Quantitation of 87 Proteins by nLC-MRM/MS in Human Plasma: Workflow for Large-Scale Analysis of Biobank Samples

Abstract

A multiple reaction monitoring (MRM) assay was developed for precise quantitation of 87 plasma proteins including the three isoforms of apolipoprotein E (APOE) associated with cardiovascular diseases using nanoscale liquid chromatography separation and stable isotope dilution strategy. The analytical performance of the assay was evaluated and we found an average technical variation of 4.7% in 4-5 orders of magnitude dynamic range (≈0.2 mg/L to 4.5 g/L) from whole plasma digest. Here, we report a complete workflow, including sample processing adapted to 96-well plate format and normalization strategy for large-scale studies. To further investigate the MS-based quantitation the amount of six selected proteins was measured by routinely used clinical chemistry assays as well and the two methods showed excellent correlation with high significance (p-value < 10e-5) for the six proteins, in addition for the cardiovascular predictor factor, APOB: APOA1 ratio (r = 0.969, p-value < 10e-5). Moreover, we utilized the developed assay for screening of biobank samples from patients with myocardial infarction and performed the comparative analysis of patient groups with STEMI (ST- segment elevation myocardial infarction), NSTEMI (non ST- segment elevation myocardial infarction) and type-2 AMI (type-2 myocardial infarction) patients.

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“…Plasma samples for the MRM analysis were prepared and analysed as previously described [11]. Briefly, 10 μL of 10 times diluted plasma from each individual was digested with sequencing grade trypsin for 17–18 h following reduction and alkylation in a 96‐well plate format.…”

“…Two different biomarker analysing methods were utilized: the commercially available Proximity Extension Assay (PEA) technique (Olink Proseek â Multiplex CVD I 96996 kit, OLink Bioscience, Uppsala, Sweden) followed by real-time polymerase chain reaction (PCR) as previously reported [9,10] and the newly developed multiple reaction monitoring (MRM) mass spectrometry assay panel as previously described [11,12]. The MRM assay was developed for quantification of 87 plasma proteins (Table S1) that are putative cardiovascular disease markers, including the three isoforms of apolipoprotein E and markers of fibrinolysis and inflammation [12].…”

“…The PEA chip assay required <10 lL of sample to measure the biomarkers that were the measured simultaneously by the binding of paired antibodies to target proteins and followed by real-time PCR quantification using the Fluidigm BioMark TM System. Plasma samples for the MRM analysis were prepared and analysed as previously described [11]. Briefly, 10 lL of 10 times diluted plasma from each individual was digested with sequencing grade trypsin for 17-18 h following reduction and alkylation in a 96-well plate format.…”

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“…The multiple reaction monitoring assay (MRM) is an analytical method that can determine and quantify a large number of proteins in a single sample by utilizing mass spectrometry. The technique was developed at Lund University by Marko-Varga and colleagues and has been previously described in detail 131,132 . Briefly, several steps are involved in the workflow of the spectrometer.…”

“…The nano spray Flex Ion source and EASY n-LC II pump were used. In study IV, 87 different proteins (Figure 8) that are putative cardiovascular disease markers, including the three isoforms of apolipoprotein E and markers of fibrinolysis and inflammation 131,132 were quantified. The protein targets from the MRM assay were quantified using concentration-balanced stable isotope standards (SIS).…”