Project PXD005726

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ECM characterization in stented porcine coronary arteries LC-MS/MS


Bare-metal (BMS) and drug-eluting stents (DES) were implanted in pig coronary arteries with an overstretch during coronary angioplasty under optical coherence tomography guidance. Arteries subjected to plain old balloon angioplasty (POBA) alone served as controls. Stented/balloon dilated segments were harvested 1, 3, 7, 14 and 28 days post-intervention for proteomics analysis. At day 28 all stented arteries showed a neointima formation covering the stent struts. The evolved neointima was separated from the media and analysed in a separate proteomics analysis. In total, 31 samples were analysed for the media by LC-MS/MS (n=3 BMS/DES at each time-point 1, 3, 7 and 28 days; n=4 POBA early [day1-day3] and n=3 POBA late [day 14 - day28]). For the neointima a total of 14 samples were analysed (n=7 BMS, n=7 DES at 28 days) including the neointima of arteries of a second cohort with 4 samples each for BMS and DES day 28. The neointima samples were run in duplicates.

Sample Processing Protocol

Protein enrichment: ECM proteins for the media samples were enriched using a three-step extraction method, as previously developed in our laboratory (Didangelos A et al, Mol Cell Proteomics. 2010;9:2048-62). In essence, the diced samples were initially washed 3 x with cold PBS supplemented with protease inhibitors and 25 mM EDTA to remove plasma contaminants. Then proteins were stepwise extracted in 0.5 M NaCl buffer to remove the loosely bound, newly synthesized proteins. Next, the tissue was incubated in 0.08% SDS buffer for decellularization to remove the intracellular proteins. Eventually, the proteins were extracted in 4M GuHCl buffer, pH 5.8, including 50 mM sodium acetate, protease inhibitors and 25 mM EDTA in a 5:1 ratio of buffer volume to tissue weight for 48h. This GuHCl extract was used for LC-MS/MS analysis. The more fragile neointima samples were extracted in a single GuHCl buffer step. Protein concentration was determined by Nano Protein deglycosylation: Next, 15 µg of protein of each GuHCl sample was mixed with 100% ethanol and stored at -20 °C overnight to remove guanidine. Precipitated proteins were pelleted by centrifugation and upon removal of ethanol the pellets were dried using a concentrator. Then proteins were resuspended in deglycosylation buffer (25 mM EDTA, 50 mM sodium acetate, 50 mM Tris, pH 6.8) and deglycosylated using the following deglycosylation enzymes (chondroitinase ABC (1:100), keratinase (1:500), heparinase II (1:500), α2-3,6,8,9-neuraminidase (1:200), ß-N-acetylglucosaminidase (1:200) and O-glycosidase (1:200)). After an initial incubation at 37 °C for 24 h, the samples were speed-vac dried and PNGase F (1:200) was added together with 18O water to identify N-linked glycosylation sites by isotopic mass-differences in the MS analysis. Samples were incubated for further 48 h. In-solution digest: 15 µg of deglycosylated proteins were denatured with 6M urea and 2M thiourea in each sample, reduced with DTT (final conc. 10 mM) for 1h and alkylated with IAA (final conc. 50 mM) for 1h at room temperature in the dark. Proteins were precipitated with ice-cold acetone (6x reaction volume) overnight at -20 °C. Protein pellets were speed-vac dried, resuspendend in 30 µl of 0.1 M TEAB buffer, pH 8.2, containing trypsin at a trypsin:protein ratio of 1:50. After overnight digestion at 37 °C in agitation, 10% TFA was added to stop the reaction. Peptide clean-up using C18: Resulting tryptic peptides were purified using a 96-well C18 spin plate (Harvard Apparatus) according to standard protocols. In brief: resin was activated using methanol, equilibrated with cleaning solution (80% acetonitrile and 0.1% TFA), washed 3x with washing solution (1% acetonitrile and 0.1% TFA), always using 200 µl of volume for each well. After sample loading and further 3 wash steps, samples were eluted with 50% acetonitrile and 0.1% TFA twice. Eluted samples were frozen at -80 °C, lyophilized in a freeze dryer at -50 °C and resuspended in 30 µl of 2% acetonitrile, 0.05% TFA in HPLC-grade H2O, resulting in a final peptide-concentration of 0.5 µg/µl. LC-MS/MS analysis: Tryptic peptides were separated using a nanoflow HPLC system (UltiMate 3000 RSLCnano, Thermo Scientific) with subsequent MS/MS analysis on an orbitrap MS (Q Exactive Plus mass spectrometer, Thermo Scientific). Peptides were separated on a reversed-phase HPLC column (Acclaim, PepMap 100 C18 peptide column, 3 µm, 100 Å, 50 cm x 75 µm, Thermo Scientific) at a flow rate of 300 nl/min and eluted with a 240 min gradient using a mobile phase solvent composition with increasing acetonitrile content. The following gradient was set: 2-10% B from 0-10 min; 10-30% B from 10-200 min; 30-40% B from 200-210 min; 99% B from 210-220 min and eventually 2% B from 220-240 min; the mobile phase solvent compositions were as follows: A = 0.1% formic acid (FA) in HPLC grade H2O; B = 80% acetonitrile, 0.1% FA in HPLC grade H2O). The sequentially eluted peptides were directly analysed by an orbitrap mass analyser using a full ion scan mode over the m/z range of 350 – 1600 and a resolution of 70,000 at 200 m/z. Data-dependent MS/MS analysis was performed using higher-energy collisional dissociation (HCD) on the 15 most abundant ions in each full MS scan with dynamic exclusion enabled for 30 sec.

