Mcw Research Foundation, Inc.

Brian D Halligan

Joseph T Barbieri

Edward A Dratz

Arthur L Haas

Lawrence M Ryan

Daniel J McCarty

Ikuko Masuda

The present invention provides a peptide identification method along with related peptide databases, method of generating the databases, computer usable media, and computer program products. The peptide identification method involves the use of qualitative amino acid composition or partial qualitative amino acid composition information of a query peptide obtained by mass spectrometry to correlate the query peptide to one or more peptides with defined amino acid sequences in a peptide database generated according to the present invention.

Peptide identification

We have cloned and sequenced the cDNA encoding the 61 kD active fragment of a unique porcine chondrocyte nucleotide pyrophosphohydrolase (NTPPHase) from a porcine chondrocyte library. Degenerate oligonucleotides, corresponding to the N-terminal amino acid sequence of this peptide were hybridized to porcine chondrocyte cDNA and used to amplify DNA encoding the N-terminal sequence of 61 kD with the polymerase chain reaction (PCR). The PCR products were then used as probes to clone the entire open reading-frame for the 61 kD fragment from a porcine chondrocyte cDNA library. The length of the cloned cDNA was 2509 bp. Translation of the open-reading-frame predicts the 61 kD fragment to be a 459 amino acid protein. BLAST and FASTA analysis confirmed that this amino acid sequence was unique and did not possess high homology to any known proteins in the non-redundant protein data base. Limited homology (17%) between the 61 kD fragment and several prokaryotic and eukaryotic ATP pyrophosphate-lyase (adenylate cyclase) was detected. Northern blot analysis of porcine chondrocyte RNA showed that the DNA encoding the 61 kD fragment hybridized to a 4.3 kbp RNA transcript. Human chondrocyte RNA also hybridized to this porcine DNA probe. Coupled in vitro transcription translation of an expression vector containing the DNA insert in frame showed the expression of a 61 kD protein.

Cloned nucleotide pyrophosphohydrolase and uses thereof

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Wauwatosa, WI, United States of America

Brian D Halligan