinking data are useful in integrative modeling workflows by providing distance restraints on the surface of folded proteins and complexes. XL-MS has been shown to be particularly powerful in combination with 3D cryo-electron microscopy.
The systematic and quantitative molecular analysis of mutant organisms that has been pioneered by studies on mutant metabolomes and transcriptomes holds great promise for improving our understanding ...of how phenotypes emerge. Unfortunately, owing to the limitations of classical biochemical analysis, proteins have previously been excluded from such studies. Here we review how technical advances in mass spectrometry-based proteomics can be applied to measure changes in protein abundance, posttranslational modifications and protein-protein interactions in mutants at the scale of the proteome. We finally discuss examples that integrate proteomics data with genomic and phenomic information to build network-centred models, which provide a promising route for understanding how phenotypes emerge.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
8.
Mass Spectrometry and Protein Analysis Domon, Bruno; Aebersold, Ruedi
Science (American Association for the Advancement of Science),
04/2006, Letnik:
312, Številka:
5771
Journal Article
Recenzirano
Mass spectrometry is a central analytical technique for protein research and for the study of biomolecules in general. Driven by the need to identify, characterize, and quantify proteins at ever ...increasing sensitivity and in ever more complex samples, a wide range of new mass spectrometry-based analytical platforms and experimental strategies have emerged. Here we review recent advances in mass spectrometry instrumentation in the context of current and emerging research strategies in protein science.
Data-independent acquisition modes isolate and concurrently fragment populations of different precursors by cycling through segments of a predefined precursor m/z range. Although these selection ...windows collectively cover the entire m/z range, overall, only a few per cent of all incoming ions are isolated for mass analysis. Here, we make use of the correlation of molecular weight and ion mobility in a trapped ion mobility device (timsTOF Pro) to devise a scan mode that samples up to 100% of the peptide precursor ion current in m/z and mobility windows. We extend an established targeted data extraction workflow by inclusion of the ion mobility dimension for both signal extraction and scoring and thereby increase the specificity for precursor identification. Data acquired from whole proteome digests and mixed organism samples demonstrate deep proteome coverage and a high degree of reproducibility as well as quantitative accuracy, even from 10 ng sample amounts.
Systems biology relies on data sets in which the same group of proteins is consistently identified and precisely quantified across multiple samples, a requirement that is only partially achieved by ...current proteomics approaches. Selected reaction monitoring (SRM)—also called multiple reaction monitoring—is emerging as a technology that ideally complements the discovery capabilities of shotgun strategies by its unique potential for reliable quantification of analytes of low abundance in complex mixtures. In an SRM experiment, a predefined precursor ion and one of its fragments are selected by the two mass filters of a triple quadrupole instrument and monitored over time for precise quantification. A series of transitions (precursor/fragment ion pairs) in combination with the retention time of the targeted peptide can constitute a definitive assay. Typically, a large number of peptides are quantified during a single LC‐MS experiment. This tutorial explains the application of SRM for quantitative proteomics, including the selection of proteotypic peptides and the optimization and validation of transitions. Furthermore, normalization and various factors affecting sensitivity and accuracy are discussed.
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