PXD000271

PXD000271 is an original dataset announced via ProteomeXchange.

Title	Mouse gastrointestinal system mucus nLC-MS/MS
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.
HostingRepository	PRIDE
AnnounceDate	2013-07-23
AnnouncementXML	Submission_2013-07-23_09:16:13.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Ana M. Rodriguez-Pineiro
SpeciesList	scientific name: Mus musculus (Mouse); NCBI TaxID: 10090;
ModificationList	iodoacetamide derivatized residue; monohydroxylated residue; acetylated residue
Instrument	LTQ Orbitrap

Revision	Datetime	Status	ChangeLog Entry
0	2013-05-30 03:38:24	ID requested
1	2013-07-23 09:14:48	announced
1	2014-07-25 01:13:43	announced
⏵ 2	2013-07-23 09:16:13	announced	Add reference

Rodr, í, guez-Pi, ñ, eiro AM, Bergstr, ö, m JH, Ermund A, Gustafsson JK, Sch, ü, tte A, Johansson ME, Hansson GC, Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins. Am J Physiol Gastrointest Liver Physiol, 305(5):G348-56(2013) [pubmed]

submitter keyword: mouse, stomach, intestine, mucus

Ana M. Rodriguez-Pineiro
contact affiliation	Biomedicinska inst
contact email	ana.rodriguez@medkem.gu.se

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