Project PXD005130

PRIDE Assigned Tags:
Biomedical Dataset
Download Project Files
Project Protein Table
Project Peptide Table
Visualize in PRIDE Inspector
Follow the next three steps to open your selected project or assay in PRIDE Inspector:

  • 1.

    Download, uncompress and open PRIDE Inspector
  • 2.

    Click in the magnifier on the left top corner, paste the project or assay that you would like to open in the search box, and hit search
  • 3.

    Click in the corresponding "Download" button to download the files and visualize them

Summary

Title

Extracellular Matrix Proteomics Identifies Molecular Signature of Atherosclerotic Plaques from Symptomatic Patients

Description

Recent findings have challenged the prevailing histology- or imaging-based definition of the “vulnerable plaque”. To investigate molecular characteristics associated with “clinical instability” of atherosclerosis, we performed a proteomics comparison of the vascular extracellular matrix and associated molecules in human carotid endarterectomy specimens from symptomatic versus asymptomatic patients. The proteomics data were integrated with gene expression profiling and an analysis of protein secretion by lipid-loaded human vascular smooth muscle cells. The molecular signature of plaques from symptomatic patients identified by proteomics and at least one of the other two approaches comprised matrix metalloproteinase-9, chitinase-3-like protein 1, S100A8/S100A9, cathepsin B, fibronectin and galectin-3-binding protein. These biomarker candidates were measured in 685 subjects of the Bruneck Study and found to significantly predict the progression to advanced atherosclerosis (as assessed by repeated carotid ultrasound) and the incidence of cardiovascular disease over a 10-year follow-up period, highlighting the strength of tissue-based proteomics for biomarker discovery.

Sample Processing Protocol

Atherosclerotic plaque samples were collected from 12 patients undergoing carotid endarterectomise. Six were obtained shortly after an acute cerebrovascular event (symptomatic stenosis with ischemic stroke in the ipsilateral carotid territory) and the remaining six samples were collected during an elective surgery (asymptomatic stenosis). Tissue were sequentially extracted using 0.5 M NaCl, 0.08% SDS, and 4M guanidine-HCl (Mol Cell Proteomics. 2010; 9: 2048-2062). The NaCl extracts were desalted, acetone precipitated and redissolved in deglycosylation buffer (150 mM NaCl, 50 mM sodium acetate pH 6.8, 10 mM EDTA and proteinase inhibitors). The guanidine extracts were ethanol precipitated, and redissolved in deglycosylation buffer. Deglycosylation was achieved using chondroitinase ABC, keratanase, and heparitinase II for 16 h at 37C. After deglycosylation the solutions were clarified with centrifugation (16.000 x g for 10 min) and protein concentration was estimated by UV absorbance at 280 nm. Thirty-five micrograms of protein per sample were denatured and reduced in sample buffer (100 mM Tris, pH 6.8, 40% glycerol, 0.2% SDS, 2% beta-mercaptoethanol and 0.02% bromphenol blue) and boiled at 96C for 10 min. Proteins were separated on 4-12% Bis-Tris gels (NuPage, Invitrogen) and stained using the PlusOne Silver staining Kit (GE Healthcare). All gel bands were excised in identical parallel positions across lanes and no empty gel pieces were left behind. Subsequently, all gel bands were subjected to in-gel tryptic digestion using an Investigator ProGest (Genomic Solutions) robotic digestion system. Tryptic peptides were separated on a nanoflow LC system (ThermoFisher Scientific UltiMate 3000, UK) and eluted with a 80 min gradient (10-25% B in 35 min, 25-40% B in 5 min, 90% B in 10 min, and 2% B in 30 min where A is 2% acetonitrile, 0.1% formic acid in HPLC H2O and B is 90% acetonitrile, 0.1% formic acid in HPLC H2O) with Replay (Advion). The column (ThermoFisher Scientific PepMap C18, 25 cm length, 75 µm ID, 3 µm particle size) was coupled to a nanospray source (Picoview). Spectra were collected from a high-mass accuracy analyzer (LTQ Orbitrap XL, ThermoFisher Scientific) using full ion scan mode over m/z 450-1600. MS/MS was performed on the top 6 ions in each MS scan using the data-dependent acquisition mode with dynamic exclusion enabled. Human vascular SMCs were obtained from explants of human aortic tissue. SMCs were cultured in M199 medium (Sigma) supplemented with 20% FBS (Sigma), 2 mM L-glutamine (Gibco), 100 U/ml penicillin and 100 mg/ml streptomycin, and used between passages 4 and 14. Lipid loaded SMC were obtained as previously described. The cell layer was lysed using RIPA buffer (Cell signalling). The serum-free conditioned media were concentrated using Amicon 3kD spin column (Millipore). Thirty micrograms of cell layer proteins or 10 µg of secreted proteins per sample were denatured and reduced in sample buffer and boiled at 96C for 10 min. Proteins were separated on 4-12% Bis-Tris gels (NuPage, Invitrogen) and stained using the PlusOne Silver staining Kit (GE Healthcare). All gel bands were excised in identical parallel positions across lanes and no empty gel pieces were left behind. Subsequently, all gel bands were subjected to in-gel tryptic digestion using an Investigator ProGest (Genomic Solutions) robotic digestion system. Samples were loaded and separated on 4-12% Bis-Tris polyacrylamide gradient gels (NuPage, Invitrogen). After electrophoresis, gels were stained using the PlusOne Silver staining Kit (GE Healthcare). All gel bands were excised in identical parallel positions across lanes and no empty gel pieces were left behind. Subsequently, all gel bands were subjected to in-gel tryptic digestion using an Investigator ProGest (Genomic Solutions) robotic digestion system. Tryptic peptides were separated on a nanoflow LC system (RSLCnano, ThermoFisher Scientific) and eluted with a 70 min gradient (10-25% B in 35 min, 25-40% B in 5 min, 99% B in 10 min, and 2% B in 20 min where A is 0.1% formic acid in HPLC H2O and B is 80% acetonitrile, 0.1% formic acid in HPLC H2O). The column (PepMap C18, 25 cm length, 75 μm ID, 3 μm particle size) was coupled to a nanospray source (Picoview). Spectra were collected from a high-mass accuracy analyzer (LTQ Orbitrap XL, Thermo Fisher Scientific) using full ion scan mode over m/z 450-1600. MS/MS was performed on the top 6 ions in each MS scan using the data-dependent acquisition mode with dynamic exclusion enabled.

