Warp lc 1

Automatic MS/MS experiments were performed using an ultrafleXtreme^TM MALDI-TOF/TOF MS (Bruker). Spot selection, automatic MS/MS mass filtering, and Mascot database searching (ver. 2.4.1, Matrix Science, Boston, MA, USA) were commanded by WARP-LC 1.3 (Bruker). Data processing and assessment of MS/MS results were performed using ProteinScape^TM 3.1 (Bruker). For measurement, MS spectra were automatically acquired on the ultrafleXtreme^TM instrument in the positive reflector mode under the control of Compass 1.3 and WARP-LC 1.3 software (Bruker), and automatic MS calibration processing was controlled by the AutoXecute function. The detection range was 1000–4000 m/z. Detected peptide compounds with a signal-to-noise ratio higher than 10 were automatically subjected to LIFT^TM-TOF/TOF experiments triggered with WARP-LC [21 (link)]. After each measurement, queries were automatically transferred to the ProteinScape 3.1 server. Database searches were also automatically achieved using an in-house Mascot server. Mascot search conditions were as follows; Database: SwissProt, Taxonomy: other Mammalia, Enzyme: none, Modifications: Acetyl (K), Delta: H (2) C (2), Methyl (K), Oxidation (M), Phospho (ST), tolerance: 50 ppm in MS, 0.8 Da in MS/MS.

Peptide samples were injected into a nano-LC system, fractionated, and spotted onto PAC (Pre-Anchored Chip) targets^{70 (link)}. Sample plates were subjected to MADLI TOF/TOF-based acquisition in the automated mode through WARP-LC 1.2 (Workflow Administration by Result driven Processing, Bruker Daltonics) software tool. The MALDI TOF/TOF instrument parameters mentioned in Kumar et al.^{70 (link)} were employed for automatic acquisition: carrier plates with samples were subjected to pre-teaching to ensure the optimum and complete acquisition of 448 sample spots. Mass list calculation was done through the WARP-LC interface. A manually updated background list containing trypsin autolysis peaks, matrix peaks, and keratin peaks was used during mass list generation. Spectral peaks (m/z) corresponding to background peaks were excluded for MS/MS measurement. Only those peaks (precursors) that have an S/N > 20 were included in the measurement. Post-acquisition processes including mass annotation, baseline subtraction, and smoothening were performed using Flex Analysis software, version 3.0, through WARP-LC. Protein identification was achieved using Biotools, version 3.2, through an in-house-licensed Mascot server (version 2.3, March 2010).

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.