Peptide sequencing data acquisition

Both these methods were further developed, taking advantage of the advanced features in data acquisition in later generations of LC-MS instruments. Negative-ion MS-MS data for phosphopeptides do not always yield useful peptide sequence information [14]. The screening based on the detection of diagnostic phosphate ions in the negative-ion mode is best combined with PSA in positive-ion ESI-MS-MS. 

Tryptic digests of RIP140 protein was subjected to LC-ESI-MS/MS. Three independent full-scan ion chromatograms from m/z 400-550, 550-750, and 750-1200 were recorded in an information-dependent acquisition (IDA) mode to acquire MS/MS data. The IDA data were searched online at MASCOT (http //www.matrixscience.com) MS/MS data search at the NCBI data bank. The MS/MS data were analyzed manually to confirm the sequence of the modified and the unmodified forms of the same peptide identified by the data bank search. The full scan chromatograms were analyzed to assign the charged state, retention time, and intensities of the peptides. 

Fig. 4. Illustration of proteomic analysis of the rat synaptic plasma-membrane fraction by combination of SDS-PAGE and gradient reversed-phase LC/ESI-MS/MS (GeLC/MS/MS) on a quadrupole ion-trap instrument [21]. (A) The developed gel is cut into bands and the bands are (i) destained, digested (trypsin), and the sample is desalted for injection into the column. (B) Base-peak chromatogram obtained from the tryptic digest of the 98-120 kDa. Data-dependent acquisition is employed, where in one acquisition cycle (ii) a full-scan mass spectrum is acquired (C), followed by (iii) CID-MS/MS of the most intense ion (m/z 915.5) in this mass spectrum (D). (However, MS/MS is not initiated, when ion intensity in the full-scan mass spectrum is below a preset threshold.) Database search (iv) matches the MS/MS to the protein(s) (Table 1, major sequence ions of the peptide are indicated in chart D).

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