Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions
Figure 5
Comparison of high resolution extracted ion chromatograms by MS1 Filtering and SWATH MS2 for the phosphorylated ErbB2 peptides DVRPQPPpSPR and GLQpSLPTHDPSPLQR. (a) MS1 Filtering is applied to the MS1 scan of data-dependent high resolution LC-MS/MS analyses. MS1 Filtering can be used to extract the ion chromatogram of the monoisotopic precursor as well as the first and second naturally occurring isotopes, [M+1] and [M+2], respectively, as shown for the ErbB2 phosphopeptide DVRPQPPpSPR. Data-independent SWATH MS2 acquisitions complement MS1 Filtering by acquiring fragment ion intensities from MS2 scans which can also be used for quantitation. (b) Since the precursor is intact, MS1 Filtering cannot differentiate between multiple potential phosphoisoforms of the ErbB2 peptide GLQpSLPTHDPSPLQR from GLQSLPpTHDPSPLQR and GLQSLPTHDPpSPLQR based on mass. SWATH MS2 acquires the MS/MS fragment ions of the peptides detected and can be reconstructed after acquisition to confirm the site of modification. Fragment ions y7, b4-98, b5-98, b6, b4-982+, b52+, and b5-982+ are all specific to the phosphoisoform GLQpSLPTHDPSPLQR.
