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

• Published in: Molecular & cellular proteomics Vol. 4; no. 10; pp. 1487 - 1502
• Main Authors: Old, William M, Meyer-Arendt, Karen, Aveline-Wolf, Lauren, Pierce, Kevin G, Mendoza, Alex, Sevinsky, Joel R, Resing, Katheryn A, Ahn, Natalie G
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 01-10-2005
• Subjects: Bias; Chromatography, Gel; Humans; K562 Cells; Mass Spectrometry; Peptides - analysis; Proteins - analysis; Proteomics - methods; RNA, Messenger - genetics; Sensitivity and Specificity; Software; Tetradecanoylphorbol Acetate - pharmacology; User-Computer Interface
• ISSN: 1535-9476; 1535-9484
• DOI: 10.1074/mcp.M500084-MCP200

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 (±2ς) 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 mitogen-activated protein kinase pathway activation. Protein changes identified with p < 0.1 showed good correlations with parallel measurements of changes in mRNA expression.