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Originally published In Press as doi:10.1074/mcp.M200020-MCP200 on July 3, 2002.
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Molecular & Cellular Proteomics 1:490-499, 2002.
© 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

High Throughput Peptide Mass Fingerprinting and Protein Macroarray Analysis Using Chemical Printing Strategies*

Andrew J. Sloane{ddagger}, Janice L. Duff{ddagger}, Nicole L. Wilson{ddagger}, Parag S. Gandhi{ddagger}, Cameron J. Hill{ddagger}, Femia G. Hopwood{ddagger}, Paul E. Smith{ddagger}, Melissa L. Thomas{ddagger}, Robert A. Cole{ddagger}, Nicolle H. Packer{ddagger}, Edmond J. Breen{ddagger}, Patrick W. Cooley§, David B. Wallace§, Keith L. Williams{ddagger} and Andrew A. Gooley{ddagger},¶

{ddagger} Proteome Systems Limited, 1/35–41 Waterloo Rd., North Ryde, Sydney, New South Wales 2113, Australia
§ Microfab Technologies, Inc., Plano, Texas 75074

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

To whom correspondence should be addressed. Tel.: 61-2-9889-1830; Fax: 61-2-9889-1805; E-mail: andrew.gooley{at}proteomesystems.com


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