Intelligent use of stable isotopes in proteomics
Abstract
Intelligent use of stable isotopes could greatly improve throughput, quantification accuracy, and peptide sequencing in proteome research. Proteins from experimental and control samples were digested into peptides, which were isotopically derivatized and mixed together. The peptide mixture was separated by multidimensional chromatography and detected in mass spectrometers, where they were quantified by measuring the abundance ratios between the isotopically coded peptides in mass spectra and sequenced if the abundance ratio exceeded the preset threshold and presented significant changes between the experimental and control samples. Isotopically coded peptides were found to be fractionated during reversed-phase chromatography (RPC). This could compromise the accuracy of the abundance ratio measurement. Using carbon 13 coding agents instead of deuterium coding eliminated this problem and enabled real-time intelligent data acquisition (IDA). Real-time IDA means selecting peptides for MS/MS experiments based on their abundance ratios and therefore focusing on significantly changed peptides. Among deuterated coding agents, resolution could be controlled by placing the deuterium atoms around hydrophilic groups. A double isotope coding scheme, i.e. code the peptide C-terminus with two oxygen-18 atoms during trypsin digestion in oxygen-18 enriched water and the N-terminus with three deuterium atoms by subsequent derivatization, was demonstrated to greatly improve peptide sequencing because of the recognition of b and y ions in MS/MS spectra based on the unique mass differences between the isotopically coded fragmented ions. A novel algorithm was developed to deconvolute overlap between isotopically coded peptides to facilitate pattern recognition and quantification, when there were not enough mass differences between the isotopically coded peptides. It is superior to the existing algorithms, which required either extra experiments or knowledge of the peptide sequences.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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