scholarly journals Quantitative cross-linking/mass spectrometry using isotope-labeled cross-linkers and MaxQuant

2016 ◽  
Author(s):  
Zhuo A. Chen ◽  
Lutz Fischer ◽  
Jüergen Cox ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Identification ◽  
Recall Rate ◽  
Charge Ratio ◽  
Cross Linking ◽  
Data Sets ◽  
Automated Quantification ◽  
Feature List ◽  
Simple Step ◽  
Cross Links

AbstractThe conceptually simple step from cross-linking/mass spectrometry (CLMS) to quantitative cross-linking/mass spectrometry (QCLMS) is compounded by technical challenges. Currently, quantitative proteomics software is tightly integrated with the protein identification workflow. This prevents automatically quantifying other m/z features in a targeted manner including those associated with cross-linked peptides. Here we present a new release of MaxQuant that permits starting the quantification process from an m/z feature list. Comparing the automated quantification to a carefully manually curated test set of cross-linked peptides obtained by cross-linking C3 and C3b with BS3 and isotope-labeled BS3-d4 revealed a number of observations: 1) Fully automated process using MaxQuant can quantify cross-links in our reference dataset with 68% recall rate and 88% accuracy. 2) Hidden quantification errors can be converted into exposed failures by label-swap replica, which makes label-swap replica an essential part of QCLMS. 3) Cross-links that failed during automated quantification can be recovered by semi-automated re-quantification. The integrated workflow of MaxQuant and semi-automated assessment provides the maximum of quantified cross-links. In contrast, work on larger data sets or by less experienced users will benefit from full automation in MaxQuant.AbbreviationsBS3Bis[sulfosuccinimidyl] suberateCLMSCross-linking/mass spectrometryMS1the initial mass-to-charge-ratio (m/z) spectrum collected for all components in a sample.QCLMSQuantitative cross-linking/mass spectrometry

scholarly journals Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

2018 ◽  
Author(s):  
Therese Dau ◽  
Kapil Gupta ◽  
Imre Berger ◽  
Juri Rappsilber
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cross Linking ◽  
Cleavage Sites ◽  
Protein Modelling ◽  
Protein Protein Interactions ◽  
Important Approach ◽  
Cross Links ◽  
Sequential Digestion

ABSTRACTCross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

scholarly journals Optimized fragmentation improves the identification of peptides cross-linked using MS-cleavable reagents

2018 ◽  
Author(s):  
Christian E. Stieger ◽  
Philipp Doppler ◽  
Karl Mechtler
Keyword(s):  
Mass Spectrometry ◽  
Quality Data ◽  
Cross Linking ◽  
High Complexity ◽  
Mass Spectrometers ◽  
Acquisition Strategies ◽  
Cross Links ◽  
The Cross ◽  
The Individual

ABSTRACTCross-linking mass spectrometry (XLMS) is becoming increasingly popular, and current advances are widening the applicability of the technique so that it can be utilized by non-specialist laboratories. Specifically, the use of novel mass spectrometry-cleavable (MS-cleavable) reagents dramatically reduces complexity of the data by providing i) characteristic reporter ions and ii) the mass of the individual peptides, rather than that of the cross-linked moiety. However, optimum acquisition strategies to obtain the best quality data for such cross-linkers with higher energy C-trap dissociation (HCD) alone is yet to be achieved. Therefore, we have carefully investigated and optimized MS parameters to facilitate the identification of disuccinimidyl sulfoxide (DSSO)- based cross-links on HCD-equipped mass spectrometers. From the comparison of 9 different fragmentation energies we chose several stepped-HCD fragmentation methods that were evaluated on a variety of cross-linked proteins. The optimal stepped-HCD-method was then directly compared with previously described methods using an Orbitrap Fusion™ Lumos™ TribridTM instrument using a high-complexity sample. The final results indicate that our stepped-HCD method is able to identify more cross-links than other methods, mitigating the need for multistage MS (MSn) enabled instrumentation and alternative dissociation techniques.

scholarly journals Mass Spectrometric Identification of a Novel Factor XIIIa Cross-Linking Site in Fibrinogen

