MALDI-TOF/TOF Mass Spectrometry Protocol

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).