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Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM Assay
KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.ORCID iD: 0000-0001-8947-2562
KTH Royal Inst Technol, Sci Life Lab, Stockholm, Sweden;KTH Royal Inst Technol, Dept Prot Sci, Stockholm, Sweden.
AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.ORCID iD: 0000-0003-1385-8869
AstraZeneca, IMED Biotech Unit, Translat Sci Cardiovasc Renal & Metab, Gothenburg, Sweden.
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2019 (English)In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 12, p. 2433-2446Article in journal (Refereed) Published
Abstract [en]

Stable isotope-labeled standard (SIS) peptides are used as internal standards in targeted proteomics to provide robust protein quantification, which is required in clinical settings. However, SIS peptides are typically added post trypsin digestion and, as the digestion efficiency can vary significantly between peptides within a protein, the accuracy and precision of the assay may be compromised. These drawbacks can be remedied by a new class of internal standards introduced by the Human Protein Atlas project, which are based on SIS recombinant protein fragments called SIS PrESTs. SIS PrESTs are added initially to the sample and SIS peptides are released on trypsin digestion. The SIS PrEST technology is promising for absolute quantification of protein biomarkers but has not previously been evaluated in a clinical setting. An automated and scalable solid phase extraction workflow for desalting and enrichment of plasma digests was established enabling simultaneous preparation of up to 96 samples. Robust high-precision quantification of 13 apolipoproteins was achieved using a novel multiplex SIS PrEST-based LC-SRM/MS Tier 2 assay in non-depleted human plasma. The assay exhibited inter-day coefficients of variation between 1.5% and 14.5% (median = 3.5%) and was subsequently used to investigate the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on these 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECT I (NCT02354976). No significant changes were observed in the OM3-CA arm, whereas treatment with fenofibrate significantly increased apoAII and reduced apoB, apoCI, apoE and apoCIV levels. The reduction in apoCIV following fenofibrate treatment is a novel finding. The study demonstrates that SIS PrESTs can facilitate the generation of robust multiplexed biomarker Tier 2 assays for absolute quantification of proteins in clinical studies. Applications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.

Place, publisher, year, edition, pages
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC , 2019. Vol. 18, no 12, p. 2433-2446
Keywords [en]
Clinical trials, assay development, targeted mass spectrometry, selected reaction monitoring, absolute quantification, apolipoproteins, fenofibrate and omega-3 carboxylic acids, NAFLD, SIS PrEST
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-400666DOI: 10.1074/mcp.RA119.001765ISI: 000501288700007PubMedID: 31591263OAI: oai:DiVA.org:uu-400666DiVA, id: diva2:1382426
Available from: 2020-01-03 Created: 2020-01-03 Last updated: 2020-01-03Bibliographically approved

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