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. 2021 Jan 11;12:254. doi: 10.1038/s41467-020-20509-1

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Experimental workflow for in-depth diGly peptide library construction (upper panel) and our single-run data-independent acquisition (DIA)-based workflow (lower panel). Protein digestion and peptide extraction are followed by basic reversed-phase (bRP) fractionation and diGly peptide enrichment. For library construction, samples were measured by data-dependent acquisition (DDA) and computationally processed (Spectronaut Pulsar). Individual samples are measured by our DIA workflow, including matching against a library for identification (Spectronaut software). b Number of identified diGly peptides in three different spectral libraries (MG132 treated HEK293 library—green, MG132 treated U2OS library—violet, U2OS library—light violet, all diGly peptides—gray). c Commonly and exclusively identified diGly peptides for different libraries (MG132 treated HEK293 library—green, MG132 treated U2OS library—violet, U2OS library—light violet). d Identified diGly sites (mean ± SEM) of MG132 treated HEK293 cells using different DIA library search strategies (n = 6, three workflow replicates measured in analytical duplicates). Source data are provided as a Source data file.