Leveraging crosslinking mass spectrometry in structural and cell biology

Structure ◽  
2021 ◽  
Author(s):  
Andrea Graziadei ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Cell Biology

scholarly journals Learning from heterogeneous data sources: an application in spatial proteomics

2015 ◽  
Author(s):  
Lisa M. Breckels ◽  
Sean Holden ◽  
David Wojnar ◽  
Claire M. Mulvey ◽  
Andy Christoforou ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Support Vector Machine ◽  
Transfer Learning ◽  
High Throughput ◽  
Cell Biology ◽  
Heterogeneous Data ◽  
Data Sources ◽  
Support Vector ◽  
Proteomics Data ◽  
Heterogeneous Data Sources

AbstractSub-cellular localisation of proteins is an essential post-translational regulatory mechanism that can be assayed using high-throughput mass spectrometry (MS). These MS-based spatial proteomics experiments enable us to pinpoint the sub-cellular distribution of thousands of proteins in a specific system under controlled conditions. Recent advances in high-throughput MS methods have yielded a plethora of experimental spatial proteomics data for the cell biology community. Yet, there are many third-party data sources, such as immunofluorescence microscopy or protein annotations and sequences, which represent a rich and vast source of complementary information. We present a unique transfer learning classification framework that utilises a nearest-neighbour or support vector machine system, to integrate heterogeneous data sources to considerably improve on the quantity and quality of sub-cellular protein assignment. We demonstrate the utility of our algorithms through evaluation of five experimental datasets, from four different species in conjunction with four different auxiliary data sources to classify proteins to tens of sub-cellular compartments with high generalisation accuracy. We further apply the method to an experiment on pluripotent mouse embryonic stem cells to classify a set of previously unknown proteins, and validate our findings against a recent high resolution map of the mouse stem cell proteome. The methodology is distributed as part of the open-source Bioconductor pRoloc suite for spatial proteomics data analysis.AbbreviationsLOPITLocalisation of Organelle Proteins by Isotope TaggingPCPProtein Correlation ProfilingMLMachine learningTLTransfer learningSVMSupport vector machinePCAPrincipal component analysisGOGene OntologyCCCellular compartmentiTRAQIsobaric tags for relative and absolute quantitationTMTTandem mass tagsMSMass spectrometry

scholarly journals Extensive non-canonical phosphorylation in human cells revealed using strong-anion exchange-mediated phosphoproteomics

2017 ◽  
Author(s):  
Gemma Hardman ◽  
Simon Perkins ◽  
Zheng Ruan ◽  
Natarajan Kannan ◽  
Philip Brownridge ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Phosphorylation ◽  
Anion Exchange ◽  
Human Cell ◽  
Cell Biology ◽  
Human Cells ◽  
Phosphopeptide Enrichment ◽  
Cell Extracts ◽  
Strong Anion Exchange ◽  
Acid Labile

Protein phosphorylation is a ubiquitous post-translational modification (PTM) that regulates all aspects of life. To date, investigation of human cell signalling has focussed on canonical phosphorylation of serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. However, mounting evidence suggests that phosphorylation of histidine also plays a central role in regulating cell biology. Phosphoproteomics workflows rely on acidic conditions for phosphopeptide enrichment, which are incompatible with the analysis of acid-labile phosphorylation such as histidine. Consequently, the extent of non-canonical phosphorylation is likely to be under-estimated. We report an Unbiased Phosphopeptide enrichment strategy based on Strong Anion Exchange (SAX) chromatography (UPAX), which permits enrichment of acid-labile phosphopeptides for characterisation by mass spectrometry. Using this approach, we identify extensive and positional phosphorylation patterns on histidine, arginine, lysine, aspartate and glutamate in human cell extracts, including 310 phosphohistidine and >1000 phospholysine sites of protein modification. Remarkably, the extent of phosphorylation on individual non-canonical residues vastly exceeds that of basal phosphotyrosine. Our study reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for exploring roles of acid-labile phosphorylation in any proteome using mass spectrometry.

Lipid Cell Biology: A Focus on Lipids in Cell Division

2018 ◽  
Vol 87 (1) ◽  
pp. 839-869 ◽  
Author(s):  
Elisabeth M. Storck ◽  
Cagakan Özbalci ◽  
Ulrike S. Eggert
Keyword(s):  
Mass Spectrometry ◽  
Cell Division ◽  
Cell Biology ◽  
Chemical Biology ◽  
Structural Integrity ◽  
Advanced Imaging ◽  
Functional Roles ◽  
Lipid Interactions ◽  
Cellular Processes ◽  
Alkyl Side Chains

Cells depend on hugely diverse lipidomes for many functions. The actions and structural integrity of the plasma membrane and most organelles also critically depend on membranes and their lipid components. Despite the biological importance of lipids, our understanding of lipid engagement, especially the roles of lipid hydrophobic alkyl side chains, in key cellular processes is still developing. Emerging research has begun to dissect the importance of lipids in intricate events such as cell division. This review discusses how these structurally diverse biomolecules are spatially and temporally regulated during cell division, with a focus on cytokinesis. We analyze how lipids facilitate changes in cellular morphology during division and how they participate in key signaling events. We identify which cytokinesis proteins are associated with membranes, suggesting lipid interactions. More broadly, we highlight key unaddressed questions in lipid cell biology and techniques, including mass spectrometry, advanced imaging, and chemical biology, which will help us gain insights into the functional roles of lipids.

