Project PXD000987

PRIDE Assigned Tags:
Biomedical Dataset

Summary

Title

Membrane proteomis profile along human colon

Description

The colonic epithelium is a highly dynamic system important for regulation of ion and water homeostasis via absorption and secretion, and in maintaining a protective barrier between the outer milieu and the inside of our body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n=4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins, while a distinct decrease in proteins associated with bacterial sensing, cation-transport and O-glycosylation in the proximal to distal direction. Proteins involved in microbial defense and anion-transport showed on the other hand an opposing gradient and increased towards the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa.

Sample Processing Protocol

The colonic epithelium is a highly dynamic system important for regulation of ion and water homeostasis via absorption and secretion, and in maintaining a protective barrier between the outer milieu and the inside of our body. These processes are known to gradually change along the length of the colon, although a complete characterization at the protein level is lacking. We therefore analyzed the membrane proteome of isolated human (n=4) colonic epithelial cells from biopsies obtained via routine colonoscopy for four segments along the large intestine: ascending, transverse, descending and sigmoid colon. Label-free quantitative proteomic analyses using high-resolution mass spectrometry were performed on enriched membrane proteins. The results showed a stable level for the majority of membrane proteins, while a distinct decrease in proteins associated with bacterial sensing, cation-transport and O-glycosylation in the proximal to distal direction. Proteins involved in microbial defense and anion-transport showed on the other hand an opposing gradient and increased towards the distal end. The gradient of ion-transporter proteins could be directly related to previously observed ion transport activities. All individual glycosyltransferases required for the O-glycosylation of the major colonic mucin MUC2 were observed and correlated with the known glycosylation variation along the colon axis. This is the first comprehensive quantitative dataset of membrane protein abundance along the human colon and will add to the knowledge of the physiological function of the different regions of the colonic mucosa.

Data Processing Protocol

Raw spectral data were converted using MaxQuant version 1.3.0.5. Search using Andromeda and searched against the human SwissProt protein database (release 2013_3, 21,324 entries) combined with a database of common contaminates concatenated with the same sequence database in reversed order for false discovery rate (FDR) estimation. The following parameters were used for searches: (i) two missed cleavages Trypsin; (ii) precursor tolerance 20 ppm first search used for recalibration, 7 ppm main search and 0.5 Da for fragment ions; (iii) carbamidomethyl cysteine (fixed), oxidized methionine and acetylated protein N-terminal (variable); (iv) a maximum of four modifications per peptide allowed; (v) match between runs was set to 2 min. Relative protein quantification was performed based on the extracted ion chromatograms over the elution time window of each identified peptide. Peptide signals were combined for all identified charge states and variable modifications. The match between run feature was used to determine if peptides also occurred in same retention time window in adjacent fractions and the total sum was used for quantification. Identifications and quantifications were combined using the identify module in MaxQuant, applying a FDR for both peptide and protein identifications of 1% based on the reversed peptide identifications (cut-off score >50.83); protein identification was based on a minimum of one unique peptide and proteins and were grouped when based on the same set of peptides. Non-unique peptides were strictly used for the quantification of the protein with the most identified peptides.

Contact

S.S van der Post, Medical Chemistry
Gunnar C Hansson, Department of Medical Biochemistry, University of Gothenburg ( lab head )

Submission Date

19/05/2014

Publication Date

31/07/2014

Tissue

colon

Cell Type

epithelial cell

Disease

disease free

Instrument

LTQ Orbitrap

Software

Not available

Experiment Type

Shotgun proteomics

Publication

    van der Post S, Hansson GC. MEMBRANE PROTEIN PROFILING OF HUMAN COLON REVEALS DISTINCT REGIONAL DIFFERENCES. Mol Cell Proteomics. 2014 Jun 2. pii: mcp.M114.040204 PubMed: 24889196