Comparison of Label-free Methods for Quantifying Human Proteins by Shotgun Proteomics

Abstract

Measurements of mass spectral peak intensities and spectral counts are promising methods for quantifying protein abundance changes in shotgun proteomic 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 versus 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 10 7 to 10 11 counts with no obvious saturation effect, and proteins in replicate samples showed variations of less than 2-fold within the 95% range (؎2) 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 in 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 mitogenactivated protein kinase pathway activation. Protein changes identified with p < 0

Increased IgG on cell‐derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation

“…This number is conveniently accessed since it is calculated as part of the protein identification process. It correlates with protein abundance and can therefore be used for relative quantitation between samples (28,29). Thus, spectral counts were used in the present study to quantitate proteins in selected samples relative to their presence in other samples.…”

Label-free quantitation: A new glycoproteomics approach

“…Changes in intensities of mass spectral peaks have been assessed by comparing different sample sets through either glycan analysis [4] or glycopeptide analysis [32]. Because signal intensities can vary between mass spectrometric samples, label-free approaches are not as commonly used for quantitative analysis [26,33].…”

“…To alleviate much of the variation due to changes in MS response among samples, normalization of data has been applied in proteomics studies [33][34][35][36]. Many proteomics researchers employ a normalization technique that involves dividing intensities of individual peaks by the total intensity from all peaks in the spectrum [33][34][35]; this approach is fundamentally similar to the approach by Wang et al, where spectra are normalized by calculating an intensity ratio for one spectrum versus another by doing pair-wise comparisons of peak intensities, for all the peaks in the spectra [36].…”

“…Many proteomics researchers employ a normalization technique that involves dividing intensities of individual peaks by the total intensity from all peaks in the spectrum [33][34][35]; this approach is fundamentally similar to the approach by Wang et al, where spectra are normalized by calculating an intensity ratio for one spectrum versus another by doing pair-wise comparisons of peak intensities, for all the peaks in the spectra [36]. In the work presented here, this concept of normalization is builtupon to produce a label-free quantitative method for glycopeptide analysis.…”

“…In the work presented here, this concept of normalization is builtupon to produce a label-free quantitative method for glycopeptide analysis. While in the proteomic field one can obtain reliably quantitative data by normalizing the data to the total ion abundance [33][34][35], this method is potentially problematic for glycopeptide analysis because the glycopeptides ionize weakly, compared with the nonglycosylated peptides that may also be present. Therefore, small changes in the presence of nonglycosylated interferents could impart large variability in a quantitative assay when the total ion current (or pairwise comparison of all the peaks) is used to normalize the ion abundances of the analytes.…”