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 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.

Cross-linking/MS analysis of TBEV and neuroblastoma cells v1

2021 ◽  
Author(s):  
Sarah Barrass ◽  
Lauri I. A. Pulkkinen ◽  
Olli Vapalahti ◽  
Suvi H. Kuivanen ◽  
Maria Anastasina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Internal Control ◽  
Limited Proteolysis ◽  
Neuroblastoma Cells ◽  
Host Protein ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cross Links ◽  
Using Data

Virus-host protein-protein interactions are central to viral infection, but are challenging to identify and characterise, especially in complex systems involving intact viruses and cells. Here, we describe a proteome-wide approach to identify virus-host interactions using chemical cross-linking coupled with mass spectrometry. We adsorbed tick-borne encephalitis virus onto metabolically-stalled neuroblastoma cells, covalently cross-linked interacting virus-host proteins, and performed limited proteolysis to release primarily the surface-exposed proteins for analysis by mass spectrometry. Proteins in the sample were identified using data-dependent acquisition mass spectrometry and cross-linked peptides were identified using the software pLink2. Cross-links are validated using the intraviral cross-links as an internal control.

scholarly journals Structure-based validation can drastically under-estimate error rate in proteome-wide cross-linking mass spectrometry studies

2019 ◽  
Author(s):  
Kumar Yugandhar ◽  
Ting-Yi Wang ◽  
Shayne D. Wierbowski ◽  
Elnur Elyar Shayhidin ◽  
Haiyuan Yu
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Error Rates ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
3D Structures ◽  
Cross Links ◽  
Assess Quality ◽  
Almost All

AbstractRecent, rapid advances in cross-linking mass spectrometry (XL-MS) has enabled detection of novel protein-protein interactions and their structural dynamics at the proteome scale. Given the importance and scale of the novel interactions identified in these proteome-wide XL-MS studies, thorough quality assessment is critical. Almost all current XL-MS studies validate cross-links against known 3D structures of representative protein complexes. However, current structure validation approach only includes cross-links where both peptides mapped to the 3D structures. Here we provide theoretical and experimental evidence demonstrating this approach can drastically underestimate error rates for proteome-wide XL-MS datasets. Addressing current shortcomings, we propose and demonstrate a comprehensive set of four metrics, including orthogonal experimental validation to thoroughly assess quality of proteome-wide XL-MS datasets.

scholarly journals MaXLinker: proteome-wide cross-link identifications with high specificity and sensitivity

2019 ◽  
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

ABSTRACTProtein-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 has the unique capability to detect protein-protein interactions at a large scale along with spatial constraints between interaction partners. However, the current cross-link search algorithms follow an “MS2-centric” approach and, as a result, suffer from a high rate of mis-identified cross-links (~15%). We address this urgent problem, by designing a novel “MS3-centric” approach for cross-link identification and implemented it as a search engine called MaXLinker. MaXLinker significantly outperforms the current state of the art search engine with up to 18-fold lower false positive rate. Additionally, MaXLinker results in up to 31% more cross-links, demonstrating its superior sensitivity and specificity. Moreover, we performed proteome-wide cross-linking mass spectrometry using K562 cells. Employing MaXLinker, we unveiled the most comprehensive set of 9,319 unique cross-links at 1% false discovery rate, comprising 8,051 intraprotein and 1,268 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.

A cross-linking/mass spectrometry workflow based on MS-cleavable cross-linkers and the MeroX software for studying protein structures and protein–protein interactions

2018 ◽  
Vol 13 (12) ◽  
pp. 2864-2889 ◽  
Author(s):  
Claudio Iacobucci ◽  
Michael Götze ◽  
Christian H. Ihling ◽  
Christine Piotrowski ◽  
Christian Arlt ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interactions ◽  
Protein Structures ◽  
Cross Linking ◽  
Protein Protein Interactions

scholarly journals Mass spectrometry-based cross-linking study shows that the Psb28 protein binds to cytochrome b559 in Photosystem II

2017 ◽  
Vol 114 (9) ◽  
pp. 2224-2229 ◽  
Author(s):  
Daniel A. Weisz ◽  
Haijun Liu ◽  
Hao Zhang ◽  
Sundarapandian Thangapandian ◽  
Emad Tajkhorshid ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Photosystem Ii ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Docking ◽  
Cross Linking ◽  
Protein Protein Interactions ◽  
Cytochrome B559 ◽  
Reaction Center Complex ◽  
Assembly Intermediate

Photosystem II (PSII), a large pigment protein complex, undergoes rapid turnover under natural conditions. During assembly of PSII, oxidative damage to vulnerable assembly intermediate complexes must be prevented. Psb28, the only cytoplasmic extrinsic protein in PSII, protects the RC47 assembly intermediate of PSII and assists its efficient conversion into functional PSII. Its role is particularly important under stress conditions when PSII damage occurs frequently. Psb28 is not found, however, in any PSII crystal structure, and its structural location has remained unknown. In this study, we used chemical cross-linking combined with mass spectrometry to capture the transient interaction of Psb28 with PSII. We detected three cross-links between Psb28 and the α- and β-subunits of cytochrome b559, an essential component of the PSII reaction-center complex. These distance restraints enable us to position Psb28 on the cytosolic surface of PSII directly above cytochrome b559, in close proximity to the QB site. Protein–protein docking results also support Psb28 binding in this region. Determination of the Psb28 binding site and other biochemical evidence allow us to propose a mechanism by which Psb28 exerts its protective effect on the RC47 intermediate. This study also shows that isotope-encoded cross-linking with the “mass tags” selection criteria allows confident identification of more cross-linked peptides in PSII than has been previously reported. This approach thus holds promise to identify other transient protein–protein interactions in membrane protein complexes.

scholarly journals Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Cross-Linking Mass Spectrometry

2021 ◽  
Author(s):  
Dmitri R. Davydov ◽  
Bikash Dangi ◽  
Guihua Yue ◽  
Bhagwat Prasad ◽  
Viktor G. Zgoda
Keyword(s):  
Mass Spectrometry ◽  
Cytochrome P450 ◽  
Protein Interactions ◽  
Human Liver ◽  
Liver Microsomes ◽  
Human Liver Microsomes ◽  
Cross Linking ◽  
Cytochrome P450 2E1 ◽  
Protein Protein Interactions ◽  
Chemical Cross Linking

This study aimed on exploration of the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) in the human liver on drug metabolism. Using membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide (BPM) and 4-(N-succinimidylcarboxy)benzophenone (BPS), we explored the array of its protein-protein interactions (proteome) in human liver microsomes (HLM) with chemical cross-linking mass spectrometry (CXMS). Exposure of bait-incorporated HLM samples to light was followed by isolation of the His-tagged bait protein and its cross-linked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the cross-linked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively cross-linked partners of CYP2E1 are cytochromes P450 2A6, 3A4, 2C9, and 4A11. We also detected the conjugates of CYP2E1 with UDP-glucuronosyltransferases (UGTs) 1A6, 1A9, 2B4, 2B15, and 2B17. These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes. Of particular interest is the observation of efficient cross-linking of CYP2E1 with CYP4A11. This enzyme plays a central role in the synthesis of vasoactive eicosanoids and its interactions with alcohol-inducible CYP2E1 may shed light on the mechanisms of alcohol-induced hypertension.

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