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. 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. The ability to accurately define protein expression in relationship to physiological changes associated with healthy or diseased states and the potential to discover novel drug targets are emerging themes of proteomic programs (1.Abbott A. A post-genomic challenge: learning to read patterns of protein synthesis.Nature. 1999; 402: 715-720Google Scholar, 2.Banks R.E. Dunn M.J. Hochstrasser D.F. Sanchez J.C. Blackstock W. Pappin D.J. Selby P.J. Proteomics: new perspectives, new biomedical opportunities.Lancet. 2000; 356: 1749-1756Google Scholar). Understanding these dynamics is rendered complex given there is often no correlation between mRNA expression levels and protein expression (3.Anderson L. Seilhamer J. A comparison of selected mRNA and protein abundances in human liver.Electrophoresis. 1997; 18: 533-537Google Scholar), and the paradigm of one gene-one protein is known not to hold (4.Wilkins M.R. Sanchez J.C. Williams K.L. Hochstrasser D.F. Current challenges and future applications for protein maps and post-translational vector maps in proteome projects.Electrophoresis. 1996; 17: 830-838Google Scholar). The rapid growth in proteomics has resulted in a technology-driven science oriented toward development of automated high throughput platforms (5.Lee K.H. Proteomics: a technology-driven and technology-limited discovery science.Trends Biot