Fig. 2. HipMHCs improve quantitation in LF and TMT-labeled samples.

a Experimental design. Five LF (orange) and six TMT (blue) technical replicates of 1 × 10⁷ cells + hipMHCs were used to compare LF and TMT quantification. b Peptide sequence and amount of hipMHC added into each sample. L⁷ denotes heavy-isotope-labeled leucine (+7). ALNEQIARL⁷ and SLEEPIGHL⁷ were used as quantification correction hipMHCs, and SVVESVKFL⁷ was titrated in across samples. For LF analysis, sample #6 was omitted. c Two thousand three hundred and sixty-nine unique LF peptides were identified across five analyses (black), 1352 of which were quantifiable (gray) via AUC quantification, and 589 quantifiable peptides which were identified in all five analyses (orange). One thousand seven hundred and fifty-four unique peptides were quantified with TMT-labeled analyses combining six TMT fractions (blue). d Ninety-two percent of TMT, 82% of LF, and 5.6% of random peptide 9-mers derived from the proteome (gray) are predicted to bind to an HLA allele in MDA-MB-231 cells with an affinity < 500 nM. e Linear fit of titrated hipMHC peptide for LF (left) and TMT (right). Raw r² = 0.48 (LF) and r² = 0.91 (TMT), hipMHC-adjusted (adj) r² = 0.99 (LF) and r² = 0.96 (TMT). f Distribution of the log₂ fold change (FC) of each peptide’s quantification (x) over the mean (μ) peptide quantification across samples for raw (left) and hipMHC adjusted (right). g Gaussian fit of the frequency distribution of log₂(FC) of (x) over (μ) for raw and hipMHC-adjusted LF and TMT samples. In all, 99.7% of variance between peptide quantitation (3× SD) is captured within a 1.65 (raw) and 1.52 (adj) FC from the mean for LF samples, and 1.30 (raw) and 1.23 (adj.) for TMT samples. h Median coefficient of variation (CV) for LF (24.27% raw, 20.99% adj) and TMT (14.00% raw, 7.48% adj). Error bars represent the interquartile range. Source data are provided in the Source data file.