scholarly journals Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

2021 ◽  
pp. 104393
Author(s):  
Keiko Masuda ◽  
Keiko Kasahara ◽  
Ryohei Narumi ◽  
Masaru Shimojo ◽  
Yoshihiro Shimizu
Keyword(s):  
Mass Spectrometry ◽  
Internal Standard ◽  
Absolute Quantification

scholarly journals Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides

2021 ◽  
Author(s):  
Keiko Masuda ◽  
Keiko Kasahara ◽  
Ryohei Narumi ◽  
Masaru Shimojo ◽  
Yoshihiro Shimizu
Keyword(s):  
Mass Spectrometry ◽  
Peptide Synthesis ◽  
Ribosomal Proteins ◽  
Ribosomal Subunit ◽  
Internal Standard ◽  
Absolute Quantification ◽  
Protein Quantification ◽  
One Pot ◽  
Free Products ◽  
Free Peptide

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis.

scholarly journals Analysis of Pembrolizumab in Human Plasma by LC-MS/HRMS. Method Validation and Comparison with Elisa

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 621
Author(s):  
Aurélien Millet ◽  
Nihel Khoudour ◽  
Jérôme Guitton ◽  
Dorothée Lebert ◽  
François Goldwasser ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Human Plasma ◽  
High Resolution Mass Spectrometry ◽  
Internal Standard ◽  
Therapeutic Drug ◽  
Limit Of Quantification ◽  
Immunoglobulin G4 ◽  
Positive Mode ◽  
Surrogate Peptide ◽  
First Time

Pembrolizumab is a humanized immunoglobulin G4-kappa anti-PD1 antibody used in the treatment of different solid tumors or haematological malignancies. A liquid chromatography coupled with a high resolution mass spectrometry (orbitrap technology) method was fully developed, optimized, and validated for quantitative analysis of pembrolizumab in human plasma. A mass spectrometry assay was used for the first time a full-length stable isotope-labelled pembrolizumab-like (Arginine 13C6-15N4 and Lysine 13C6-15N2) as an internal standard; the sample preparation was based on albumin depletion and trypsin digestion and, finally, one surrogate peptide was quantified in positive mode. The assay showed good linearity over the range of 1–100 μg/mL, a limit of quantification at 1 μg/mL, excellent accuracy from 4.4% to 5.1%, and also a between-day precision below 20% at the limit of quantification. In parallel, an in-house ELISA was developed with a linearity range from 2.5 to 50 µg/mL. Then, results were obtained from 70 plasma samples of cancer patients that were treated with pembrolizumab and quantified with both methods were compared using the Passing-Bablok regression analysis and Bland-Altman plotting. The LC-MS/HRMS method is easy to implement in the laboratory for use in the context of PK/PD studies, clinical trials, or therapeutic drug monitoring.

scholarly journals Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient

2020 ◽  
Vol 58 (9) ◽  
pp. 1461-1468 ◽  
Author(s):  
Jean-Claude Alvarez ◽  
Pierre Moine ◽  
Isabelle Etting ◽  
Djillali Annane ◽  
Islam Amine Larabi
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Half Life ◽  
Limit Of Detection ◽  
Treated Patient ◽  
Internal Standard ◽  
Therapeutic Drug ◽  
Active Metabolites ◽  
Limit Of Quantification ◽  
Positive Mode

AbstractObjectivesA method based on liquid chromatography coupled to triple quadrupole mass spectrometry detection using 50 µL of plasma was developed and fully validated for quantification of remdesivir and its active metabolites GS-441524.MethodsA simple protein precipitation was carried out using 75 µL of methanol containing the internal standard (IS) remdesivir-13C6 and 5 µL ZnSO4 1 M. After separation on Kinetex® 2.6 µm Polar C18 100A LC column (100 × 2.1 mm i.d.), both compounds were detected by a mass spectrometer with electrospray ionization in positive mode. The ion transitions used were m/z 603.3 → m/z 200.0 and m/z 229.0 for remdesivir, m/z 292.2 → m/z 173.1 and m/z 147.1 for GS-441524 and m/z 609.3 → m/z 206.0 for remdesivir-13C6.ResultsCalibration curves were linear in the 1–5000 μg/L range for remdesivir and 5–2500 for GS-441524, with limit of detection set at 0.5 and 2 μg/L and limit of quantification at 1 and 5 μg/L, respectively. Precisions evaluated at 2.5, 400 and 4000 μg/L for remdesivir and 12.5, 125, 2000 μg/L for GS-441524 were lower than 14.7% and accuracy was in the [89.6–110.2%] range. A slight matrix effect was observed, compensated by IS. Higher stability of remdesivir and metabolite was observed on NaF-plasma. After 200 mg IV single administration, remdesivir concentration decrease rapidly with a half-life less than 1 h while GS-441524 appeared rapidly and decreased slowly until H24 with a half-life around 12 h.ConclusionsThis method would be useful for therapeutic drug monitoring of these compounds in Covid-19 pandemic.

scholarly journals Calcineurin Activity Assay Measurement by Liquid Chromatography–Tandem Mass Spectrometry in the Multiple Reaction Monitoring Mode

2014 ◽  
Vol 60 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Lynn Carr ◽  
Anne-Laure Gagez ◽  
Marie Essig ◽  
François-Ludovic Sauvage ◽  
Pierre Marquet ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Mononuclear Cells ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Activity Assay ◽  
Peripheral Blood Mononuclear ◽  
Blood Concentrations ◽  
Transplant Patients

Abstract BACKGROUND Blood concentrations of the calcineurin inhibitors (CNIs) cyclosporine and tacrolimus are currently measured to monitor immunosuppression in transplant patients. The measurement of calcineurin (CN) phosphatase activity has been proposed as a complementary pharmacodynamic approach. However, determining CN activity with current methods is not practical. We developed a new method amenable to routine use. METHODS Using liquid chromatography–multiple reaction monitoring mass spectrometry (LC-MRM-MS), we quantified CN activity by measuring the dephosphorylation of a synthetic phosphopeptide substrate. A stable isotope analog of the product peptide served as internal standard, and a novel inhibitor cocktail minimized dephosphorylation by other major serine/threonine phosphatases. The assay was used to determine CN activity in peripheral blood mononuclear cells (PBMCs) isolated from 20 CNI-treated kidney transplant patients and 9 healthy volunteers. RESULTS Linearity was observed from 0.16 to 2.5 μmol/L of product peptide, with accuracy in the 15% tolerance range. Intraassay and interassay recoveries were 100.6 (9.6) and 100 (7.5), respectively. Michaelis–Menten kinetics for purified CN were Km = 10.7 (1.6) μmol/L, Vmax = 2.8 (0.3) μmol/min · mg, and for Jurkat lysate, Km = 182.2 (118.0) μmol/L, Vmax = 0.013 (0.006) μmol/min · mg. PBMC CN activity was successfully measured in a single tube with an inhibitor cocktail. CONCLUSIONS Because LC-MRM-MS is commonly used in routine clinical dosage of drugs, this CN activity assay could be applied, with parallel blood drug concentration monitoring, to a large panel of patients to reevaluate the validity of PBMC CN activity monitoring.

Absolute Quantification of E1, Ubiquitin-Like Proteins and Nedd8–MLN4924 Adduct by Mass Spectrometry

2013 ◽  
Vol 67 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Xiaofeng Yang ◽  
James E. Brownell ◽  
Qing Xu ◽  
Fengying Zhu ◽  
Jingya Ma ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Absolute Quantification
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