Mass Spectrometry Identification of Proteins

Selected spots (1-mm) were excised from the gels and submitted to trypsin proteolysis as described [17] (link). In brief, gel spots were incubated at 37°C for 30 min in 50 mM NH₄HCO₃, dehydrated twice for 5 min each in 100-µl acetonitrile and dried, and then, in-gel proteins were digested at 37°C for 6 h with 10 µl of trypsin solution (1% trypsin in 25 mM ammonium bicarbonate). After digestion, 1 µl of peptide mixture was directly spotted onto a MALDI-TOF-MS/MS target plate with 1 µl of alpha-cyano-4-hydroxycinnamic acid matrix solution (5 mg/ml in 50% acetonitrile). Peptides were analyzed by using a MALDI-TOF/TOF ABI 4800 Proteomics Analyzer (Applied Biosystems). The Applied Biosystems software package included the 4000 Series Explorer (v. 3.6 RC1) with Oracle Database Schema Version (v. 3.19.0) and Data Version (3.80.0) to acquire and analyze MS and MS/MS spectral data. The instrument was operated in a positive ion reflectron mode with the focus mass set at 1700 Da (mass range: 850–3000 Da). For MS data, 1000–2000 laser shots were acquired and averaged from each protein spot. Automatic external calibration was performed by using a peptide mixture with the reference masses 904.468, 1296.685, 1570.677, and 2465.199. MALDI MS/MS was performed on several (5–10) abundant ions from each protein spot. A 1-kV positive ion MS/MS method was used to acquire data under post-source decay (PSD) conditions. The instrument precursor selection window was +/− 3 Da. Automatic external calibration was performed by using reference fragment masses 175.120, 480.257, 684.347, 1056.475, and 1441.635 (from precursor mass 1570.700).
Applied Biosystems GPS Explorer™ (v. 3.6) software was used in conjunction with MASCOT to search the respective protein database by using both MS and MS/MS spectral data for protein identification. Protein match probabilities were determined by using expectation values and/or MASCOT protein scores. The MS peak filtering included the following parameters: a mass range of 800 Da to 3000 Da, minimum S/N filter = 10, mass exclusion list tolerance = 0.5 Da and mass exclusion list for trypsin and keratin-containing compounds included masses 842.51, 870.45, 1045.56, 1179.60, 1277.71, 1475.79, and 2211.1. The MS/MS peak filtering included the following parameters: minimum S/N filter = 10, maximum missed cleavages = 1, fixed modification of carbamidomethyl (C), variable modifications due to oxidation (M), precursor tolerance = 0.2 Da, MS/MS fragment tolerance = 0.3 Da, mass = monoisotopic, and peptide charges = +1. The significance of a protein match, based on the peptide mass fingerprint (PMF) in the MS and the MS/MS data from several precursor ions, is presented as expectation values (p<0.001).