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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Relative, label-free protein quantitation: spectral counting error statistics from nine replicate MudPIT samples.
Bret Cooper1, Jian Feng, Wesley M Garrett
1Soybean Genomics and Improvement Laboratory, USDA-ARS, Beltsville, Maryland 20705, USA. bret.cooper@ars.usda.gov
Journal of the American Society for Mass Spectrometry
|June 15, 2010
Summary
Label-free spectral counting accurately quantifies proteins. Combining replicate samples increases protein identification and spectral counts correlate linearly with protein amounts, confirming reliability.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Quantitative proteomics is crucial for understanding biological processes.
- Mass spectrometry-based proteomics offers high-throughput protein identification.
- Accurate relative protein quantitation is essential for comparative studies.
Purpose of the Study:
- To evaluate the accuracy and reliability of label-free spectral counting for relative protein quantitation.
- To assess the impact of combining replicate samples on protein identification.
- To investigate the influence of spectral counting thresholds on quantitation accuracy.
Main Methods:
- Analysis of soybean leaf peptides spiked with bovine apotransferrin peptides using multidimensional protein identification technology (MudPIT).
- Statistical evaluation of spectral counts for protein identification and quantitation.
- Comparison of different spectral counting approaches and threshold settings using PANORAMICS software.
Main Results:
- Combining spectra from replicate samples increased the number of identifiable proteins.
- Summed spectral counts increased linearly with increasing protein molar amounts.
- Statistical analysis showed minimal run-to-run variation, confirming data reliability.
- Empirical setting of counting thresholds improved relative quantitation accuracy.
Conclusions:
- Label-free spectral counting is a reliable method for relative protein quantitation.
- Combining replicate samples enhances protein identification in mass spectrometry.
- Optimizing spectral counting thresholds is key to improving quantitative accuracy in proteomics.
