Serum protein quantitation and identification by liquid chromatography–mass spectrometry (LC-MS)

LC-MS was used to analyze serum proteins. Serum supernatants (after 20-fold dilution) were mixed with acetone at a 2:1 (v/v) ratio and centrifuged at 10,000 g for 10 minutes. The resulting pellets were suspended in lysis buffer [0.25% (w/v) sodium dodecyl sulfate, 50 mM Tris-HCl, pH 9.0], and then the protein concentration was measured via Lowry et al.’s method (1951) using bovine serum albumin as the protein concentration standard. All pooled protein samples were prepared by mixing equal amounts of protein from individual serum protein samples.

To reduce disulfide bonds, protein samples (5 μg) were reduced using 5 mM dithiothreitol in 10 mM ammonium bicarbonate at 60°C for 1 hour. Sulfhydryl groups were alkylated by incubation in 15 mM iodoacetamide in 10 mM ammonium bicarbonate for 45 minutes in the dark at room temperature. Subsequently, the protein samples were mixed with sequencing-grade trypsin (Promega, Mannheim, Germany) at a ratio of 1:20 and incubated at 37°C overnight. The resulting tryptic peptides were dried and protonated using 0.1% formic acid and then injected into an Ultimate 3000 Nano/Capillary LC system (Dionex Ltd., UK) coupled to an HCTUltra mass spectrometer (Bruker Daltonics, Billerica, MA), and peptides were analyzed by electrospray ionization at a flow rate of 300 nl/minute using a 100-mm PepSwift monolithic nanocolumn with 50-mm internal diameter. Mobile phase solvent A consisted of 0.1% formic acid, and solvent B consisted of 80% acetonitrile and 0.1% formic acid. Peptides were eluted using a linear gradient of 4%–70% solvent B over the period 0–20 minutes (the time point of retention), followed by 90% solvent B from 20 to 25 minutes to remove all peptides retained on the column. A final elution was performed using 10% solvent B from 25 to 40 minutes to remove any remaining salts. Mass spectra of the peptide fragments were acquired in data-dependent AutoMS (2) mode over the scanning range 300–1,500 m/z. Three averages were taken, and up to five precursor ions were selected over the MS scan range 50−3,000 m/z.

DeCyder MS Differential Analysis software (DeCyderMS, GE Healthcare) was used to quantify the proteins in individual samples, and the Mascot search engine was used to correlate the MS/MS spectra to the Macaca protein database maintained by UniProt (Johansson et al., 2006; Thorsell et al., 2007). Mascot’s standard settings were used, which included a maximum of three missed cleavages, a peptide tolerance of 1.2 Da, an MS/MS tolerance of 0.6 Da, trypsin as the digesting enzyme, cysteine carbamidomethylation as the fixed modification, methionine oxidation as the variable modification, and peptide charge states. Protein levels in each sample were expressed as log2 values.