Identification of the Ptyr 155-kD protein in p210^bcr/abl-expressing cells by mass spectrophotometric analysis. The tryptic digest mixture was passed over an RP micro-tip and the peptides batch fractionated into a 16% and 30% pool. Each pool was individually analyzed by MALDI reflectron-TOF MS (16% fraction shown in [A]; 30% fraction in [B]) and by continuous flow ESI (JaFIS) triple quadrupole MS/MS (Q1 scan of 30% fraction shown in [C]); only the relevant portions of the spectra are shown. Both types of MS analysis served to independently identify this 155-kD protein as SHIP2 (EMBL Y14385). MALDI-reTOF mass spectra were obtained by averaging 150 scans under constant irradiance. The 17 most prominent peaks (from both pools combined) are labeled in (A) and (B); the corresponding m/z values were taken, from spectra analyzed in the presence of calibrants, to query a nonredundant protein sequence database (NRDB) for pattern matches, using the PeptideSearch program. With a requirement of 17 matches of 17, at a mass accuracy of 40 ppm or better, and a maximum of two missed cleavage sites per peptide, a single protein was retrieved (18% sequence coverage). The ESI-MS (Q1) spectrum of the 30% fraction obtained by a JaFIS-generated continuous flow of 4 nL/min, and averaging 100 scans; (C) contained several peaks corresponding to those observed by MALDI-reTOF mass analysis of the same pool (B). One peptide (1601.69²⁺ [C]) was then selected, by appropriate tuning of Q1, for collision-induced dissociation and subsequent analysis of fragment spectra (in Q3), as shown in (D). A short sequence was assigned, based on the presence of a contiguous y" ion series, enabling positive identification of SHIP2 by SequenceTag (peptide molecular weight [M_r], 1,602 ± 2; [536.0]PFS(IL)EE[1238.6]) based searching of the NRDB database.