Dataset Information

The putative 'link' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

ABSTRACT: The existence of a discrete 'link' peptide in epithelial mucins has been debated for many years. There is evidence that at least some mucins contain a specific 'link' peptide (or glycopeptide) that enhances mucin polymerization by forming disulphide bridges to large mucin glycoprotein subunits. A major difficulty has been to know whether the reported differences in putative 'link' components represent artifacts generated by inter-laboratory differences in technical procedures used in mucin purification. The present paper outlines the results of a collaborative study involving five laboratories and 53 samples of purified gastrointestinal mucins (including salivary, gastric, small-intestinal and colonic mucins) prepared by five techniques from four different animal species. An early step in mucin purification in all cases was the addition of proteinase inhibitors. Representative mucins were analysed for their composition, electrophoretic mobility in SDS/polyacrylamide-gel electrophoresis before and after disulphide-bond reduction, and for their reactivity with monospecific antibodies developed against the 118 kDa putative 'link' glycopeptide isolated from either rat or human small-intestinal mucins. Our results indicate that, despite differences in laboratory techniques, preparative procedures, organs and species, each of the purified mucins contained a 'link' component that was released by disulphide-bond reduction and produced a band on SDS/polyacrylamide-gel electrophoresis at a position of approx. 118 kDa. After electroelution and analyses, the 118 kDa bands from the different mucins were found to have similar amino acid profiles and to contain carbohydrate. It would appear therefore that a 'link' glycopeptide of molecular mass approx. 118 kDa is common to all of the gastrointestinal mucins studied.

SUBMITTER: Roberton AM

PROVIDER: S-EPMC1138871 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Comparison of mucus proteins from 6 different segments (from stomach to distal colon). 6 biological replicates, 2 MS replicates. The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the tract. We combined collection of mucus from explant tissues with FASP processing and single-shot mass spectrometry in an LTQ-Orbitrap system, to characterize the proteome of the murine mucus from stomach to distal colon. We identified ~1,300 proteins in the mucus, and found no differences in the protein composition or abundance between genders, but clear differences in the different gastrointestinal locations. Qualitatively, there was a relatively stable core proteome (~80% of the total proteins identified). Quantitatively, we found significant differences (~40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of proteins that correlated with the main intestinal mucin, Muc2 (e.g. Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system. Bioinformatics pipeline: Data was acquired in a hybrid LTQ-Orbitrap XL instrument (Thermo Scientific) in dependent mode, measuring full MS in the Orbitrap and selecting the 8 most abundant multiply charged ions for collision (CID, 30% normalized collision energy) and acquisition in the LTQ. Full MS scans were performed in the m/z 350-2,000 range, with internal calibration by lock mass (m/z 371.1012, siloxane), and resolution of 60,000. MS/MS scans were set at a target value of 100,000, with isolation width of 3 amu. The raw files obtained were analyzed in the MaxQuant 1.2.2.5 environment. Data were searched with the Andromeda search engine integrated in MaxQuant against an in-house database containing all the mucin sequences available (http://www.medkem.gu.se/mucinbiology/databases/), the UniProt-SwissProt mouse database (version 1203, reviewed sequences), and the standard MaxQuant contaminant database. Oxidation of methionines and acetylation of the protein N-terminus were set as variable modifications, and carbamidomethylation of cysteines as fixed; enzyme cleavage rules were defined for trypsin/P, with a maximum of 2 missed cleavages. Tolerances were limited as maximum 5 modifications per peptide, 20 ppm error for the first search and 6 ppm for the main search. Isoleucine and leucine were considered indistinguishable. The false discovery rate was calculated from searches against a reversed database, and set to 0.01 for proteins, peptides and modified sites. The identification rate was improved by matching between runs through remapped retention time (window of 2 min). The resulting data were loaded as protein groups into Perseus 1.3.0.4. Protein quantities were calculated as intensity values (from the extracted ion current: XIC) and normalized in ppm to the total XIC for the standard labeled peptides (AQUA) in each run.

Dataset Information

The putative 'link' glycopeptide associated with mucus glycoproteins. Composition and properties of preparations from the gastrointestinal tracts of several mammals.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets