KTH Uhlen Lab - APO-project (Effect1)

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

  • Organism: Homo sapiens
  • Instrument: 6490 Triple Quadrupole LC/MS
  • SpikeIn: Yes
  • Keywords: human, apolipoprotein, omega-3, fenofibrate, hypertriglyceridemia
  • Lab head: Fredrik Edfors
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.
Experiment Description
72 patients between 40 and 75 years of age, with a body mass index of 25–40 kg/m2, a serum triglyceride level of 1.7 mM (150 mg/dl) or higher, and a liver proton density fat fraction (PDFF) measured by MRI > 5.5% were subjected to a 12-week, multicenter, randomized, placebo-controlled, double-blind, double-dummy, three-armed, parallel-group trial, called EFFECT I. For each patient, a plasma sample was collected before the study and at the end of the study. These samples were subjected to analysis by LC-SRM/MS. The samples were spiked with a master batch of SIS PrESTs (stable isotope-labeled standards), in levels that had been adjusted to represent a 1:1 ratio to the endogenous peptides of a plasma pool from healthy individuals, before tryptic digestion. The dataset includes the following files: 20190312_ce-opt_2019-03-22_17-52-43.sky.zip - Collision Energy optimization for all selected peptides. 20190221_extended-repeatability_2019-02-26_15-38-15.sky.zip - Extended repeatability over 13 months of storage. 20181106_prest_transitions_selection_2018-11-06_11-29-31.sky.zip - Selection of peptides and transitions from the evaluated QPrESTs corresponding to endogenous peptides. 20170913_screening_2018-11-02_11-34-24.sky.zip - Screening of QPrEST peptides. 20180130_repeatability_final_2018-06-20_11-28-05.sky.zip - Evaluation of 3 by 5 sample digestions. 20180420_effect1_final_2018-06-20_11-03-21.sky.zip - Complete assay with case/control plasma samples spiked with QPrEST standards. 20190523_LOQ_2019-07-15_16-46-47.sky.zip - Standard curves for determining LLOQ and LOD for each SIS PrEST. 20190708_apoa1_2019-09-02_14-48-45.sky.zip - Results from benchmarking of SIS PrESTs to SIL protein and SIS peptides.
Sample Description
Two microliters human plasma was spiked into a pooled SIS PrEST mixture that was mixed with 12 µL 9 M Urea (Sigma-Aldrich), 300 mM Tris buffer, pH 8.0, 15 mM TCEP and incubated for 30 min at 37 °C. Subsequently, 12 µL of 75 mM IAA dissolved in water was added to the mixture and the solution was incubated for 30 min, shielded from light. The solution was subsequently diluted by adding 165 μL of 100 mM Tris (pH 8.0) in water. Trypsin (Sigma-Aldrich; St Louis, MO; USA) was dissolved in 100 mM Tris buffer (pH 8.0) at the concentration of 0.250 µg/µL and 22 µL was added, resulting in an enzyme to substrate ratio of approximately 1:15. The digestion was performed at 37 °C for 17 hours and was stopped by quenching with 5 µL of concentrated FA. The solid phase extraction (SPE) was performed using the Bravo AssayMAP liquid handler robot (Agilent Technologies; Santa Clara, CA, USA) utilizing reversed-phase cartridges (RPS, #65496-60033, Agilent Technologies; Santa Clara, CA, USA). The cartridges were activated with 100 µL of 0.1% trifluoroacetic acid (TFA, Sigma Aldrich; St Louis, MO, USA) in 50% ACN using a flow rate of 300 µL/min. The cartridges were equilibrated with 50 µL 0.1% TFA in water using a flow rate of 10 µL/min. 100 µg of tryptic peptides were loaded onto the cartridge using a flow rate of 10 µL/min. The cartridge was then washed with 50 µL of 0.1% TFA in water using a flow rate of 10 µL/min. The syringe was washed with 50 µL 0.1% TFA in 50% ACN using a flow rate of 300 µL/min and subsequently, the samples were eluted in 10 µL of 0.1% TFA in 70% ACN using a flow rate of 5 µL/min. The peptides were eluted into a collection plate (Eppendorf PCR plate, Cat no. 0030129300) where the wells contained 90 µL of 0.1% FA in water, giving a ten times dilution of the eluate and a total peptide concentration of 1 μg/μl.
Created on 10/8/19, 10:56 AM
Clustergrammer Heatmap
Flag FileDownloadCreatedProteinsPeptidesPrecursorsTransitionsReplicates
20180420_effect1_final_2018-06-20_11-03-21.sky.zip2019-10-08173467180171
20180130_repeatability_final_2018-06-20_11-28-05.sky.zip2019-10-0817356918945
20190221_extended_repeatability_2019-02-26_15-38-15.sky.zip2019-10-0817356926749
20170913_screening_2018-11-02_11-21-25.sky.zip2019-10-0818434317519
20190312_ce-opt_2019-03-22_17-52-43.sky.zip2019-10-081945451752
20181106_prest_transitions_selection_2018-11-07_11-01-36.sky.zip2019-10-0829931241,1880
20190523_LOQ_2019-07-15_17-06-32.sky.zip2019-10-08143059161138
20190708_apoa1_2019-09-02_14-48-45.sky.zip2019-10-0817144245

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