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

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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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