Quantification of proteins using data-independent analysis (MSE) in simple andcomplex samples: a systematic evaluation.

A MS-based method for the quantification of proteins termed data-independent analysis (or MS(E)) has been introduced recently. Although this method has been applied to the analysis of various types of biological samples, a thorough evaluation to assess the performance of this approach has yet to be conducted. Presented here is the first systematic and comprehensive study investigating the MS(E) approach for quantitative analysis of low-, medium-, and high-complexity samples. We demonstrate that this method has a linear dynamic range spanning three orders of magnitude with a limit of quantification of 61 amol/uL in low-complexity samples and 488 amol/uL in high-complexity samples. In addition, comprehensive sequence coverage was obtained and accurate quantification achieved for expression ratios ranging from 1:1.5 to 1:6. However, underestimation of ratios was detected independent of sample type, consistent with other quantitative proteomic methods. The present study provides validation of the MS(E) approach for accurate quantitative proteomic analysis of biological samples while, at the same time, proving high sequence coverage of target proteins.