35-3 |
Use of mass spectrometry to characterize bio-polymers |
T. KEOUGH, M. P. Lacey, and R. S. Youngquist. Miami Valley Labs., Procter & Gamble Co., PO Box 538707, Cincinnati, OH 45253 Knowledge of protein and peptide sequences is fundamentally important for understanding many physiological and biochemical processes at the molecular level. Electrospray mass spectrometry has recently emerged as an essential tool for high-sensitivity peptide sequencing because it produces doubly protonated peptide ions, and doubly protonated tryptic peptides fragment extensively on the mass spectrometry time frame. MALDI mass spectrometry, on the other hand, has proven to be much less reliable for peptide sequencing because it produces mainly singly protonated molecules, which are quite stable in the gas-phase. The objective of this research was to develop high-sensitivity, user-friendly derivatization chemistry to enable reliable de novo peptide sequencing using MALDI mass spectrometry methods. New water-compatible peptide sulfonation reagents and greatly simplified derivatization protocols have been developed to facilitate high-throughput peptide sequencing using MALDI mass spectrometry. We previously have successfully sequenced sulfonic acid derivatized peptides using reflector time-of-flight mass spectrometers. Recently, we evaluated the performance of these derivatives with an atmospheric pressure MALDI source, which was interfaced to the widely available Finnigan LCQ ion trap mass spectrometer. Convenient, “one tube” derivatization methods have been developed and optimized. They are applicable to tryptic peptides, which are terminated with either Arg- or Lys-residues. The new reagents and current derivatization protocol enable simple, multiplexed sample preparation at a rate of one sample per minute. Derivatized peptides were sequenced using an AP-MALDI ion trap instrument. Several advantages of ion trap mass analyzers, relative to current reflector time-of-flight mass spectrometers, were demonstrated. They include: improved parent ion selection, MSn and automated unattended peptide sequencing. The implications of these developments for proteomics research will be highlighted.
Session 35, Applications of proteomics to food science
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