Post-Translational Modification Analysis | Phosphorylation Analysis by MALDI-TOF MS (2)

Sequence Analysis of Phosphopeptides by Seamless PSD

Recently, mass spectrometry has been applied to the analysis of protein phosphorylation and phosphorylation sites. The phosphorylation sites are determined by a variety of types of MS/MS.

A method of analyzing the phosphorylation modification sites using seamless PSD (sPSD) with the MALDI method is described below.
Collision-induced dissociation (CID) is commonly used for amino acid sequence analysis by MALDI-MS/MS. However, due to the dissociation of the fragile phosphate groups caused by collisions with the inert gas, it is difficult to obtain information on phosphorylation sites using this method.
MS/MS measurements using seamless PSD (sPSD) require no inert gas and can obtain MS/MS spectra with some suppression of phosphate group dissociation.

Fig. 1 shows the seamless PSD (sPSD) spectrum of a doubly phosphorylated peptide obtained using DHB as the matrix. From the fragment ion assignment results in Fig. 1, it is apparent that y-ions, which are the C-terminal fragments, are formed preferentially.
While phosphate group dissociation from the fragment ions was observed, the intensity is low (y7-98 in diagram). The MS/MS spectrum obtained is adequate to decode the entire sequence from the masses of the phosphorylated amino acid residues.

Fig. 1 sPSD Spectrum of MAP Kinase Fragment 177-189 (a doubly-phosphorylated peptide)

This effect is known to be amplified when DHB is used, but conventional PSD was not practical due to insufficient fragment ion detection sensitivity. A major feature of sPSD is its superior fragment ion generation efficiency to PSD. This practical level of sensitivity permits MS/MS measurements of phosphopeptides.

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