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Submitted on March 22, 2005
Dept. of Chemistry & Biochemistry, University of Colorado, Boulder, CO 80309
Corresponding Author: William.Old{at}colorado.edu
Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomics analyses. We describe SERAC, software developed to evaluate the ability of each method to quantify relative changes in protein abundance. Dynamic range and linearity using a three-dimensional ion trap were tested using standard proteins spiked into a complex sample. Linearity and good agreement between observed vs. expected protein ratios were obtained after normalization and background subtraction of peak area intensity measurements and correction of spectral counts to eliminate discontinuity in ratio estimates. Peak intensity values useful for protein quantitation ranged from 107 to 1011 counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (± 2
Revised on June 22, 2005
Accepted on June 23, 2005
Comparison of label free methods for quantifying human proteins by shotgun proteomics
) when = 3 peptides/protein were shared between samples. Protein ratios were determined with high confidence from spectral counts when maximum spectral counts were = 4 spectra/protein, and replicates showed equivalent measurements well within 95% confidence limits. In further tests, complex samples were separated by gel exclusion chromatography, quantifying changes in protein abundance between different fractions. Linear behavior of peak area intensity measurements was obtained for peptides from proteins in different fractions. Protein ratios determined by spectral counting agreed well with those determined from peak area intensity measurements, and both agreed with independent measurements based on gel staining intensities. Overall, spectral counting proved to be a more sensitive method for detecting proteins that undergo changes abundance, whereas peak area intensity measurements yielded more accurate estimates of protein ratios. Finally, these methods were used to analyze differential changes in protein expression in human erythroleukemia K562 cells, stimulated under conditions that promote cell differentiation by MAP kinase pathway activation. Protein changes identified with p<0.1 showed good correlations with parallel measurements of changes in mRNA expression.
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