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Fig. 1. Identification of biotin-BMCC-conjugated peptides, using precursor ion scanning. (a) Chemical structure of biotin-BMCC. (B) Detection of the conjugate between glutathione and biotin-BMCC (mass = 841.35), using a Q-TOF mass spectrometer. (C) Fragmentation of GSH-biotin-BMCC into daughter ions by MS/MS. (D) Identification of GSH-biotin-BMCC as the parent ion, using a precursor ion scan.

voltage) produced three distinct daughter ions (Fig. 1C). The largest is derived from fragmentation at the peptide bond between the glutamic acid and serine at the N terminus of GSH (m/z = 712.3). The loss of glutamic acid is noted as an "E" in Fig. 1C. Two additional fragments are created by fragmentation within the linker region between the biotin and the maleimide groups (m/z = 315 and m/z — 298). On occasion, we have also observed a fragment with an m/z ratio of 226 (biotin moiety of BMCC). The specific fragmentation and intensity of each daughter ion can be optimized by increasing the R02 voltage settings to -80, -95, and -105 V for the m/z 315,298, and 226 ions, respectively. The daughter ions with m/z values of 315 and 298 were then used as specific diagnostic indicators to identify peptides containing a cysteine residue modified by BMCC. This is accomplished by the use of precursor ion scanning (available on mass spectrometers equipped with triple quadrupole mass filters). BMCC-GSH conjugate is detected in the first quadrupole for parent ions, while holding the third quadrupole at a constant m/z value (315, daughter ion) after subjecting all ions to CAD in the second quadrupole. Using this scan mode, the conjugate between GSH- and biotin-BMCC with an m/z value of 841 is readily identified as the parent ion (Fig. ID). We have also performed experiments to detect the presence of the BMCC-GSH modification in complex peptide mixtures. Using these scanning techniques, at least 100 fmol of BMCC-GSH conjugate parent ion (m/z — 841) can be detected in the presence of a 10-pmol mixture of tryptic peptides derived from an in-gel digest, using a nanoelectrospray ionization source (data not shown). Precursor ion scanning for m/z = 315 is most specific for the presence of the BMCC-GSH conjugate in complex peptide mixtures. In principle, this approach could be used to identify the position of biotin-BMCC-tagged sulfhydryls in any protein, including integrin.

Acknowledgment

This work was supported by Grants HL-58925, CA-69306, and CA-30199 from the NIH.

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