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

By W. Old, K. Meyer-Arendt, L. Aveline-Wolf, K. Pierce, A. Mendoza, Joel R. Sevinsky, Katheryn Resing, Natalie G. Ahn. Molecular & Cellular Proteomics, 2005

1,232 citations recorded by the source corpus.

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 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 (±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.

Subfields: Molecular Biology, Spectroscopy

View Paper: https://www.semanticscholar.org/paper/11b24116b08a1b4f1f36381d87500c8e7c7c5660

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Molecular Biology

In Spectroscopy

Citations in the corpus

Label-free, normalized quantification of complex mass spectrometry data for proteomics analysis (2009, neutral): “and log 2 (protein ratio) from spectral counts (Rsc”
Mass Spectrometry-Based Label-Free Quantitative Proteomics (2009, background): “Relative quantification by spectral count has been widely applied in different biological complex, including analysis of urine sample from healthy donors and patients with acute inflammation [32], finding biomarkers in human saliva proteome in type-2 diabetes [33], comparison of protein expression i”
A protocol for exosome isolation and characterization: evaluation of ultracentrifugation, density-gradient separation, and immunoaffinity capture methods. (2015, methodology): “2), as previously described [32] based on studies by [33, 34].”
Sorting Signals, N-Terminal Modifications and Abundance of the Chloroplast Proteome (2008, methodology): “G-test was performed across three technical and across three biological replicates of chloroplast stroma preparations as described in [17,40] and in [39].”
Computational prediction of proteotypic peptides for quantitative proteomics (2007, neutral): “For quantitative proteomic analysis, label-free method”
Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures (2009, background): “The number of identified peptides to a protein is the basis for some LC-MS spectral counting-based absolute quantification algorithms [43, 59–62], which can bias the results obtained by this method, for the above reason.”
Less label, more free: Approaches in label‐free quantitative mass spectrometry (2011, methodology): “org [87] Serac AUC – [51] SpecArray AUC http://sourceforge.”
Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. (2007, neutral): “For example, comparison of phosphopeptide numbers for c-Met with the levels of phosphorylated c- Met protein observed by western analysis are in good agreement (Gilchrist et al., 2006; Old et al., 2005; Zybailov et al., 2005) (see Figure 1D).”