Protein identification and quantitation

Labelled peptides were analyzed by LC-MS/MS using a C-18 reversed phase nano-column (75 µm I.D. × 50 cm, 2 µm particle size, Acclaim PepMap RSLC, 100 C18; Thermo Fisher Scientific, Waltham, MA, USA) in a continuous acetonitrile gradient consisting of 0–30% B in 360 min, 50–90% B in 3 min (A = 0.1% formic acid; B = 90% acetonitrile, 0.1% formic acid). A flow rate of 200 nL/min was used to elute peptides from the nano-column to an emitter nanospray needle for real time ionization and peptide fragmentation on a Q-Exactive mass spectrometer (Thermo Fisher). An enhanced FT-resolution spectrum (resolution = 70,000) followed by the MS/MS spectra from the 15 most intense parent ions were analyzed along the chromatographic run. Dynamic exclusion was set at 40 s. For peptide identification, all spectra were analyzed with Proteome Discoverer (version 1.4.0.29, Thermo Fisher Scientific) using SEQUEST-HT (Thermo Fisher Scientific). For database searching at the Uniprot database containing all sequences from human and crap contaminants (March 06, 2013; 70024 entries), the parameters were selected as follows: trypsin digestion with 2 maximum missed cleavage sites, precursor and fragment mass tolerances of 2 Da and 0.02 Da, respectively, carbamidomethyl cysteine, iTRAQ modifications at N-terminal and Lys residues as fixed modifications, and methionine oxidation as dynamic modification. Peptide identification was validated using the probability ratio method⁴⁸. False discovery rate (FDR) was calculated using inverted databases, and the refined method⁴⁹ with an additional filtering for precursor mass tolerance of 12 ppm⁵⁰. Only peptides with a confidence of at least 95% were used to quantify the relative abundance of each peptide determined as previously described. Protein quantification from reporter ion intensities and statistical analysis of quantitative data to identify significant proteins were performed using QuiXoT based on a statistical model previously described⁵¹. In this model protein log2-ratios are expressed in form of the standardized variables, i.e., in units of standard deviation according to their estimated variances (Zq values). Individual protein responses to albuminuria development were evaluated. An average of Zq value of the four biological replicates was calculated per condition. Ratio of change between two conditions was expressed as the difference in average Zq values between compared groups (dnA/N and MHA/N).Those proteins with ratio of change between groups ≥|2|, and a minimum of 3 peptides identified were selected for further analysis.