Study on the synthesis and characterization of peptides containing phosphorylated tyrosine

Lynda Bonewald, L. Bibbs, S. A. Kates, A. Khatri, J. S. McMurray, K. F. Medzihradszky, S. T. Weintraub

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

Phosphorylation and dephosphorylation are key events in receptor- mediated and post-receptor-mediated signal transduction. Synthetic phosphopeptides have been shown to have dramatic agonist or antagonist effects in several of these signaling pathways. For its 1997 study, the Association of Biomolecular Resource Facilities (ABRF) Peptide Synthesis Research Group assessed the ability of member laboratories to synthesize phosphotyrosine peptides. Participating laboratories were requested to synthesize and submit the following crude peptide, H-Glu-Asp-Tyr-Glu- Tyr(PO3H2)-Thr-Ala-Arg-Phe-NH2, for evaluation by amino acid analysis, sequence analysis, RPHPLC, MALDI-TOF and ESI mass spectrometry. Prior to analysis of submitted peptides from ABRF members, the Peptide Synthesis Research Group synthesized and characterized the nonphosphorylated form of the peptide, the doubly phosphorylated form and the peptides singly phosphorylated on either the first or the second tyrosine. These peptide standards were separated easily by HPLC and capillary electrophoresis and the phosphotyrosine was detected readily by Edman degradation sequence analysis. No differences were seen by amino acid analysis and the expected masses were observed by mass spectrometry. The two singly phosphorylated peptides were easily distinguished by MALDI-PSD. Analysis of the peptides submitted from member facilities revealed that all but four of the 33 samples contained the correct product as determined by HPLC and mass spectrometry. HPLC analysis indicated that 20 of the 33 submitted samples contained greater than 75% correct product, five contained less than 50% correct product and four did not contain any correct product. By ESI/MS, an additional singly charged ion at m/z 535.5 was detected in five of the 33 submitted samples; this ion was subsequently shown to represent Ac-TARF-NH2. No correlation was found to exist between coupling time and percentage correct product; however, a correlation may exist between a greater percentage of correct product and the use of non-protected phosphotyrosine.

Original languageEnglish (US)
Pages (from-to)161-169
Number of pages9
JournalJournal of Peptide Research
Volume53
Issue number2
DOIs
StatePublished - 1999
Externally publishedYes

Fingerprint

Tyrosine
Peptides
Phosphotyrosine
Mass spectrometry
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
High Pressure Liquid Chromatography
Sequence Analysis
Mass Spectrometry
Ions
Amino Acids
Phosphopeptides
Capillary electrophoresis
Signal transduction
Phosphorylation
Electrospray Ionization Mass Spectrometry
Capillary Electrophoresis
Research
Signal Transduction
Degradation

Keywords

  • Electrospray
  • MALDI
  • MALDI- PSD
  • Phosphopeptide
  • Phosphorylation
  • Phosphotyrosine

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

Cite this

Bonewald, L., Bibbs, L., Kates, S. A., Khatri, A., McMurray, J. S., Medzihradszky, K. F., & Weintraub, S. T. (1999). Study on the synthesis and characterization of peptides containing phosphorylated tyrosine. Journal of Peptide Research, 53(2), 161-169. https://doi.org/10.1034/j.1399-3011.1999.00014.x

Study on the synthesis and characterization of peptides containing phosphorylated tyrosine. / Bonewald, Lynda; Bibbs, L.; Kates, S. A.; Khatri, A.; McMurray, J. S.; Medzihradszky, K. F.; Weintraub, S. T.

In: Journal of Peptide Research, Vol. 53, No. 2, 1999, p. 161-169.

Research output: Contribution to journalArticle

Bonewald, L, Bibbs, L, Kates, SA, Khatri, A, McMurray, JS, Medzihradszky, KF & Weintraub, ST 1999, 'Study on the synthesis and characterization of peptides containing phosphorylated tyrosine', Journal of Peptide Research, vol. 53, no. 2, pp. 161-169. https://doi.org/10.1034/j.1399-3011.1999.00014.x
Bonewald, Lynda ; Bibbs, L. ; Kates, S. A. ; Khatri, A. ; McMurray, J. S. ; Medzihradszky, K. F. ; Weintraub, S. T. / Study on the synthesis and characterization of peptides containing phosphorylated tyrosine. In: Journal of Peptide Research. 1999 ; Vol. 53, No. 2. pp. 161-169.
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