Development and Validation of Multiple Reaction Monitoring (MRM) Assays for Clinical Applications

Kontostathi, Georgia; Makridakis, Manousos; Bitsika, Vasiliki; Tsolakos, Nikolaos; Vlahou, Antonia; Zoidakis, Jérôme

doi:10.1007/978-1-4939-9164-8_14

Cited by 17 publications

(13 citation statements)

References 33 publications

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“…Equal volume (2 μL) of plasma samples containing approximately 100 μg of total protein were used for LC-MRM-MS analysis as previously described 89,90 . Briefly, after protein denaturation (8 M urea), reduction (10 mM dithioerythritol) and alkylation (50 mM iodoacetamide) the samples were digested with trypsin [(1:100 enzyme: protein ratio (w/w)] for 16 hours in the dark (RT).…”

Section: Sample Preparation and Liquid Chromatography-multiple Reactimentioning

confidence: 99%

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

Makridakis

Kontostathi

Petra

et al. 2020

Sci Rep

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Current diagnostic measures for Chronic Kidney Disease (CKD) include detection of reduced estimated glomerular filtration rate (eGFR) and albuminuria, which have suboptimal accuracies in predicting disease progression. The disease complexity and heterogeneity underscore the need for multiplex quantification of different markers. The goal of this study was to determine the association of six previously reported CKD-associated plasma proteins [B2M (Beta-2-microglobulin), SERPINF1 (Pigment epithelium-derived factor), AMBP (Protein AMBP), LYZ (Lysozyme C), HBB (Hemoglobin subunit beta) and IGHA1 (Immunoglobulin heavy constant alpha 1)], as measured in a multiplex format, with kidney function, and outcome. Antibody-free, multiple reaction monitoring mass spectrometry (MRM) assays were developed, characterized for their analytical performance, and used for the analysis of 72 plasma samples from a patient cohort with longitudinal follow-up. The MRM significantly correlated (Rho = 0.5-0.9) with results from respective ELISA. Five proteins [AMBP, B2M, LYZ, HBB and SERPINF1] were significantly associated with eGFR, with the three former also associated with unfavorable outcome. The combination of these markers provided stronger associations with outcome (p < 0.0001) compared to individual markers. Collectively, our study describes a multiplex assay for absolute quantification and verification analysis of previously described putative CKD prognostic markers, laying the groundwork for further use in prospective validation studies.Chronic kidney disease (CKD), defined as reduced kidney function and/or evidence of kidney damage, is a major public health problem throughout the world. Major health problems around the globe, with consistent prevalence rates of 10-13% have been reported (depending on reference group and stage) 1,2 . Disease management is characterized by excessive financial costs (with expenses associated with CKD treatment, exceeding 100 B € in total, annually in Europe) 2,3 . Common risk factors for CKD include ageing of the population and increased rates of diabetes and hypertension 2,4 . Of note, patients with CKD have an overall 30-fold increased risk for suffering from Cardio Vascular Disease (CVD) complications, this being the main cause of CKD-associated deaths. Specifically, ~45% of patients with CKD stage 4-5 die from CVD 5 and risk of CVD increases with CKD severity, which is already significantly higher in early CKD compared to non-CKD 6,7 . Early identification of CKD and addressing modifiable risk factors is recommended, as it can reduce the risk of kidney failure and CVD by up to 50% 8 . Early detection or prediction of complications enable early intervention, thus could increase the chances for higher treatment efficacy 9-11 . CKD diagnosis is currently based on the detection of reduced estimated glomerular filtration rate (eGFR) and/or albuminuria, as indicators of renal dysfunction 12,13 . However, these markers have substantial suitable number of k-nearest neighbors was conducted iteratively as ...

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Section: Sample Preparation and Liquid Chromatography-multiple Reactimentioning

confidence: 99%

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

Makridakis

Kontostathi

Petra

et al. 2020

Sci Rep

Self Cite

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“…The drugs of interest were determined by monitoring the abundance of a single precursor/product ion transition against a precursor/product ion transition for an internal standard. This represents the conventional method for quantitation of drugs of abuse, multiple reaction monitoring (MRM,•⇒•) in which two or more transitions are monitored . A few precursor/product ion transitions can be used to confirm the detected analyte while a single transition is used as quantifier.…”

