Assessing the precision of a detergent‐assisted cartridge precipitation workflow for non‐targeted quantitative proteomics

Abstract

AbstractDetergent‐based workflows incorporating sodium dodecyl sulfate (SDS) necessitate additional steps for detergent removal ahead of mass spectrometry (MS). These steps may lead to variable protein recovery, inconsistent enzyme digestion efficiency, and unreliable MS signals. To validate a detergent‐based workflow for quantitative proteomics, we herein evaluate the precision of a bottom‐up sample preparation strategy incorporating cartridge‐based protein precipitation with organic solvent to deplete SDS. The variance of data‐independent acquisition (SWATH‐MS) data was isolated from sample preparation error by modelling the variance as a function of peptide signal intensity. Our SDS‐assisted cartridge workflow yield a coefficient of variance (CV) of 13%–14%. By comparison, conventional (detergent‐free) in‐solution digestion increased the CV to 50%; in‐gel digestion provided lower CVs between 14% and 20%. By filtering peptides predicting to display lower precision, we further enhance the validity of data in global comparative proteomics. These results demonstrate the detergent‐based precipitation workflow is a reliable approach for in depth, label‐free quantitative proteome analysis.