Phosphopeptides phosphatase treatment

Phosphatase treatment The phosphopeptides in the mixture can be identified by recording MS (usually MALDl-MS) spectra before and after the treatment with alkaline phosphatase or phosphoprotein phosphatase. A net mass differential of 80 Da is caused by the addition of a phosphate group to Ser, Thr or Tyr residue. A prior knowledge of the amino acid sequence of the peptide facilitates the identification of the phosphorylation site. 

Phosphatase Treatment The specificity of phosphatases for the removal of the phosphate group has also been exploited to identify phosphopeptides and proteins selectively [38]. The molecular mass of phosphopeptides decreases by 80 Da for each phospho unit after the phosphatase treatment. The reaction is monitored by MALDI-MS [39]. The peptide maps can be compared before and after phosphatase treatment to identify phosphopeptides in the digest [40]. Sensitivity is a major issue in such experiments. One way to improve sensitivity is to perform dephosphorylation on the MALDI target [40]. Another way is to use immobilized phosphatase packed in a smaU-diameter colnmn in-line with an LC/ESI-MS or CE/ESI-MS system [39,41],... 

Figure 4 Phosphopeptide identification by MALDI-TOF-MS mapping combined with alkaline phosphatase treatment. (A) The MALDI-TOF-MS spectrum of a proteolytic digest. Phospho-peptides are indicated by peaks shifted by multiples of 80 Da (HPO3 = 80Da) relative to predicted unphosphorylated peptide masses. (B) The disappearance of such peaks upon treatment with a phosphatase confirms their identity as phosphopeptides. (Reprinted with permission from McLachlin DT and Chart BT (2001) Analysis of posphorylated proteins and peptides by mass spectrometry (review). Current Opinion in Chemicai Bioiogy5 5) 591-602 Elsevier.)...

Similarly, phosphatases are used to detect and identify phosphopeptides in MALDl-MS. Phosphatase treatment leads to a mass decrease of 80 Da for phosphopeptides [7, 50-52]. 

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