(A) to (D) is the MS/MS spectrum of MPB/BIAM-labeled two synthetic peptides ion precursors (charge 2). The ratio of mass to charge (m/z) is 527.7726 (A),474.2459 (B), 956.8995 (C), and 858.3426 (D), respectively, corresponding to oligopeptide LTTAIC from calcium-transporting ATPase (AT4G00900) and oligopeptideSYLCGDEFNSVR from calmodulin binding IQ-domain 33 protein (AT5G35670). The site-specific labeling occurs at the C-terminus of LTTAIC and the middle ofSYLCGDEFNSVR, respectively.
(E) and (F) illustrates the chemical reaction of MPB and BIAM with the thiol moiety of cysteine residue, respectively.
(G) is the antibiotin immune-blotting analysis of the biotin-tagged alkylation of the total cellular protein, which was isolated from 14N - (L) and 15N-coded
(H) wild typeplant, respectively.(H) illustrates an MS1 spectrum example of biotin-tagged alkylating reagents labeled peptide from mixed 14N - and 15N-labeled plant samples. The differentially stableisotope-labeled total proteins were mixed at a ratio of 1:1. Forward and Reciprocal refers to the order of mixing 14N-coded control (C) protein with 15N- coded treated (T) protein and vice versa, respectively. Superscript BIAM marks the cysteine residues labeled by BIAM. M/z represents the ratio of the ion mass to the charge ofeach PTM peptide.
