Automated Assignment of MS/MS Cleavable Cross-Links in Protein 3D-Structure Analysis

Götze, Michael; Pettelkau, Jens; Fritzsche, Romy; Ihling, Christian; Schäfer, Matthias; Sinz, Andrea

doi:10.1007/s13361-014-1001-1

Cited by 227 publications

(228 citation statements)

References 52 publications

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“…Peptides were separated on nanoRP-HPLC coupled to an ESI source on a Q-Exactive mass spectrometer (Thermo) with a gradient of 5-60% B (solvent A, 0.2% formic acid; solvent B, 0.2% formic acid, 80% (v/v) acetonitrile) (22). Cross-linked products were identified using the StavroX software tool (version 3.5.1) (23). MS data files were searched for cross-linked peptides using the following settings: mass tolerance of the precursor ion of 2 ppm; tolerance for fragment ions 20 ppm; cleavage C terminus of Trp; Tyr, Phe, and Leu for chymotrypsin as cleaving enzyme; cleavage N terminus of Asp and cleavage C terminus of Trp; Tyr, Phe, and Leu for combined chymotrypsin/AspN proteolysis (for the analysis of CysD oligomers) up to two missed cleavages were allowed; carbamidomethylation of Cys as fixed modification; oxidized methionine as variable modification; ammonia loss for cross-linked products.…”

Section: In-gel/in-solution Digestion and Mass Spectrometric Analysesmentioning

confidence: 99%

The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide Bonds

Recktenwald

Hansson

2016

Journal of Biological Chemistry

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The main structural component of the mucus in the gastrointestinal tract is the MUC2 mucin. It forms large networks that in colon build the loose outer mucous layer that provides the habitat for the commensal flora and the inner mucous layer that protects the epithelial cells by being impenetrable to bacteria. The epithelial cells in mice lacking MUC2 are not adequately protected from bacteria, resulting in inflammation and the development of colon cancer as found in human ulcerative colitis. Correct processing of the MUC2 mucin is the basis for the building of these protective networks. During the biosynthesis of the MUC2 mucin, post-translational modifications are formed resulting in reduction-insensitive bonds between MUC2 monomers. By the use of ␥-glutamyltranspeptidase and isopeptidase activity in leech saliva, we could show that the molecular nature of these reduction-insensitive bonds is isopeptide bonds formed between side chains of lysine and glutamine. Transglutaminase 2 has an affinity to the MUC2 CysD2 domain in the nanomolar range and can catalyze its cross-linking. By using mass spectrometry, we identified MUC2 residues involved in this cross-linking. This shows for the first time that transamidation is not only stabilizing the skin and the fibrin clot, but is also important for the correct intracellular processing of MUC2 to generate protective mucus.Protection of epithelial cells from the environment in the intestinal tract is maintained by the mucus, a gelatinous protein network. The MUC2 mucin is the major constituent of the twolayered mucous structure in the colon with a stratified inner layer that is attached to the epithelium and is impenetrable to bacteria, whereas the outer layer provides the habitat for the commensal flora (1, 2). The MUC2 monomer is made up of more than 5,000 amino acids with three complete domains and one partial von Willebrand domain (vWD) 2 at the N terminus followed by the first CysD domain, two regions with a high amount of proline, threonine, and serine the so-called PTS domains that are separated by the second CysD domain, and the C-terminal part harbors the fourth vWD, a vWC, and a cysteine knot domain (Fig. 1a) (3). The primarily translation product forms C-terminal dimers via disulfide bonds in the endoplasmic reticulum (4). During their transport in the Golgi apparatus, the MUC2 dimers become heavily O-glycosylated thereby shifting their mass to Ϸ5 MDa. In the trans-Golginetwork MUC2 is sorted in the regulatory pathway and experiences disulfide bond-based trimerization of the dimers in their vWD3 domain (5). In the later stages of the secretory pathway, around or after this trimerization, reduction-insensitive bonds are formed (6). At that time MUC2 becomes insoluble in regular buffers, including SDS-containing ones when the biosynthesis was studied in LS174T cells (6). The more mature and secreted MUC2 mucin from the intestine is also insoluble in chaotropic salts (like guanidinium chloride) what was first discovered by Carlstedt and co-workers (7,8). Ever ...

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Section: In-gel/in-solution Digestion and Mass Spectrometric Analysesmentioning

confidence: 99%

The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide Bonds

Recktenwald

Hansson

2016

Journal of Biological Chemistry

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“…One alternate approach uses MS/MS cleavable cross-linkers analyzed by so-called label-free CXMS analysis in which cross-linked peptides can be identified by interpretation of MS/MS spectra. During MS/MS fragmentation, a MS/MS cleavable group that was incorporated into the structure of the cross-linker, will be fragmented to generate a neutral loss or cross-link specific product ions [13,[53][54][55][56][57]. Unlike the isotope-labeling experiments, this label-free technique is beneficial in Fig.…”

Section: Detection Of Ms/ms Cleavable Groups (Label-free)mentioning

confidence: 99%

Advances in protein complex analysis by chemical cross-linking coupled with mass spectrometry (CXMS) and bioinformatics

Tran

Goodlett

Goo

2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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“…StavroX provides an easy-to-use graphical interface and shortens the processing time by only processing the MS/MS spectra of the precursor ions that match to the theoretical masses of cross-linked peptide candidates. MeroX [19], is designed for CID-MS/MS cleavable cross-links with a self-explanatory graphical user interface, similar to StavroX. BLinks [20] is designed for the PIR cross-linking applications.…”

Section: Cross-linking Identification Algorithmsmentioning

confidence: 99%

Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions

Xu¹

2015

J Proteomics Bioinform

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Automated Assignment of MS/MS Cleavable Cross-Links in Protein 3D-Structure Analysis

Cited by 227 publications

References 52 publications

The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide Bonds

The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide Bonds

Advances in protein complex analysis by chemical cross-linking coupled with mass spectrometry (CXMS) and bioinformatics

Chemical Cross-linking Mass Spectrometry for Profiling Protein Structures and Protein-Protein Interactions

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