Section: Resultsmentioning

confidence: 99%

“…This represents the conventional method for quantitation of drugs of abuse, multiple reaction monitoring (MRM,•⇒•) in which two or more transitions are monitored. 30 A few precursor/product ion transitions can be used to confirm the detected analyte while a single transition is used as quantifier. This "scan" is rarely used in instruments other than triple quadrupoles; however, pseudo-MRM methods have been implemented in other instruments where the single individual transitions of interest can be extracted from a product ion scan.…”

Section: ■ Resultsmentioning

confidence: 99%

2D MS/MS Spectra Recorded in the Time Domain Using Repetitive Frequency Sweeps in Linear Quadrupole Ion Traps

et al. 2020

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Ion trap mass spectrometers have emerged as powerful on-site analytical platforms, in spite of limited mass resolution, due to their compatibility with ambient ionization methods and ready implementation of tandem mass spectrometry (MS/MS). When operated at constant trapping voltage, ions can be activated at their secular frequencies and all MS/MS experiments can be performed, including the two-dimensional tandem mass scan (2D MS/MS scan) in which all precursor ions and their subsequent product ions are both identified and correlated. In the new method of performing this 2D MS/MS experiment presented here, the precursor ions are excited by a nonlinear (inverse Mathieu q) frequency sweep while the resulting product ions are identified by their ejection time within a repeating orthogonally applied nonlinear (inverse Mathieu q) frequency sweep. This resulting compact representation contains the total fragmentation behavior of a collection of ionized compounds and captures detailed chemical information efficiently (typically in 1 s). The approach is implemented using a simple single mass analyzer instrument. This methodology was tested on three different multicomponent mixtures: drugs of abuse, peptides, and fentanyl analogs. The data are compared with those obtained by more common MS/MS scan methods.

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“…Screening approaches are also required to distinguish low abundance peptide biomarker signal - typically presented as peak height or area - from peptide contaminants and MS noise that can produce false-positive signals. SNR and sample/IS peak area ratios from negative control sample(s) are often used to set minimum thresholds for positive signal 40 , 45 , 46 , but are not sufficient to reliably distinguish target peaks from noise at low target concentrations. Interference peaks with high SNRs can arise from contaminant peptides that have mass-to-charge ratios (m/z) similar to target peptides, but different amino acid sequences 43 , or from electronic noise detected during sample analysis.…”

Section: Discussionmentioning

confidence: 99%

Assay design for unambiguous identification and quantification of circulating pathogen-derived peptide biomarkers

Shu¹,

Liu²,

Alonzi³

et al. 2022

Theranostics

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Rationale: Circulating pathogen-derived proteins can serve as useful biomarkers for infections but may be detected with poor sensitivity and specificity by standard immunoassays due to masking effects and cross-reactivity. Mass spectrometry (MS)-read immunoassays for biomarker-derived peptides can resolve these issues, but lack standard workflows to select species-specific peptides with strong MS signal that are suitable for antibody generation. Methods: Using a Mycobacterium tuberculosis ( Mtb ) protein as an example, candidate peptides were selected by length, species-specificity, MS intensity, and antigenicity score. MS data from spiked healthy serum was employed to define MS feature thresholds, including a novel measure of internal MS data correlation, to produce a peak detection algorithm. Results: This algorithm performed better in rejecting false positive signal than each of its criteria, including those currently employed for this purpose. Analysis of an Mtb peptide biomarker (CFP-10pep) by this approach identified tuberculosis cases not detected by microbiologic assays, including extrapulmonary tuberculosis and tuberculosis cases in children infected with HIV-1. Circulating CFP-10pep levels measured in a non-human primate model of tuberculosis distinguished disease from asymptomatic infection and tended to correspond with Mtb granuloma size, suggesting that it could also serve as a surrogate marker for Mtb burden and possibly treatment response. Conclusions: These biomarker selection and analysis approach appears to have strong potential utility for infectious disease diagnosis, including cryptic infections, and possibly to monitor changes in Mtb burden that may reflect disease progression or a response to treatment, which are critical needs for more effective disease control.

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Development and Validation of Multiple Reaction Monitoring (MRM) Assays for Clinical Applications

Cited by 17 publications

References 33 publications

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

Multiplexed MRM-based protein quantification of putative prognostic biomarkers for chronic kidney disease progression in plasma

2D MS/MS Spectra Recorded in the Time Domain Using Repetitive Frequency Sweeps in Linear Quadrupole Ion Traps

Assay design for unambiguous identification and quantification of circulating pathogen-derived peptide biomarkers

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