Data Processing Protocol

Spectral raw files were searched using Proteome DiscovererTM software (version 1.4, Thermo Scientific) against a custom-made hybrid database using Mascot (version 2.3.01, Matrix Science). The custom-made database contained a comprehensive porcine ECM protein list with a human proteome background (UniProtKB/Swiss-Prot, release 2014_06, 20220 protein entries). A custom-made porcine ECM database was generated to achieve best peptide sequence coverage and improve protein identification and quantification. For this purpose, all previously reported cardiovascular ECM proteins were searched for their porcine sequences in the UniProt protein database. The non-annotated uncharacterized porcine proteins were retrieved after blasting them against the human sequence. ECM proteins with no matching sequences in UniProt were deduced from public nucleotide databases. The final database contained a total of 270 manually included porcine ECM protein sequences. The mass tolerance was set at 10ppm for the precursor ions and at 20 mmu (0.02 Da) for fragment ions. Carbamidomethylation of cysteines due to alkylation with IAA was chosen as a fixed modification. Oxidation of methionine, lysine and proline as well as deamidation of asparagine with 18O water (+2.99 Da) were defined as variable modifications. Only tryptic peptides were included in the analysis. Two missed cleavages were allowed. Scaffold (version 4.3.2, Proteome Software Inc., US) was used to validate MS/MS-based peptide and protein identifications and quantification. Peptide identifications were accepted if they were established with greater than 95% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they were established at greater than 99% probability with at least 2 unique peptides. The normalised total ion current (TIC) of MS/MS spectra of the peptides from the same protein was used for quantification.


Xiaoke Yin, Cardiovascular Division, King's College London
Manuel Mayr, King's College London ( lab head )

Submission Date


Publication Date



Q Exactive


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Experiment Type

Shotgun proteomics

Assay count



    Suna G, Wojakowski W, Lynch M, Barallobre-Barreiro J, Yin X, Mayr U, Baig F, Lu R, Fava M, Hayward R, Molenaar C, White SJ, Roleder T, Milewski K, Gasior P, Buszman PP, Buszman PE, Jahangiri M, Shanahan C, Hill JM, Mayr M. Extracellular Matrix Proteomics Reveals Interplay of Aggrecan and Aggrecanases in Vascular Remodeling of Stented Coronary Arteries. Circulation. 2017 PubMed: 29030347


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# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 73429 File Name_ 10_des_day3.raw (F028676).mzid 5197 18853 8586 15494 0
2 73428 File Name_ 11_poba_day3.raw (F028677).mzid 5429 20677 9187 16393 0
3 73427 File Name_ 12_bms_day3.raw (F028678).mzid 6668 21363 10669 17222 0
4 73426 File Name_ 13_bms_day3.raw (F028679).mzid 6531 20955 10533 17291 0
5 73425 File Name_ 14_poba_day3.raw (F028680).mzid 5597 21941 9801 17241 0
6 73469 File Name_ 15_des_day3.raw (F028681).mzid 3965 15572 6610 12548 0
7 73424 File Name_ 16_bms_day3.raw (F028682).mzid 5453 17616 8547 13940 0
8 73468 File Name_ 17_des_day7.raw (F028683).mzid 5434 19125 9047 15639 0
9 73423 File Name_ 18_bms_day7.raw (F028684).mzid 5160 20405 8985 16438 0
10 73467 File Name_ 19_bms_day7.raw (F028685).mzid 5848 20121 9470 16462 0