Quantitative Performance of Internal Standard Platforms for Absolute Protein Quantification Using Multiple Reaction Monitoring-Mass Spectrometry

2015 ◽  
Vol 87 (8) ◽  
pp. 4429-4435 ◽  
Author(s):  
Kerry Bauer Scott ◽  
Illarion V. Turko ◽  
Karen W. Phinney
Keyword(s):  
Mass Spectrometry ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Protein Quantification ◽  
Reaction Monitoring ◽  
Quantitative Performance

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.

scholarly journals Clinical Assay for AFP-L3 by Using Multiple Reaction Monitoring–Mass Spectrometry for Diagnosing Hepatocellular Carcinoma

2018 ◽  
Vol 64 (8) ◽  
pp. 1230-1238 ◽  
Author(s):  
Hyunsoo Kim ◽  
Areum Sohn ◽  
Injoon Yeo ◽  
Su Jong Yu ◽  
Jung-Hwan Yoon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Hepatocellular Carcinoma ◽  
Multiple Reaction Monitoring ◽  
False Negative ◽  
Internal Standard ◽  
Analytical Sensitivity ◽  
Reaction Monitoring ◽  
Clinical Implementation ◽  
Clinical Value ◽  
Serum Samples

Abstract BACKGROUND Lens culinaris agglutinin-reactive fraction of α-fetoprotein (AFP-L3) is a serum biomarker for hepatocellular carcinoma (HCC). AFP-L3 is typically measured by liquid-phase binding assay (LiBA). However, LiBA does not always reflect AFP-L3 concentrations because of its low analytical sensitivity. Thus, we aimed to develop an analytically sensitive multiple reaction monitoring–mass spectrometry (MRM-MS) assay to quantify AFP-L3 in serum. METHODS The assay entailed the addition of a stable isotope-labeled internal standard protein analog, the enrichment of AFP using a monoclonal antibody, the fractionation of AFP-L3 using L. culinaris agglutinin lectin, deglycosylation, trypsin digestion, online desalting, and MRM-MS analysis. The performance of the MRM-MS assay was compared with that of LiBA in 400 human serum samples (100 chronic hepatitis, 100 liver cirrhosis, and 200 HCC). Integrated multinational guidelines were followed to validate the assay for clinical implementation. RESULTS The lower limit of quantification of the MRM-MS assay (0.051 ng/mL) for AFP-L3 was less than that of LiBA (0.300 ng/mL). Thus, AFP-L3, which was not observed by LiBA in HCC samples (n = 39), was detected by the MRM-MS assay, improving the clinical value of AFP-L3 as a biomarker by switching to a more analytical sensitive platform. The method was validated, meeting all the criteria in integrated multinational guidelines. CONCLUSIONS Because of the lower incidence of false-negative findings, the MRM-MS assay is more suitable than LiBA for early detection of HCC.

scholarly journals Development and Validation of an HPLC-MS/MS Method for Rapid Simultaneous Determination of Cefprozil Diastereomers in Human Plasma

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Guodong He ◽  
Liping Mai ◽  
Xipei Wang
Keyword(s):  
Mass Spectrometry ◽  
Antimicrobial Activity ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Reverse Phase ◽  
Development And Validation ◽  
Positive Ion ◽  
Sensitive Method

Background. Both cis- and trans-cefprozil have antimicrobial activity, but their potencies are quite different. It is therefore necessary to develop a sensitive method to simultaneously determine both isomers for pharmacokinetic and bioequivalence studies. Methods. An LC-MS/MS method, using stable isotope-labeled cefprozil as the internal standard, was developed and validated. The analytes were extracted from plasma by protein precipitation and separated on a reverse-phase C18 column using a gradient program consisting of 0.5% formic acid and acetonitrile within 4 min. The mass spectrometry acquisition was performed with multiple reaction monitoring in positive ion mode using the respective [M+H]+ ions, m/z 391.2→114.0 for cefprozil and 395.0→114.5 for cefprozil-D4. Results. The calibration curves were linear over the ranges of 0.025–15 μg/mL for cis-cefprozil and 0.014–1.67 μg/mL for trans-cefprozil. The accuracies for the cis and trans isomers of cefprozil were 93.1% and 103.0%, respectively. The intra- and interassay precisions for the QC samples of the isomers were < 14.3%. The intra- and interassay precisions at the LLOQ were < 16.5%. Conclusions. The method was sensitive and reproducible and was applied in a pilot pharmacokinetic study of healthy volunteers.

