Dataset Information

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

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 upon 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 utilized to investigate the effects of omega-3 carboxylic acids (OM3-CA) andfenofibrate onthese 13 apolipoproteins in human plasma samples from a randomized placebo-controlled trial, EFFECTI (NCT02354976). No significant changes were observed in the OM3-CA arm, while 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.

ORGANISM(S): Homo Sapiens

SUBMITTER: Andreas Hober

PROVIDER: PXD013314 | panorama | Thu Oct 10 00:00:00 BST 2019

REPOSITORIES: PanoramaPublic

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Publications

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.

Hober Andreas A Edfors Fredrik F Ryaboshapkina Maria M Malmqvist Jonas J Rosengren Louise L Percy Andrew J AJ Lind Lars L Forsström Björn B Uhlén Mathias M Oscarsson Jan J Miliotis Tasso T

Molecular & cellular proteomics : MCP 20191007 12

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 ...[more]

PMID: 31591263

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Project description:Targeted proteomics has become the method of choice for biomarker validation in human biopsies due to its capacity to measure a set of peptides in multiple samples with high sensitivity, reproducibility, accuracy and precision. However, for targeted proteomics technologies to be transferred to clinical routine there is the need to reduce its complexity and make its procedures simpler, increase its throughput and improve its analytical performance. Biomarker peptide quantification with high accuracy and precision is one of the steps that still remains a challenge in clinical routine, mainly due to the difficulty to account for matrix effects (i.e. signal variation due to biological patient variability) and to establish a valid range of quantification for each analyte in each individual sample. Research-grade proteomics laboratories perform targeted peptide quantification with stable isotopically-labelled peptides (one per analyte), which are added to the samples and used as internal standards. This strategy, known as single-point calibration, enables to confidently assign the endogenous peptide and infer its quantity by direct comparison to the internal standard. This approach however assumes that both the endogenous and the internal standard peptides are within the linear range of quantification, which is not always granted as the linear dependency between peptide areas and concentrations only occurs in a limited sample-dependent range of concentrations. Although standard peptide abundances are adjusted to endogenous levels in low-throughput projects, this is not feasible when dealing with large cohorts of patients in clinical routine that exhibit a high variability of peptide abundances and different matrix effects. External multi-point standard curves in their different forms (calibrated, reverse) have been used to alleviate part of these limitations, but the requirement of a representative blank matrix and the fact that they do not account for matrix effects in individual samples nor establish a valid range of quantification for each individual sample limit their application. Here we present the Isotopologue Multiple-point Calibration (ImCal) quantification strategy, which uses a mix of isotopologue peptides to generate internal multiple-point calibration curves for each individual sample and to accurately quantify biomarker peptides in clinical applications without the need of expert supervision. ImCal relies on the use of five different isotopically-labelled peptides of different nominal mass―ranging from 10 to 39 Da mass shifts—mixed at different concentrations to be used as internal calibration curve for each endogenous peptide of interest

Project description:Targeted mass spectrometry-based assays are proven to offer sensitive, selective quantification of proteins in a variety of translational applications, including cell lines, plasma/serum, dried blood spots and tissue biopsies. Recent efforts have successfully transferred targeted mass spectrometry methodologies, specifically multiplexed multiple reaction monitoring (MRM)-based assays, into the clinical environment. Yet, if MRM-based assays are to be utilized in the clinical space, quality control (QC) metrics are needed to control specifically for protease digestion, the sample processing step with the highest variability, thus ensuring optimal, reliable, and reproducible protein quantification. In this study, we evaluate the utility of monitoring extended stable isotope labeled internal standards (SIS) that are digested in situ within native protein lysates to survey the extent of sample protease digestion. Over 500 extended SIS peptides corresponding to targets of two separate multiplexed MRM assays, a direct-MRM and an immuno-MRM assay, were spiked into cell lysates which then underwent a series of systematically designed robustness experiments that stressed efficient protease digestion, including a trypsin digestion time-course as well as different protease digestion stressors. By simultaneously measuring levels of the extended and tryptic forms of the SIS peptide, the peak area ratio of extended SIS (hE) to tryptic SIS (hT), or hE/hT, was shown to effectively monitor perturbations in digestion performance. From time-course experiments, observed rates of extended SIS peptide digestion were calculated and hE/hT ratios uncovered an optimum time needed for protease digestion. When the endogenous peptides were quantified in these stressor and time-course experiments, this hE/hT QC metric reflected the sensitivity and fidelity of the MRM assays. This study demonstrates the effectiveness of monitoring extended SIS peptides within targeted, multiplexed MRM assays as a QC metric for protease digestion to ensure reliable, robust quantification of the endogenous protein targets.

Dataset Information

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

Publications

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.

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