Development of novel mass spectrometric protocols for rapid protein characterization
King, Daniel Allen
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Mass spectrometry (MS) has found an indispensable roll in the analysis of biological molecules. In the wake of the completion of the human genome, proteomics work in academic, government, and pharmaceutical laboratories has grown dramatically and the need for fast protein analyzing protocols is a high priority. Mass spectrometry is often the final instrument of analysis after a long and arduous series of separation and processing steps. Although mass spectrometry is often a very quick technique, the analysis time is limited by the slow separation, purification, and processing procedures. Presented here are novel mass spectrometric procedures for characterizing proteins that attempt to increase the speed of analysis and provide complementary approaches to standard protein protocols. New on-probe procedures have been developed that provide an expedient alternative to standard protocols. For the identification of a protein, a proteolytic digestion must be performed. These enzymatic digestions can typically require 10-20 hours under standard conditions. The performance of proteolytic digestions directly on a MALDI probe has proven in the past to be an effective way to shorten the digestion time to less than an hour. Presented here are two digestion procedures that provide some unique advantages over the typical trypsin digest in addition to their increased speed. Pepsin is shown to work in the presence of MALDI matrix and can be used after previous mass spectra have been generated, and acid hydrolysis is shown to generate very valuable sequence ladders. Initially used to study protein folding, amide exchange-MS is continually being adapted to the study of new types of protein systems. Presented here is the first adaptation of amide exchange-MS to the study of a protein-carbohydrate binding system. Aside from the speed of amide exchange-MS, the many difficulties that are often encountered in x-ray crystallography and nuclear magnetic resonance (NMR) are avoided. Amide exchange-MS is less sensitive to protein size, limited sample quantities, and varying solubility and flexibility properties of protein and carbohydrate components. Amide exchange-MS is shown to be an essential, complementary technique to x-ray crystallography and NMR.