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

• Published in: Journal of proteome research Vol. 16; no. 9; pp. 3242 - 3254
• Main Authors: Rezeli, Melinda, Sjödin, Karin, Lindberg, Henrik, Gidlöf, Olof, Lindahl, Bertil, Jernberg, Tomas, Spaak, Jonas, Erlinge, David, Marko-Varga, György
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.09.2017
• Subjects: Aged; Amino Acid Sequence; Apolipoprotein A-I - blood; Apolipoprotein B-100 - blood; Apolipoproteins E - blood; Basic Medicine; Biological Specimen Banks; Blood Proteins - analysis; Cell and Molecular Biology; Cell- och molekylärbiologi; Chromatography, Liquid - methods; Diagnosis, Differential; Female; human plasma; Humans; isotope labeled standards; Isotope Labeling - methods; large-scale analysis; Male; Mass Spectrometry - methods; Medical and Health Sciences; Medicin och hälsovetenskap; Medicinska och farmaceutiska grundvetenskaper; Middle Aged; multiple reaction monitoring; Non-ST Elevated Myocardial Infarction - blood; Non-ST Elevated Myocardial Infarction - diagnosis; Non-ST Elevated Myocardial Infarction - physiopathology; Protein Isoforms - blood; protein quantitation; ST Elevation Myocardial Infarction - blood; ST Elevation Myocardial Infarction - diagnosis; ST Elevation Myocardial Infarction - physiopathology; isotope labeled standards; multiple reaction monitoring; protein quantitation; large-scale analysis; human plasma
• ISSN: 1535-3893; 1535-3907; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00235

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.