On the excessive use of coefficient of variation as a metric of quantitation quality in proteomics

Abstract

The coefficient of variation (CV) is often used in proteomics as a proxy to characterize the performance of a quantitation method and/or the related software. In this note, we question the excessive reliance on this metric in quantitative proteomics that may result in erroneous conclusions. We support this note using a ground‐truth Human‐Yeast‐E. coli dataset demonstrating in a number of cases that erroneous data processing methods may lead to a low CV which has nothing to do with these methods’ performances in quantitation.

Reanalysis of Orbitrap Astral DIA data demonstrates the capabilities of MS/MS-free proteomics to reveal new biological insights in disease-related samples

“…At the same time, we believe that the popular reporting purely the number of “identified and quantified” proteins, as well as the coefficients of variation provide no warranty for better quantitation results in comparable methods, or tell much about the perspectives of obtaining biologically important information about regulated proteins and/or cellular processes. 3,6…”

“…At the same time, we believe that the popular reporting purely the number of "identified and quantified" proteins, as well as the coefficients of variation provide no warranty for better quantitation results in comparable methods, or tell much about the perspectives of obtaining biologically important information about regulated proteins and/or cellular processes. 3,6 Figure 1. Overview of datasets and results obtained using different proteomic analysis methods.The number of differentially expressed proteins for DIA results and all numbers for DirectMS1 reanalysis were obtained in this study.…”

“…And finally, there are many situations which could lead to wrong estimation of LFQ values even for true peptide/protein identifications. 6 The Orbitrap Astral provides parallelized acquisition of MS1 and MS/MS spectra at the MS/MS scanning rate of ~200 Hz and the isolation window down to 2 Th in DIA implementation, that positions this instrument as a method of choice in quantitative proteomics. 7 However, the MS/MS-based quantitation, typically employed in DIA, has limitations, especially in the ultrashort LC gradient implementations and may miss, or underscore the important biological insights of the samples under study due to extremely high MS/MS signal interferences.…”

“…And finally, there are many situations which could lead to wrong estimation of LFQ values even for true peptide/protein identifications. 6…”

Reanalysis of DIA Data Demonstrates the Capabilities of MS/MS-Free Proteomics to Reveal New Biological Insights in Disease-Related Samples

“…Finally, there are many cases which could lead to incorrect estimation of the labelfree quantitation (LFQ) values even for the true peptide/protein identifications. 6 The recently introduced Orbitrap Astral provides parallelized acquisition of MS1 and MS/MS spectra at the MS/MS scanning rate of ∼200 Hz and an isolation window down to 2 Th in DIA implementation, which positions this instrument as a method of choice in quantitative proteomics. 7 However, the MS/MS-based quantitation, typically employed in DIA, has limitations, especially in ultrashort LC gradient implementations and may miss or underscore the important biological insights of the samples under study due to extremely high MS/MS signal interferences.…”