The ability of living systems to adapt to changing conditions originates from their capacity to change their molecular constitution. This is achieved by multiple mechanisms that modulate the ...quantitative composition and the diversity of the molecular inventory. Molecular diversification is particularly pronounced on the proteome level, at which multiple proteoforms derived from the same gene can in turn combinatorially form different protein complexes, thus expanding the repertoire of functional modules in the cell. The study of molecular and modular diversity and their involvement in responses to changing conditions has only recently become possible through the development of new 'omics'-based screening technologies. This Review explores our current knowledge of the mechanisms regulating functional diversification along the axis of gene expression, with a focus on the proteome and interactome. We explore the interdependence between different molecular levels and how this contributes to functional diversity. Finally, we highlight several recent techniques for studying molecular diversity, with specific focus on mass spectrometry-based analysis of the proteome and its organization into functional modules, and examine future directions for this rapidly growing field.
Protein complexes are the main functional modules in the cell that coordinate and perform the vast majority of molecular functions. The main approaches to identify and quantify the interactome to ...date are based on mass spectrometry (MS). Here I summarize the benefits and limitations of different MS-based interactome screens, with a focus on untargeted interactome acquisition, such as co-fractionation MS. Specific emphasis is given to the discussion of discovery- versus hypothesis-driven data analysis concepts and their applicability to large, proteome-wide interactome screens. Hypothesis-driven analysis approaches, i.e., complex- or network-centric, are highlighted as promising strategies for comparative studies. While these approaches require prior information from public databases, also reviewed herein, the available wealth of interactomic data continuously increases, thereby providing more exhaustive information for future studies. Finally, guidance on the selection of interactome acquisition and analysis methods is provided to aid the reader in the design of protein-protein interaction studies.
Protein ubiquitination is involved in virtually all cellular processes. Enrichment strategies employing antibodies targeting ubiquitin-derived diGly remnants combined with mass spectrometry (MS) have ...enabled investigations of ubiquitin signaling at a large scale. However, so far the power of data independent acquisition (DIA) with regards to sensitivity in single run analysis and data completeness have not yet been explored. Here, we develop a sensitive workflow combining diGly antibody-based enrichment and optimized Orbitrap-based DIA with comprehensive spectral libraries together containing more than 90,000 diGly peptides. This approach identifies 35,000 diGly peptides in single measurements of proteasome inhibitor-treated cells - double the number and quantitative accuracy of data dependent acquisition. Applied to TNF signaling, the workflow comprehensively captures known sites while adding many novel ones. An in-depth, systems-wide investigation of ubiquitination across the circadian cycle uncovers hundreds of cycling ubiquitination sites and dozens of cycling ubiquitin clusters within individual membrane protein receptors and transporters, highlighting new connections between metabolism and circadian regulation.
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.
Abstract
Tumor necrosis factor (TNF) is one of the few cytokines successfully targeted by therapies against inflammatory diseases. However, blocking this well studied and pleiotropic ligand can cause ...dramatic side-effects. Here, we reason that a systems-level proteomic analysis of TNF signaling could dissect its diverse functions and offer a base for developing more targeted therapies. Therefore, we combine phosphoproteomics time course experiments with subcellular localization and kinase inhibitor analysis to identify functional modules of protein phosphorylation. The majority of regulated phosphorylation events can be assigned to an upstream kinase by inhibiting master kinases. Spatial proteomics reveals phosphorylation-dependent translocations of hundreds of proteins upon TNF stimulation. Phosphoproteome analysis of TNF-induced apoptosis and necroptosis uncovers a key role for transcriptional cyclin-dependent kinase activity to promote cytokine production and prevent excessive cell death downstream of the TNF signaling receptor. This resource of TNF-induced pathways and sites can be explored at
http://tnfviewer.biochem.mpg.de/
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To a large extent functional diversity in cells is achieved by the expansion of molecular complexity beyond that of the coding genome. Various processes create multiple distinct but related proteins ...per coding gene - so-called proteoforms - that expand the functional capacity of a cell. Evaluating proteoforms from classical bottom-up proteomics datasets, where peptides instead of intact proteoforms are measured, has remained difficult. Here we present COPF, a tool for COrrelation-based functional ProteoForm assessment in bottom-up proteomics data. It leverages the concept of peptide correlation analysis to systematically assign peptides to co-varying proteoform groups. We show applications of COPF to protein complex co-fractionation data as well as to more typical protein abundance vs. sample data matrices, demonstrating the systematic detection of assembly- and tissue-specific proteoform groups, respectively, in either dataset. We envision that the presented approach lays the foundation for a systematic assessment of proteoforms and their functional implications directly from bottom-up proteomic datasets.
Reproducibility in research can be compromised by both biological and technical variation, but most of the focus is on removing the latter. Here we investigate the effects of biological variation in ...HeLa cell lines using a systems-wide approach. We determine the degree of molecular and phenotypic variability across 14 stock HeLa samples from 13 international laboratories. We cultured cells in uniform conditions and profiled genome-wide copy numbers, mRNAs, proteins and protein turnover rates in each cell line. We discovered substantial heterogeneity between HeLa variants, especially between lines of the CCL2 and Kyoto varieties, and observed progressive divergence within a specific cell line over 50 successive passages. Genomic variability has a complex, nonlinear effect on transcriptome, proteome and protein turnover profiles, and proteotype patterns explain the varying phenotypic response of different cell lines to Salmonella infection. These findings have implications for the interpretation and reproducibility of research results obtained from human cultured cells.
Machine learning and in particular deep learning (DL) are increasingly important in mass spectrometry (MS)-based proteomics. Recent DL models can predict the retention time, ion mobility and fragment ...intensities of a peptide just from the amino acid sequence with good accuracy. However, DL is a very rapidly developing field with new neural network architectures frequently appearing, which are challenging to incorporate for proteomics researchers. Here we introduce AlphaPeptDeep, a modular Python framework built on the PyTorch DL library that learns and predicts the properties of peptides ( https://github.com/MannLabs/alphapeptdeep ). It features a model shop that enables non-specialists to create models in just a few lines of code. AlphaPeptDeep represents post-translational modifications in a generic manner, even if only the chemical composition is known. Extensive use of transfer learning obviates the need for large data sets to refine models for particular experimental conditions. The AlphaPeptDeep models for predicting retention time, collisional cross sections and fragment intensities are at least on par with existing tools. Additional sequence-based properties can also be predicted by AlphaPeptDeep, as demonstrated with a HLA peptide prediction model to improve HLA peptide identification for data-independent acquisition ( https://github.com/MannLabs/PeptDeep-HLA ).
The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry ...(MS)-based proteomics has identified and quantified tens of thousands of posttranslational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, disordered regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis, which revealed examples of spatial coregulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.
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