Supplementary Data: A multiplex protein panel assay for severity prediction and outcome prognosis in patients with COVID-19: An observational multi-cohort study
Plasma of two observational cohorts of inpatients with SARS-COV2 were measured using a targeted liquid chromatography - multiple reaction monitoring mass spectrometry assay (LC-MRM). The assay captures peptides that are associated with COVID-19 severity, and that are prognostic about COVID-19 outcome. Provided are the the LC-MRM measurements for the respective cohorts, as well as custom code used to predict COVID-19 outcome and severity. Details, and corresponding meta data linking to the measurement data are available in the original publication.
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The full description on sample preparation, measurements, as well as data processing are described in the original publication. Sample preparation: Samples from Cohort 2 as well as calibration lines were prepared as follows: 5 µl of citrate plasma were added to 55 µl of denaturation buffer, composed of 50 µl 8 M Urea, 100 mM ammonium bicarbonate, 5 µl 50 mM dithiothreitol (DTT) and internal standard mix. The samples were incubated for 1 h at room temperature (RT) before addition of 5 µl of 100 mM iodoacetamide (IAA). After a 30 min incubation at RT the samples were diluted with 340 µl of 100 mM ammonium bicarbonate and digested overnight with 22.5 µl of 0.1 µg/µl trypsin at 37 ℃. The digestion was quenched by adding 50 µl of 10% v/v formic acid. The resulting tryptic peptides were purified on a 96-well C18-based solid phase extraction (SPE) plate (BioPureSPE Macro 96-well, 100mg PROTO C18, The Nest Group). The purified samples were resuspended in 120 µl of 0.1% formic acid and 20 µl were injected. Samples in Cohort 3 were prepared as described above, with modifications: EDTA plasma was used instead of citrate plasma, and internal standards were digested separately and added to pre-digested clinical and calibration line samples before their injection into the LC-MS/MS system. LC-MRM measurements Samples were analysed on the Agilent 6495C mass spectrometer, coupled to an Agilent 1290 Infinity II UHPLC system. Prior to MS analysis, samples were chromatographically separated on an Agilent InfinityLab Poroshell 120 EC-C18 1.9 µm, 2.1x50 mm column heated to 45 °C and with a flow rate of 800 µl/min. Linear gradients employed were as follows (time, % of mobile phase B): 0 min, 3%; 1 min, 3%; 7.5 min, 35%; 8 min 98%; 8.5 min, 98%; 8.6 min, 3%; 10 min, 3% where mobile phase A & B are 0.1 % formic acid in water and 0.1 % formic acid in acetonitrile respectively. The 6495C mass spectrometer was controlled by Agilent’s MassHunter Workstation software (LC-MS/MS Data Acquisition for 6400 series Triple Quadrupole, Version 10.1) and was operated in positive electrospray ionisation mode with the following parameters: 3500 V capillary voltage (positive), 0 V nozzle voltage (positive), 12 L/min sheath gas flow at a temperature of 280 °C, 17 L/min gas flow at a temperature of 170 °C, 40 psi nebulizer pressure, 166 V fragmentor voltage, 5 V cell accelerator potential. Samples were analysed in dynamic MRM mode with both quadrupoles operated in unit resolution. All other parameters are provided in the original publication. Processing LC-MRM data was processed using MassHunter Quantitative Analysis, v10.1. Peptide concentration (ng/ml) was determined from calibration curves, constructed with native and SIL peptide standards. Linear regression analysis of each calibration curve was performed in R (with 1/x weighting). Matching of native peptides and internal standards is detailed in the Supplementary Methods of the original publication.