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. 2021 Feb 4;24(3):102135. doi: 10.1016/j.isci.2021.102135

Figure 3.

Figure 3

MRM validation of the host proteins upregulated in COVID-19-positive samples

The MRM analysis was performed on 6 COVID-negative and 16 COVID-positive patient swab samples. 1 μg peptide from each sample was injected into a Vanquish HPLC coupled to a TSQ Altis™ triple quadrupole and run against a transition list of peptides belonging to important clinical markers. The list of transitions was prepared for unique peptides of these selected proteins using Skyline (Version 20.2.1.286). This list included a spiked-in synthetic heavy peptide (THCLYTHVCDAIK) used for monitoring the consistency of the mass spectrometry runs. For identification of the sensitivity of the peptide detection, we performed serial dilution of two crude synthetic peptides (heavy and light). The concentration of peptide was calculated using the Scopes method from its O.D. value at 205 nm and 280 nm. Different concentrations of the peptides starting from 25 to 125 ng were run in TSQ Altis™ Triple Quadrupole Mass Spectrometer.

(A) Peak shapes of representative peptides for clinical marker proteins validated using MRM. The statistically significant change in the expression of the proteins was observed between COVID-19-positive and COVID-19-negative patient samples (T-test, ∗∗p < 0.05; Fold change > 1.5 at a confidence interval of 99% - determined by Skyline).

(B) Box plots of representative peptides for clinical marker proteins validated using MRM.

(C) Standard curve of heavy synthetic peptide HSGFEDELSEVLENQSSQAELK. The crude heavy synthetic peptide was diluted in the range of 25 to 125 ng concentration. The standard curve for this peptide was plotted using the peak area against the concentration of the peptide. The intensity of the peak area was proportional to the amount of the synthetic peptide. The lowest amount of synthetic peptide detected was at 30.9 ng.

(D) Standard curve of light synthetic peptide HSGFEDELSEVLENQSSQAELK. The crude light synthetic peptide was diluted in the range of 25 to 125 ng concentration. The standard curve for this peptide was plotted using the peak area against the concentration of the peptide. The intensity of the peak area was proportional to the amount of the synthetic peptide. The lowest amount of synthetic peptide detected was at 19.8 ng.