Because much interest is currently focused on post-translational modifications, especially phosphorylation and dephosphorylation, the Committee chose to introduce phosphopeptide synthesis to its member laboratories for evaluation. It had also been speculated that potential phosphate shifts could occur between tyrosine residues during synthesis and cleavage. A peptide was designed to investigate these two aims and was requested from the member facilities: E-D-Y-E-pY-T-A-R-F-amide.
In preparation for analyzing the peptides submitted by study participants, the Committee synthesized, purified, and characterized the following test peptides: the non-phosphorylated peptide, the peptide phosphorylated on the tyrosine in the third postion, the test peptide phosphorylated on the tyrosine in the fifth position, and the doubly phosphorylated peptide. The four peptides were characterized by AAA, HPLC, CE, and electrospray and MALDI-TOF mass spectrometry.
The test peptides were readily separated by CE and HPLC and were easily quantified. The peptides gave the calculated masses by electrospray and MALDI-TOF. The peptide phosphorylated on the tyrosine in the third position could easily be distinquished from the peptide phosphorylated on the tyrosine in the fifth position by sequence analysis as determined by both MALDI-PSD and by Edman degradation sequence analysis.
The Committee has received 33 samples from member facilities to date, and all but five contain the correct peptide. Of the samples analyzed so far, 20 have greater than 75% of the correct product, 4 have less than 50% of the correct product. There is no evidence of a phosphate shift occurring in these peptides. No correlation appears at this time between coupling time and yield, but a correlation may exist between the use of non-protected phosphotyrosine and yield. The average percent correct product was greater in samples synthesized with a non-protected phosphotyrosine. Of the five samples lacking the correct product, one was synthesized with threonine residues in place of the tyrosines, and one was synthesized in the reverse direction and appeared to be cleaved at the position where the phosphotyrosine was expected. Overall, the quality of the peptides was quite good, 61% of the samples containing greater than 75% correct peptide and 73% of the samples containing greater than 50% correct peptide.
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