PMC

Schematic of experimental null (EN) method and its utilities in guiding experiment design/development and FADR estimation.

Estimation of FADR by EN method using concocted proteomic samples. (A) Details of the designs of the concocted samples. Sample set A is for technical replicates and sample set B is for mimic biological replicates. (B) Correlation of the FADR estimated by EN method versus actual FADR (calculated based on known true-positives/negatives). Details are shown in the Methods section.

Assessment of the underlying assumption of EN method by comparing the null distribution measured by EN method versus the true null distribution (negative distribution) in the case-vs-contol sample set for (A) sample set A and (B) sample set B. Excellent agreement was observed for both sample sets.

Use of EN method to evaluate the effects of different experimental strategies and parameters on precision (i.e., within-group variability, CV %) and quantitative accuracy(i.e., deviation from true ratio, 0 at log 2 scale). (A,B) Effects of different quantitative strategies including ion current (IC)-based methods (median-IC, LMM-IC, and Sum-IC) and MS2-based methods (MS2-TIC and SpC). (C,D) Effects of the quality of chromatographic separation and (E,F) effects of database selection.

EN method to determine the effect of sample size and experimental design on the capacity to determine protein abundance changes. Histograms of the log 2 ratio of the quantified proteins under different sample sizes for analyzing (A) technical replicates, (C) biological replicates, and (E) different experimental design (true value = 0 at log 2 scale). (B,D,F) Plot for protein % above log 2 ratios. Dashed vertical line denotes the rational ratio cut off for reliable quantification, which is defined as the ratio, where 95% of the proteins quantified in the EN data set fall below.