Proteomes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 43
Author(s):  
Mariya E. Semkova ◽  
J. Justin Hsuan
Keyword(s):  
Mass Spectrometry ◽  
Protein Function ◽  
Peptide Identification ◽  
Mass Spectrometric ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Spectrometric Analysis ◽  
Peptide Analysis ◽  
Linear Peptide ◽  
Cross Links

Transglutaminases are a class of enzymes that catalyze the formation of a protein:protein cross-link between a lysine and a glutamine residue. These cross-links play important roles in diverse biological processes. Analysis of cross-linking sites in target proteins is required to elucidate their molecular action on target protein function and the molecular specificity of different transglutaminase isozymes. Mass-spectrometry using settings designed for linear peptide analysis and software designed for the analysis of disulfide bridges and chemical cross-links have previously been employed to identify transglutaminase cross-linking sites in proteins. As no control peptide with which to assess and improve the mass spectrometric analysis of TG cross-linked proteins was available, we developed a method for the enzymatic synthesis of a well-defined transglutaminase cross-linked peptide pair that mimics a predicted tryptic digestion product of collagen I. We then used this model peptide to determine optimal score thresholds for correct peptide identification from y- and b-ion series of fragments produced by collision-induced dissociation. We employed these settings in an analysis of fibrinogen cross-linked by the transglutaminase Factor XIIIa. This approach resulted in identification of a novel cross-linked peptide in the gamma subunit. We discuss the difference in behavior of ions derived from different cross-linked peptide sequences and the consequent demand for a more tailored mass spectrometry approach for cross-linked peptide identification compared to that routinely used for linear peptide analysis.

scholarly journals Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

2019 ◽  
Author(s):  
Tamar Tayri-Wilk ◽  
Moriya Slavin ◽  
Joanna Zamel ◽  
Ayelet Blass ◽  
Shon Cohen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Atom ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Atomic Structures ◽  
Cross Links ◽  
Structured Proteins ◽  
Structural Insights ◽  
Current Mechanism

AbstractFormaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

scholarly journals MaXLinker: Proteome-wide Cross-link Identifications with High Specificity and Sensitivity

2019 ◽  
Vol 19 (3) ◽  
pp. 554-568 ◽  
Author(s):  
Kumar Yugandhar ◽  
Ting-Yi Wang ◽  
Alden King-Yung Leung ◽  
Michael Charles Lanz ◽  
Ievgen Motorykin ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Search Engine ◽  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Cross Linking ◽  
Interaction Patterns ◽  
Protein Protein Interactions ◽  
Cross Link ◽  
Wide Cross ◽  
Cross Links

Protein-protein interactions play a vital role in nearly all cellular functions. Hence, understanding their interaction patterns and three-dimensional structural conformations can provide crucial insights about various biological processes and underlying molecular mechanisms for many disease phenotypes. Cross-linking mass spectrometry (XL-MS) has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. The inception of MS-cleavable cross-linkers enabled the MS2-MS3 XL-MS acquisition strategy that provides cross-link information from both MS2 and MS3 level. However, the current cross-link search algorithm available for MS2-MS3 strategy follows a “MS2-centric” approach and suffers from a high rate of mis-identified cross-links. We demonstrate the problem using two new quality assessment metrics [“fraction of mis-identifications” (FMI) and “fraction of interprotein cross-links from known interactions” (FKI)]. We then address this problem, by designing a novel “MS3-centric” approach for cross-link identification and implementing it as a search engine named MaXLinker. MaXLinker outperforms the currently popular search engine with a lower mis-identification rate, and higher sensitivity and specificity. Moreover, we performed human proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we identified a comprehensive set of 9319 unique cross-links at 1% false discovery rate, comprising 8051 intraprotein and 1268 interprotein cross-links. Finally, we experimentally validated the quality of a large number of novel interactions identified in our study, providing a conclusive evidence for MaXLinker's robust performance.

scholarly journals Peptidoglycan Cross-Linking Activity of L,D-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by β-Lactams

