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. 2023 Jun 16;14:3599. doi: 10.1038/s41467-023-39347-y

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© The Author(s) 2023

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 Description of the hybrid-DIA acquisition method. The figure represents a scheme of three acquisition cycles. A hybrid-DIA acquisition scan cycle starts with a full-scan or MS1 scan that it is acquired at high resolution. The hybrid-DIA API scans the MS1 for the precursor m/z values in the inclusion list that match the specific retention time window. If any is found (e.g., Mass 1) a targeted scan of this IS precursor m/z will be triggered as part of the subsequent MS/MS scan cycle (red box). The API will on-the-fly match the obtained fragments from that PRM scan (red box) to the IS peptide fragment values in the inclusion list. If the fragments match the expected values, then a targeted multiplexed scan (MSx) will be added to the cycle (purple box). This MSx scan (purple box) will simultaneously analyze the heavy (IS) and the endogenous (ENDO) peptide using non-synchronous injection times. Subsequently, the predefined DIA windows of the MS method will be acquired. Once the complete scan cycle is done, a new one will start and the process will be repeated in each cycle when heavy peptides are found in the MS1 scan. The box width reflects the injection times used for each scan, but it is not set to scale. b Example of the increase in sensitivity by using differential injection time in the multiplexed (MSx) scans. In red, spiked-in heavy stable isotopically labeled peptide standard (IS), and in green the triggered endogenous peptide (ENDO). c Example of a DIA scan and an IS/ENDO MSx scan from the same cycle where the heavy peptide carrying the G12S mutation, as well as its endogenous counterpart were isolated and fragmented. The top 3 y-fragments of each peptide are highlighted, in red the heavy fragments and in green the endogenous fragments. d Signal-to-noise (S/N) measured in the top 3 y-fragments in the endogenous G12S peptide in a DIA window (green) and in the MSx IS/ENDO scan (purple) in the same acquisition cycle. Height of the bars is the average S/N of three replicates, error bars are the standard deviation. RT: retention time; IT: injection time; m/z: mass to charge. Source data are provided as a Source Data file.