Screening combinatorial libraries by mass spectrometry. 2. Identification of optimal substrates of protein tyrosine phosphatase SHP-1.
Protein tyrosine phosphatases (PTPs) are a large family of enzymes that catalyze the hydrolytic removal of the phosphoryl group from phosphotyrosyl (pY) proteins. In this work, we have developed a novel combinatorial library method, termed "enzyme-catalyzed loss of isotope peak signal enhancement (ECLIPSE)", to determine the substrate specificity of PTPs. This method involves partial labeling of pY at a nonbridging phosphate oxygen atom with 50% (18)O ((16)O/(18)O = 1:1). A 361-member solution-phase peptide library with randomization at the -1 and -2 positions (relative to pY), RNNXXpYA-NH(2) (X = 19 alpha-amino acids except for Cys), was synthesized with the partially (18)O-labeled pY by the split-synthesis method. Each member of the resulting pY peptide library appeared as a doublet peak in the mass spectrum (m/z m and m + 2.0043). Limited treatment of the library with a PTP removed the mass-degenerate phosphoryl group from the most preferred substrates to generate products as singlet peaks, which were readily identified and sequenced by tandem mass spectrometry. Screening of the pY library against the catalytic domain of SHP-1 revealed that SHP-1 prefers an acidic residue at the -2 position, with aspartic acid being slightly better than glutamic acid. At the -1 position, SHP-1 also prefers an acidic residue, although a variety of other amino acids are also tolerated. On the other hand, positively charged residues at these positions render the corresponding peptides very poor substrates of SHP-1. Several selected peptides were individually synthesized and assayed against SHP-1, and the kinetic data confirmed the screening results. These results demonstrate that ECLIPSE is a viable method for studying the substrate specificity of PTPs.
Wang
Peng
P
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210, USA.
Fu
Hua
H
Snavley
Donald F
DF
Freitas
Michael A
MA
Pei
Dehua
D
D000595:Amino Acid Sequence; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D015195:Drug Design; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry; D019151:Peptide Library; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D047908:Intracellular Signaling Peptides and Proteins; D009842:Oligopeptides; D019151:Peptide Library; D011994:Recombinant Proteins; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; D017027:Protein Tyrosine Phosphatases