Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates
Figure 1
Experimental outline of the label-free protein quantitation approach to assess the acid stress response between the unlabeled stressed culture (S) and the unlabeled reference culture (R) with the [15N]-labeled culture as control (C). The procedures are divided into six stages (I–VI). Briefly, equal amounts of protein extract from the S culture triplicates were pooled. Equal amounts of protein extract from the R culture triplicates were also pooled. Into these two pooled unlabeled protein samples, an equal amount of protein extract from the C culture was added. This resulted in the two pooled samples; that is, and . The proteins differentially expressed between the S and R cultures were determined based on comparison of the abundances of the unlabeled proteins that is, and , between samples and . For the purpose of false discovery rate assessment, the abundances of the [15N]-labeled proteins that is, and , were quantified and compared between and in the same way as between and . The proteins found differentially expressed between and were considered positives, because they reflected the difference between the S and R cultures. The proteins found differentially expressed between and in the labeled form were false positives, because difference was not expected from the identical C sample that was run concurrently with two unlabeled samples in separate runs.