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Laetitia Sütterlin ◽  
Zainab Edoo ◽  
Jean-Emmanuel Hugonnet ◽  
Jean-Luc Mainardi ◽  
Michel Arthur
Keyword(s):  
Mass Spectrometry ◽  
Clostridium Difficile ◽  
Cross Linking ◽  
Content Type ◽  
Catalytic Activities ◽  
Cross Links ◽  
Covalent Adducts ◽  
The Stability ◽  
Hydrolysis Of ◽  
Insight Into

ABSTRACT In most bacteria, the essential targets of β-lactam antibiotics are the d , d -transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l , d -transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l , d -transpeptidase paralogues Ldt Cd1 , Ldt Cd2 , and Ldt Cd3 , which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt Cd2 and Ldt Cd3 catalyzed the formation of 3→3 cross-links ( l , d -transpeptidase activity), the hydrolysis of the C-terminal d -Ala residue of the disaccharide-tetrapeptide substrate ( l , d -carboxypeptidase activity), and the exchange of the C-terminal d -Ala for d -Met. Ldt Cd1 displayed only l , d -carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt Cd1 and Ldt Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt Cd1 and Ldt Cd2 was higher for the carbapenems (480 to 6,600 M −1 s −1 ) than for ampicillin and ceftriaxone (3.9 to 82 M −1 s −1 ). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt Cd1 and Ldt Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt Cd1 and Ldt Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.

scholarly journals High-density chemical cross-linking for modeling protein interactions

2019 ◽  
Vol 117 (1) ◽  
pp. 93-102 ◽  
Author(s):  
Julian Mintseris ◽  
Steven P. Gygi
Keyword(s):  
Mass Spectrometry ◽  
Method Development ◽  
Conformational Dynamics ◽  
Complex Function ◽  
Computational Cost ◽  
Dimensional Structure ◽  
Cross Linking ◽  
Proteasome Subunits ◽  
Cross Links ◽  
Chemical Cross Linking

Detailed mechanistic understanding of protein complex function is greatly enhanced by insights from its 3-dimensional structure. Traditional methods of protein structure elucidation remain expensive and labor-intensive and require highly purified starting material. Chemical cross-linking coupled with mass spectrometry offers an alternative that has seen increased use, especially in combination with other experimental approaches like cryo-electron microscopy. Here we report advances in method development, combining several orthogonal cross-linking chemistries as well as improvements in search algorithms, statistical analysis, and computational cost to achieve coverage of 1 unique cross-linked position pair for every 7 amino acids at a 1% false discovery rate. This is accomplished without any peptide-level fractionation or enrichment. We apply our methods to model the complex between a carbonic anhydrase (CA) and its protein inhibitor, showing that the cross-links are self-consistent and define the interaction interface at high resolution. The resulting model suggests a scaffold for development of a class of protein-based inhibitors of the CA family of enzymes. We next cross-link the yeast proteasome, identifying 3,893 unique cross-linked peptides in 3 mass spectrometry runs. The dataset includes 1,704 unique cross-linked position pairs for the proteasome subunits, more than half of them intersubunit. Using multiple recently solved cryo-EM structures, we show that observed cross-links reflect the conformational dynamics and disorder of some proteasome subunits. We further demonstrate that this level of cross-linking density is sufficient to model the architecture of the 19-subunit regulatory particle de novo.

scholarly journals Using XlinkCyNET to visualize protein cross-links in Cytoscape software

2020 ◽  
Author(s):  
Diogo Borges Lima ◽  
Ying Zhu ◽  
Fan Liu
Keyword(s):  
Mass Spectrometry ◽  
Complex Networks ◽  
Open Source ◽  
Protein Interaction ◽  
High Performance ◽  
Protein Interaction Networks ◽  
Interaction Networks ◽  
Cross Linking ◽  
Interactive Interface ◽  
Cross Links

Abstract We present a high-performance app for Cytoscape to visualize cross-linking mass-spectrometry (XL-MS) data. XlinkCyNET is an open-source Java plugin that generates residue-to-residue connections provided by XL-MS in protein interaction networks. Importantly, it provides an interactive interface for the exploration of cross-links and offers various options to display protein domains. XlinkCyNET works well in complex networks containing thousands of proteins.

scholarly journals Targeted in situ cross-linking mass spectrometry and integrative modeling reveal the architectures of Nsp1, Nsp2, and Nucleocapsid proteins from SARS-CoV-2

