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.
Highlights
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
As an alternative rapid high throughput approach, we demonstrate dispensing of trypsin onto a macroarray of human plasma proteins on a polyvinylidene fluoride (PVDF) membrane that had been adhered to a MALDI-TOF target plate (Fig. 2A)
DNA microarrays have been heralded as a revolution in genomic studies, the role of protein microarrays in proteomic studies is not as simple, principally because of the challenges of developing high throughput strategies for preparing proteins prior to their deposition onto chip surfaces
Summary
2-DE, two-dimensional gel electrophoresis; BSA, bovine serum albumin; CFR, curved field reflectron; ESI, electrospray ionization; FITC, fluorescein isothiocyanate; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MS, mass spectrometry; OGP, n-octyl -D-glucopyranoside; PBS-TAC, phosphate-buffered saline containing 0.1% (v/v) Tween 20, 0.05% (w/v) NaN3, and 0.5% (w/v) casein, pH 7.4; pmf, peptide mass fingerprinting; PSL, Proteome Systems Limited; PVDF, polyvinylidene fluoride; TB, tuberculosis. The chemical printer represents a powerful tool for identification of novel protein targets for biomedical and diagnostic purposes
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