High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.
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.
Sloane
Andrew J
AJ
Proteome Systems Limited, 1/35-41 Waterloo Rd., North Ryde, Sydney, New South Wales 2113, Australia.
Duff
Janice L
JL
Wilson
Nicole L
NL
Gandhi
Parag S
PS
Hill
Cameron J
CJ
Hopwood
Femia G
FG
Smith
Paul E
PE
Thomas
Melissa L
ML
Cole
Robert A
RA
Packer
Nicolle H
NH
Breen
Edmond J
EJ
Cooley
Patrick W
PW
Wallace
David B
DB
Williams
Keith L
KL
Gooley
Andrew A
AA
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
D007136:Immunoglobulins; D020543:Proteome; D014357:Trypsin