scholarly journals Structure elucidation of colibactin and its DNA cross-links

Science ◽  
2019 ◽  
Vol 365 (6457) ◽  
pp. eaax2685 ◽  
Author(s):  
Mengzhao Xue ◽  
Chung Sub Kim ◽  
Alan R. Healy ◽  
Kevin M. Wernke ◽  
Zhixun Wang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Mass Spectrometry ◽  
Cell Biology ◽  
Structure Elucidation ◽  
Isotope Labeling ◽  
Tandem Mass ◽  
Physiological Effects ◽  
Human Gut ◽  
Structural Assignment ◽  
Cross Links

Colibactin is a complex secondary metabolite produced by some genotoxic gut Escherichia coli strains. The presence of colibactin-producing bacteria correlates with the frequency and severity of colorectal cancer in humans. However, because colibactin has not been isolated or structurally characterized, studying the physiological effects of colibactin-producing bacteria in the human gut has been difficult. We used a combination of genetics, isotope labeling, tandem mass spectrometry, and chemical synthesis to deduce the structure of colibactin. Our structural assignment accounts for all known biosynthetic and cell biology data and suggests roles for the final unaccounted enzymes in the colibactin gene cluster.

scholarly journals Mass spectrometry: From plasma proteins to mitochondrial membranes

2019 ◽  
Vol 116 (8) ◽  
pp. 2814-2820 ◽  
Author(s):  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Cell Biology ◽  
Protein Complexes ◽  
Intact Protein ◽  
Exchange Reactions ◽  
Soft Landing ◽  
Mass Spectrometers ◽  
Radical Method ◽  
Key Steps ◽  
Soluble Complexes

In this Inaugural Article, I trace some key steps that have enabled the development of mass spectrometry for the study of intact protein complexes from a variety of cellular environments. Beginning with the preservation of the first soluble complexes from plasma, I describe our early experiments that capitalize on the heterogeneity of subunit composition during assembly and exchange reactions. During these investigations, we observed many assemblies and intermediates with different subunit stoichiometries, and were keen to ascertain whether or not their overall topology was preserved in the mass spectrometer. Adapting ion mobility and soft-landing methodologies, we showed how ring-shaped complexes could survive the phase transition. The next logical progression from soluble complexes was to membrane protein assemblies but this was not straightforward. We encountered many pitfalls along the way, largely due to the use of detergent micelles to protect and stabilize complexes. Further obstacles presented when we attempted to distinguish lipids that copurify from those that are important for function. Developing new experimental protocols, we have subsequently defined lipids that change protein conformation, mediate oligomeric states, and facilitate downstream coupling of G protein-coupled receptors. Very recently, using a radical method—ejecting protein complexes directly from native membranes into mass spectrometers—we provided insights into associations within membranes and mitochondria. Together, these developments suggest the beginnings of mass spectrometry meeting with cell biology.

scholarly journals Mass spectrometry–based proteomics in cell biology

2010 ◽  
Vol 190 (4) ◽  
pp. 491-500 ◽  
Author(s):  
Tobias C. Walther ◽  
Matthias Mann
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Analysis ◽  
High Resolution ◽  
Sample Preparation ◽  
Cell Biology ◽  
Computational Analysis ◽  
Global Analysis ◽  
Protein Composition ◽  
Broad Community

The global analysis of protein composition, modifications, and dynamics are important goals in cell biology. Mass spectrometry (MS)–based proteomics has matured into an attractive technology for this purpose. Particularly, high resolution MS methods have been extremely successful for quantitative analysis of cellular and organellar proteomes. Rapid advances in all areas of the proteomic workflow, including sample preparation, MS, and computational analysis, should make the technology more easily available to a broad community and turn it into a staple methodology for cell biologists.

The protein microscope: incorporating mass spectrometry into cell biology

2007 ◽  
Vol 4 (10) ◽  
pp. 783-784 ◽  
Author(s):  
Alexander W Bell ◽  
Tommy Nilsson ◽  
Robert E Kearney ◽  
John J M Bergeron
Keyword(s):  
Mass Spectrometry ◽  
Cell Biology

scholarly journals The human proteome co-regulation map reveals functional relationships between proteins

2019 ◽  
Author(s):  
Georg Kustatscher ◽  
Piotr Grabowski ◽  
Tina A. Schrader ◽  
Josiah B. Passmore ◽  
Michael Schrader ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Cell Biology ◽  
Mammalian Cells ◽  
Data Matrix ◽  
Peroxisomal Membrane ◽  
Isotope Labelling ◽  
Functional Relationships ◽  
Human Proteins ◽  
Functional Context

The annotation of protein function is a longstanding challenge of cell biology that suffers from the sheer magnitude of the task. Here we present ProteomeHD, which documents the response of 10,323 human proteins to 294 biological perturbations, measured by isotope-labelling mass spectrometry. Using this data matrix and robust machine learning we create a co-regulation map of the cell that reflects functional associations between human proteins. The map identifies a functional context for many uncharacterized proteins, including microproteins that are difficult to study with traditional methods. Co-regulation also captures relationships between proteins which do not physically interact or co-localize. For example, co-regulation of the peroxisomal membrane protein PEX11β with mitochondrial respiration factors led us to discover a novel organelle interface between peroxisomes and mitochondria in mammalian cells. The co-regulation map can be explored atwww.proteomeHD.net.

scholarly journals Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

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