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

Abstract

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 ...

A time-resolved proteomic and prognostic map of COVID-19

“…Released abundantly by activated platelets leading to pro-inflammatory M1-like macrophage polarization ( Hilt et al, 2019 ). Increase of B2M has been associated with death in patients with chronic kidney disease ( Makridakis et al., 2020 ). C1R: Complement C1r : Initiator of the classical complement pathway ( Hajishengallis et al, 2017 ).…”

“…The proteins increased in patients receiving RRT mainly reflect impaired kidney function and have been associated with RRT before (AMBP, B2M, CST3, LYZ, RBP4, Figure S7 ) ( Shao et al., 2015 ). Of note, increased levels of AMBP, B2M and LYZ have been associated with death in chronic kidney disease ( Makridakis et al., 2020 ). Levels of CFD and APOH, both involved in the complement system, were also increased ( McDonnell et al., 2020 ; Volanakis and Narayana, 1996 ).…”

A time-resolved proteomic and diagnostic map characterizes COVID-19 disease progression and predicts outcome

“…SF6) 75 . Of note, increased levels of AMBP, B2M and LYZ have been associated with death in chronic kidney disease 39 . Levels of CFD and APOH, both involved in the complement system, were also increased 41,76 .…”

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“…Moreover, an aptamer-based proteomic analysis (SOMAscan) has enabled multiplexed, highly-sensitive, and specific protein detection in human blood and other biomatrices, and applied to profile EVs ( Welton et al, 2017 ). Other high-throughput MS approaches, such as multiple/parallel reaction monitoring MS (MRM/PRM-MS) enable rapid and accurate identification and quantification of protein biomarkers in broad dynamic range ( Anwar et al, 2019 ; Makridakis et al, 2020 ). Indeed, MRM-MS has been used for identification of Mycobacterium tuberculosis peptides in patient derived exosomes ( Kruh-Garcia et al, 2014 ), highlighting the sensitivity and specificity of this approach in EVs.…”