scholarly journals Simultaneous Quantification of Apolipoprotein A-I and Apolipoprotein B by Liquid-Chromatography–Multiple- Reaction–Monitoring Mass Spectrometry

2010 ◽  
Vol 56 (12) ◽  
pp. 1804-1813 ◽  
Author(s):  
Sean A Agger ◽  
Luke C Marney ◽  
Andrew N Hoofnagle
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Human Plasma ◽  
Apolipoprotein B ◽  
Multiple Reaction Monitoring ◽  
Internal Standard ◽  
Reaction Monitoring ◽  
Normal Flow ◽  
Apolipoprotein A ◽  
Deming Regression

BACKGROUND If liquid-chromatography–multiple-reaction–monitoring mass spectrometry (LC-MRM/MS) could be used in the large-scale preclinical verification of putative biomarkers, it would obviate the need for the development of expensive immunoassays. In addition, the translation of novel biomarkers to clinical use would be accelerated if the assays used in preclinical studies were the same as those used in the clinical laboratory. To validate this approach, we developed a multiplexed assay for the quantification of 2 clinically well-known biomarkers in human plasma, apolipoprotein A-I and apolipoprotein B (apoA-I and apoB). METHODS We used PeptideAtlas to identify candidate peptides. Human samples were denatured with urea or trifluoroethanol, reduced and alkylated, and digested with trypsin. We compared reversed-phase chromatographic separation of peptides with normal flow and microflow, and we normalized endogenous peptide peak areas to internal standard peptides. We evaluated different methods of calibration and compared the final method with a nephelometric immunoassay. RESULTS We developed a final method using trifluoroethanol denaturation, 21-h digestion, normal flow chromatography-electrospray ionization, and calibration with a single normal human plasma sample. For samples injected in duplicate, the method had intraassay CVs &lt;6% and interassay CVs &lt;12% for both proteins, and compared well with immunoassay (n = 47; Deming regression, LC-MRM/MS = 1.17 × immunoassay − 36.6; Sx|y = 10.3 for apoA-I and LC-MRM/MS = 1.21 × immunoassay + 7.0; Sx|y = 7.9 for apoB). CONCLUSIONS Multiplexed quantification of proteins in human plasma/serum by LC-MRM/MS is possible and compares well with clinically useful immunoassays. The potential application of single-point calibration to large clinical studies could simplify efforts to reduce day-to-day digestion variability.

Quantitative analysis of genetically modified soya using multiple reaction monitoring mass spectrometry with endogenous peptides as internal standards

2018 ◽  
Vol 25 (1) ◽  
pp. 50-57 ◽  
Author(s):  
Shobha Devi ◽  
Yi-Cheng Lin ◽  
Yen-Peng Ho
Keyword(s):  
Mass Spectrometry ◽  
Genetically Modified ◽  
Multiple Reaction Monitoring ◽  
Linear Regression Analysis ◽  
Exchange Chromatography ◽  
Protein Quantification ◽  
Reaction Monitoring ◽  
Label Free ◽  
Internal Standards ◽  
Phosphate Synthase

A simple label-free method was developed for the quantification of the herbicide-resistant gene-related protein 5-enolpyruvylshikimate-3-phosphate synthase using multiple reaction monitoring liquid chromatography–mass spectrometry. Sample pretreatment procedures including ion exchange chromatography and CaCl2 precipitation were used to purify the 5-enolpyruvylshikimate-3-phosphate synthase protein. Quantification of various percentages of genetically modified soya (0.5–100%) was performed by selecting suitable endogenous soybean peptides as internal standards. Results indicated that Gly P (QGDVFVVPR) and Lec P (LQLNK) are useful internal standards for the quantification of low and high percentages of genetically modified soya, respectively. Linear regression analysis of both calibration curves yielded good linearity with R2 of 0.99. This approach is a convenient and accurate quantification method for genetically modified soya at a level as low as 0.5% (less than the current EU threshold for labeling genetically modified soya).

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