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. 2023 May 9;10:261. doi: 10.1038/s41597-023-02158-3

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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. 3 — Isotopologue peptide internal reference standards to determine consistency of LC-MS/MS platform. Each of the CSF samples were spiked with a six-peptide, 5 isotopologue concentration LC-MS/MS Peptide Reference Mix from Promega (50 fmol/µL). (a) Extracted ion chromatogram for the 6 peptide (1pmol) mixture illustrating the wide range of retention times due to their hydrophobicity. (b) The raw peak areas in 423 injections over 5 days were used to determine the label-free CV for each peptide isotopologue estimating the lowest limits of detection to be between 1–10 fmole for each peptide. (c) The 5 unique isotopologues are used to assess the dynamic range across the gradient profile and each peptide demonstrates linearity across 3–4 orders of magnitude in the batch of 423 injections. Error bars represent the standard deviation across 423 injections.