title: High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.
abstract: 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.
authors.affiliation: Proteome Systems Limited, 1/35-41 Waterloo Rd., North Ryde, Sydney, New South Wales 2113, Australia.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Sloane
authors.forename: Janice L
authors.initials: JL
authors.lastname: Duff
authors.forename: Nicole L
authors.initials: NL
authors.lastname: Wilson
authors.forename: Parag S
authors.initials: PS
authors.lastname: Gandhi
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Hill
authors.forename: Femia G
authors.initials: FG
authors.lastname: Hopwood
authors.forename: Paul E
authors.initials: PE
authors.lastname: Smith
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Thomas
authors.forename: Robert A
authors.initials: RA
authors.lastname: Cole
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: Edmond J
authors.initials: EJ
authors.lastname: Breen
authors.forename: Patrick W
authors.initials: PW
authors.lastname: Cooley
authors.forename: David B
authors.initials: DB
authors.lastname: Wallace
authors.forename: Keith L
authors.initials: KL
authors.lastname: Williams
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Gooley
mesh_terms: D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000737:chemistry; D004563:Electrochemistry; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000295:instrumentation* / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods; D014357:Trypsin / Q000378:metabolism
chemical_list: D007136:Immunoglobulins; D020543:Proteome; D014357:Trypsin