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; Edfors, Fredrik; Ryaboshapkina, Maria; Malmqvist, Jonas; Rosengren, Louise; Percy, Andrew J.; Lind, Lars; Uhlén, Mathias; Oscarsson, Jan; Miliotis, Tasso

doi:10.1074/mcp.ra119.001765

Cited by 13 publications

(21 citation statements)

References 63 publications

(109 reference statements)

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“…This demonstrates that other material losses occurring during the sample preparation steps are significant. Thus, a correction factor is required in any case, even with a digestion time of 0.5 h. Although the peptide-based calibration approach is widely applied thanks to its ease of use and reduced cost [47][48][49][50][51][52], our results agree with a significant quantitative bias using this approach, as reported in other studies [53][54][55].…”

Section: Evaluation Of the Use Of A Correction Factor For The Quantification Of Pctsupporting

confidence: 87%

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

et al. 2021

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The quantification of low abundant proteins in complex matrices by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) remains challenging. A measurement procedure based on optimized antibody-free sample preparation and isotope dilution coupled to LC-MS/MS was developed to quantify procalcitonin (PCT) in human serum at sub-microgram per liter level. A combination of sodium deoxycholate-assisted protein precipitation with acetonitrile, solid-phase extraction, and trypsin digestion assisted with Tween-20 enhanced the method sensitivity. Linearity was established through peptide-based calibration curves in the serum matrix (0.092-5.222 μg/L of PCT) with a good linear fit (R2 ≥ 0.999). Quality control materials spiked with known amounts of protein-based standards were used to evaluate the method's accuracy. The bias ranged from −2.6 to +4.3%, and the intra-day and inter-day coefficients of variations (CVs) were below 2.2% for peptide-based quality controls. A well-characterized correction factor was determined and applied to compensate for digestion incompleteness and material loss before the internal standards spike. Results with metrological traceability to the SI units were established using standard peptide of well-characterized purity determined by peptide impurity corrected amino acid analysis. The validated method enables accurate quantification of PCT in human serum at a limit of quantification down to 0.245 μg/L (bias −1.9%, precision 9.1%). The method was successfully applied to serum samples obtained from patients with sepsis. Interestingly, the PCT concentration reported implementing the isotope dilution LC-MS/MS method was twofold lower than the concentration provided by an immunoassay.

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Section: Evaluation Of the Use Of A Correction Factor For The Quantification Of Pctsupporting

confidence: 87%

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

et al. 2021

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“…Addition of PrESTs show very robust with interday coefficient of variations around 5%. 88 The PrEST technology has been used to quantify more than 100 proteins in human plasma 89 with addition‐only protocols. This has introduced a new and more precise quantification rationale of proteins present in body fluids with good quantitative accuracy (<15%) 90 if compared with fully labeled protein standards.…”

Section: Technologies For Plasma Proteomicsmentioning

confidence: 99%

“…In contrast to spiked peptides, spiked SIS proteins or protein fragments generate multiple proteotypic peptides to be added before the trypsin cleavage, ensuring that uncleaved endogenous peptides will not affect the quantification, as long as the digestion efficiency of the protein standard is the same as that of the endogenous protein target. Addition of PrESTs show very robust with interday coefficient of variations around 5% 88 . The PrEST technology has been used to quantify more than 100 proteins in human plasma 89 with addition‐only protocols.…”

Section: Technologies For Plasma Proteomicsmentioning

confidence: 99%

Proteomics in thrombosis research

Edfors

Iglesias

Butler

et al. 2022

Research and Practice in Thrombosis and Haemostasis

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A State of the Art lecture titled “Proteomics in Thrombosis Research” was presented at the ISTH Congress in 2021. In clinical practice, there is a need for improved plasma biomarker‐based tools for diagnosis and risk prediction of venous thromboembolism (VTE). Analysis of blood, to identify plasma proteins with potential utility for such tools, could enable an individualized approach to treatment and prevention. Technological advances to study the plasma proteome on a large scale allows broad screening for the identification of novel plasma biomarkers, both by targeted and nontargeted proteomics methods. However, assay limitations need to be considered when interpreting results, with orthogonal validation required before conclusions are drawn. Here, we review and provide perspectives on the application of affinity‐ and mass spectrometry‐based methods for the identification and analysis of plasma protein biomarkers, with potential application in the field of VTE. We also provide a future perspective on discovery strategies and emerging technologies for targeted proteomics in thrombosis research. Finally, we summarize relevant new data on this topic, presented during the 2021 ISTH Congress.

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“…A previously established LC-SRM/MS [16] analysis was done using an Ultimate 3000 equipped with a trap cartridge (catalog no. 160438, Thermo Fisher Scientific, MA, USA) and a 15-cm-long analytical EASY-Spray column (catalog no.…”

Section: Quantification Of Sis-prests Using Lc-srmmentioning

confidence: 99%

Targeted Proteomics Analysis of Plasma Proteins Using Recombinant Protein Standards for Addition Only Workflows

et al. 2021

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Targeted proteomics is an attractive approach for the analysis of blood proteins. Here, we describe a novel analytical platform based on isotope-labeled recombinant protein standards stored in a chaotropic agent and subsequently dried down to allow storage at ambient temperature. This enables a straightforward protocol suitable for robotic workstations. Plasma samples to be analyzed are simply added to the dried pellet followed by enzymatic treatment and mass spectrometry analysis. Here, we show that this approach can be used to precisely (coefficient of variation <10%) determine the absolute concentrations in human plasma of hundred clinically relevant protein targets, spanning four orders of magnitude, using simultaneous analysis of 292 peptides. The use of this next-generation analytical platform for high-throughput clinical proteome profiling is discussed.

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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

Cited by 13 publications

References 63 publications

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

Development of an antibody-free ID-LC MS method for the quantification of procalcitonin in human serum at sub-microgram per liter level using a peptide-based calibration

Proteomics in thrombosis research

Targeted Proteomics Analysis of Plasma Proteins Using Recombinant Protein Standards for Addition Only Workflows

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