2021 ◽  
Author(s):  
Moriya Slavin ◽  
Joanna Zamel ◽  
Keren Zohar ◽  
Siona Eliyahu ◽  
Merav Braitbard ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Full Length ◽  
Integrative Model ◽  
Cross Linking ◽  
Cross Link ◽  
Integrative Modeling ◽  
Cellular Context ◽  
Cross Links ◽  
The Cross

AbstractAtomic structures of several proteins from the coronavirus family are still partial or unavailable. A possible reason for this gap is the instability of these proteins outside of the cellular context, thereby prompting the use of in-cell approaches. In situ cross-linking and mass spectrometry (in situ CLMS) can provide information on the structures of such proteins as they occur in the intact cell. Here, we applied targeted in situ CLMS to structurally probe Nsp1, Nsp2, and Nucleocapsid (N) proteins from SARS-CoV-2, and obtained cross-link sets with an average density of one cross-link per twenty residues. We then employed integrative modeling that computationally combined the cross-linking data with domain structures to determine full-length atomic models. For the Nsp2, the cross-links report on a complex topology with long-range interactions. Integrative modeling with structural prediction of individual domains by the AlphaFold2 system allowed us to generate a single consistent all-atom model of the full-length Nsp2. The model reveals three putative metal binding sites, and suggests a role for Nsp2 in zinc regulation within the replication-transcription complex. For the N protein, we identified multiple intra- and inter-domain cross-links. Our integrative model of the N dimer demonstrates that it can accommodate three single RNA strands simultaneously, both stereochemically and electrostatically. For the Nsp1, cross-links with the 40S ribosome were highly consistent with recent cryo-EM structures. These results highlight the importance of cellular context for the structural probing of recalcitrant proteins and demonstrate the effectiveness of targeted in situ CLMS and integrative modeling.

scholarly journals Data mining in mass spectrometry-based proteomics studies

2020 ◽  
Vol 2 (4) ◽  
pp. 258-276
Author(s):  
Vu Anh Le ◽  
Cam Quyen Thi Phan ◽  
Thuy Huong Nguyen
Keyword(s):  
Mass Spectrometry ◽  
Data Mining ◽  
Biomedical Research ◽  
Protein Level ◽  
Protein Identification ◽  
Biomarker Discovery ◽  
Data Sets ◽  
Biological Processes ◽  
Data Mining Techniques ◽  
Analytical Technique

The post-genomic era consists of experimental and computational efforts to meet the challenge of clarifying and understanding the function of genes and their products. Proteomic studies play a key role in this endeavour by complementing other functional genomics approaches, encompasses the large-scale analysis of complex mixtures, including the identification and quantification of proteins expressed under different conditions, the determination of their properties, modifications and functions. Understanding how biological processes are regulated at the protein level is crucial to understanding the molecular basis of diseases and often highlights the prevention, diagnosis and treatment of diseases. High-throughput technologies are widely used in proteomics to perform the analysis of thousands of proteins. Specifically, mass spectrometry (MS) is an analytical technique for characterizing biological samples and is increasingly used in protein studies because of its targeted, nontargeted, and high performance abilities. However, as large data sets are created, computational methods such as data mining techniques are required to analyze and interpret the relevant data. More specifically, the application of data mining techniques in large proteomic data sets can assist in many interpretations of data; it can reveal protein-protein interactions, improve protein identification, evaluate the experimental methods used and facilitate the diagnosis and biomarker discovery. With the rapid advances in mass spectrometry devices and experimental methodologies, MS-based proteomics has become a reliable and necessary tool for elucidating biological processes at the protein level. Over the past decade, we have witnessed a great expansion of our knowledge of human diseases with the adoption of proteomic technologies based on MS, which leads to many interesting discoveries. Here, we review recent advances of data mining in MS-based proteomics in biomedical research. Recent research in many fields shows that proteomics goes beyond the simple classification of proteins in biological systems and finally reaches its initial potential – as an essential tool to aid related disciplines, notably biomedical research. From here, there is great potential for data mining in MS-based proteomics to move beyond basic research, into clinical research and diagnostics.

Sign in / Sign up

Export Citation Format

Share Document