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. 2021 Apr 9;20:100080. doi: 10.1016/j.mcpro.2021.100080

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© 2021 The Authors. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology.

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Fig. 2 — Application of sample-specific spectral libraries for matching immunopeptidomics DIA data. HLA-I peptides purified from RA957 cells were measured by DIA in duplicates as normal and 3× and 5× diluted samples and matched against a library constructed from three DDA replicates, all measured sequentially (A). The number of peptides identified in each of the DDA measurements included in the RA957-specific library and the number of peptides identified in each of the DIA measurements following matching to this library are reported. The orange bar refers to the number of peptides identified in three DDA runs, and the blue bar refers to the total number of peptides included in the DIA library. Length distribution of peptides identified in RA957 DIA samples (B). Representative correlation between measured RT in the DIA data and the predicted RT calculated by Spectronaut (C). Deconvolution of the consensus binding motifs of RA957 DIA immunopeptidomics samples (D). The number of peptides and the HLA restriction assigned to each motif are reported. Two subspecificities were detected for HLA-A∗68:01. HLA-I peptide samples purified from JY cells were measured by DIA in triplicates and matched against a JY-specific library constructed from three DDA replicates (E). The length distribution of peptides identified in JY DIA samples (F) and a representative correlation between measured RT and the predicted RT (G) are provided. Deconvolution of the consensus binding motifs of JY DIA immunopeptidomics samples (H). HLA-I peptide samples purified from OD5P cells were measured by DIA in triplicates and matched against a OD5P-specific library constructed from four DDA replicates (I). The length distribution of peptides identified in OD5P DIA samples (J) and a representative correlation between measured RT and the predicted RT (K) are provided. Deconvolution of the consensus binding motifs of OD5P DIA immunopeptidomics samples (L). DDA, data-dependent MS/MS acquisition; DIA, data-independent acquisition; HLA, human leukocyte antigen; RT, retention time.