Sequential abundant ion fragmentation analysis (SAIFA): an alternative approach for phosphopeptide identification using an ion trap mass spectrometer

Abstract

Phosphorylation has been the most studied of all the posttranslational modifications of proteins. Mass spectrometry has emerged as a powerful tool for phosphomapping on proteins/peptides. Collision-induced dissociation (CID) of phosphopeptides leads to the loss of phosphoric or metaphosphoric acid as a neutral molecule, giving an intense neutral loss product ion in the mass spectrum. Dissociation of the neutral loss product ion identifies peptide sequence. This method of data-dependent constant neutral loss (DDNL) scanning analysis has been commonly used for mapping phosphopeptides. However, preferential losses of groups other than phosphate are frequently observed during CID of phosphopeptides. Ions that result from such losses are not identified during DDNL analysis due to predetermined scanning for phosphate loss. In this study, we describe an alternative approach for improved identification of phosphopeptides by sequential abundant ion fragmentation analysis (SAIFA). In this approach, there is no predetermined neutral loss molecule, thereby undergoing sequential fragmentation of abundant peak, irrespective of the moiety lost during CID. In addition to improved phosphomapping, the method increases the sequence coverage of the proteins identified, thereby increasing the confidence of protein identification. To the best of our knowledge, this is the first report to use SAIFA for phosphopeptide identification.

Figure 1

Sequential Abundant Ion Fragmentation Analysis: The method is unbiased towards neutral loss molecule and fragments the most abundant ion in the spectrum. Excitation is continued up to four stages, unless the signal intensity reaches the defined threshold.

Figure 2

Identification of diphosphopeptide KELITCPTPGCDGSGHVTGDYASHR using SAIFA method. a) Total ion chromatogram of MS run, b) Full scan MS depicting m/z 993.6 that is fragmented in next scan, c) CID of m/z 993.6 giving an abundant ion at m/z 940.4 due to neutral loss of 106 mass units, d) CID of m/z 940.4 losing the first phosphate group to give an abundant ion at m/z 891.3, e) CID of m/z 891.3 losing the second phosphate group giving an abundant ion at m/z 842.3, and f) Final stage excitation of m/z 842.3 is giving the daughter ion spectrum that is used for the sequence identification.

Figure 3

Identification of alpha-casein peptide HIQKEDVPSER using SAIFA method. a) Full scan MS depicting m/z 690.7 that is fragmented in next scan, b) CID of m/z 690.7 giving an abundant ion at m/z 669.1 due to nuetral loss of 43 mass units, c) CID of m/z 669.1 is used for the sequence identification.

Figure 4

Plot representing the average number of peptides and average number of scans each protein is identified from using the SAIFA method and DDNL approach. The two methods are compared by the analysis of membrane proteins from HepG2 cells to validate the method for the analysis of complex protein mixtures.