Data Processing Protocol

MS/MS peaklists were generated by extract_msn.exe and matched to human database (UniProtKB version 2013_8, 88378 protein entries) using Mascot (version 2.3.01, Matrix Science). Carboxyamidomethylation of cysteine was chosen as fixed modification and oxidation of methionine, lysine and proline were chosen as variable modifications. The mass tolerance was set at 10ppm for the precursor ions and at 0.8Da for fragment ions. Two missed cleavages were allowed. Scaffold (version 4.3.2, Proteome Software Inc., Portland, OR) was used to calculate the normalized spectral counts, and to validate peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability with at least two independent peptides. ECM proteins identified for the first time by proteomics in the vasculature as well as ECM proteins with low spectral counts were further examined to ensure the quality of the identified spectra.

Contact

Xiaoke Yin, Cardiovascular Division, King's College London
Manuel Mayr, King's British Heart Foundation Centre, King's College London, London, United Kingdom ( lab head )

Submission Date

11/10/2016

Publication Date

20/04/2017

Instrument

LTQ Orbitrap

Software

Not available

Experiment Type

Gel-based experiment

Assay count

936

Publication

    Langley SR, Willeit K, Didangelos A, Matic LP, Skroblin P, Barallobre-Barreiro J, Lengquist M, Rungger G, Kapustin A, Kedenko L, Molenaar C, Lu R, Barwari T, Suna G, Yin X, Iglseder B, Paulweber B, Willeit P, Shalhoub J, Pasterkamp G, Davies AH, Monaco C, Hedin U, Shanahan CM, Willeit J, Kiechl S, Mayr M. Extracellular matrix proteomics identifies molecular signature of symptomatic carotid plaques. J Clin Invest. 2017 Apr 3;127(4):1546-1560 PubMed: 28319050

Assay

Page 1 2 3 4 5 ... 94
Page size 10 20 50 100
Showing 1 - 10 of 936 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 69721 AS1_01.DAT-pride.xml 124 372 0 1657 0
2 69720 AS1_01_100220033851.DAT-pride.xml 108 298 0 1587 0
3 69725 AS1_02.DAT-pride.xml 54 279 0 1813 0
4 69724 AS1_02_100220045201.DAT-pride.xml 50 286 0 1770 0
5 69723 AS1_03.DAT-pride.xml 118 466 0 1786 0
6 69722 AS1_03_100220060516.DAT-pride.xml 118 461 0 1827 0
7 69729 AS1_04.DAT-pride.xml 169 588 0 1972 0
8 69728 AS1_04_100220071826.DAT-pride.xml 156 593 0 2009 0
9 69727 AS1_05.DAT-pride.xml 150 579 0 2153 0
10 69726 AS1_05_100220083139.DAT-pride.xml 140 559 0 2210 0