Proteinscape 2

Dried peptides were concentrated and de-salted using Zip-Tips C18 (microbed) (Millipore, Billerica, MA) according to the manufacturer’s instructions.
After elution from the Zip-Tip 0.5 µL of the de-salted peptides were spotted with α-cyano-4-hydroxycinnamic acid onto a ground steel MALDI target plate (Bruker Daltonics, Bremen, Germany). MALDI-TOF/TOF mass spectrometry (Ultraflex II, Bruker Daltonics, Bremen, Germany) was used for spectra acquisition in MS and MS/MS modes. Spectra processing and peak annotation were carried out using FlexAnalysis 3.0 and Biotools 3.2 (both Bruker Daltonics, Bremen, Germany).
Processed spectra were searched via an in-house Mascot server version 2.4.1 (Matrix Science, Boston, MA) and the software ProteinScape 2.1 (Bruker Daltonics, Bremen, Germany) in the UniProt database of sus scrofa using the following search parameters: global modification carbamidomethylation on cysteine; variable modifications oxidation on methionine; deamidation on asparagine and glutamine as well as formation of pyroglutamic acid; enzyme specificity trypsin; charge state z = 1; MS tolerance 100 ppm; MS/MS tolerance 1 Da; two missed cleavages allowed; significance threshold p < 0.05.

Protein identification and quantification was performed using Protein Scape 2.1 and WARP-LC 1.2 (Bruker). Proteins were identified using Mascot (Matrix Science, London, UK) on the SwissProt human protein database. MS/MS spectra were searched with a 1.5 Da precursor mass tolerance, 0.5 Da fragment tolerance, 1 missed cleavage maximum trypsin specificity, cysteine carbamidomethylation set as fixed modification and methionine oxidation and the N-terminal and Lys and Arg SILAC labels as variable modifications. Positive identification criterion was set as an individual Mascot score for each peptide MS/MS spectrum above the homology threshold score. False positive rates for Mascot protein identification were measured by searching a randomized decoy database [67 (link)], and estimated to be under 4%. For relative protein quantification, H/L ratios were calculated averaging the measured H/L ratio for the observed peptides, after discarding outliers. For selected proteins of interest, quantitative data obtained from the automated Protein Scape analysis were manually curated. For further protein analysis, UniProtKB (http://www.uniprot.org) and GeneCards databases (http://www.genecards.org) were used.