Abstract
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.
Highlights
The first application of stable isotope-labeled recombinant protein fragments internal standards (SIS PrEST) on clinical samples
We established a robust Stable isotope-labeled standard (SIS) PrEST-based SRM assay for absolute quantification of apolipoproteins
We applied this assay to assess the effects of omega-3 carboxylic acids (OM3-CA) and fenofibrate on apolipoprotein levels in a clinical cohort [44]
Summary
The first application of stable isotope-labeled recombinant protein fragments internal standards (SIS PrEST) on clinical samples. Author’s Choice los 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*□S. The SIS PrEST methodology demonstrated better accuracy than WiSIL and SIS peptides compared with gold standard with known concentration of isotopically labeled alpha-synuclein, which indicated that SIS PrESTs better account for differences arising during sample preparation (including the digestion efficiency) Their data revealed that SIS PrESTs could be accurate alternatives for SIS proteins because recombinant SIS PrESTs may mimic the structural features of proteins and better account for the actual digestion conditions present in a sample. We subsequently analyzed the 13 apolipoproteins in nondepleted plasma samples from a randomized placebo-controlled, parallel-arm study comparing the effects of 12 weeks of daily treatment with 4 g free omega-3 carboxylic acids (OM-3CA) and 200 mg fenofibrate in hypertriglyceridemic patients with non-alcoholic fatty liver disease (NAFLD) [44], a patient population with high cardiovascular risk [45]
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