FIG. 2.

The accuracy and precision of iodoTMT-based multiplexed quantification. (A) Schematics of the sample preparation strategy used to evaluate the performance of iodoTMT-based quantification as mentioned in text. After tryptic digestion, the anti-TMT affinity enriched peptides were subjected to liquid chromatography (LC)-MS/MS analysis, which resulted in identification of over 2000 iodoTMT-labeled peptides representing an enrichment specificity of ∼75% (B). A representative MS/MS spectrum of an iodoTMT-labeled peptide is shown in (C), with its major sequence-informative b/y fragment ions fully annotated and the narrow mass region containing the TMT reporter ions (marked by an arrow) further expanded in the inset. Deviation of the experimentally derived reporter ion ratio values from the predicted ratios, namely 0.6, 0.2, and 2.0, for m/z 127, 128, and 129 to 126, respectively, for all 1669 quantifiable iodoTMT-labeled peptides as shown by Boxplot in (D), was taken as a measure of the quantification accuracy. CV, coefficient of variation.