Fig. 4.

Illustration of the eVLP quantitation method workflow and calculations. a VLP digests are resuspended in 120 µL and four twofold serial dilutions performed. Each dilution is mixed 1:1 with a solution containing 200 fmol/µL of each of the four isotopically labeled AQUA peptides and run in triplicate using 2 µL injections. b Method used for calculating the GP₁ concentration in the rGP₁ standard at each dilution. Average XIC area counts from the 2+ and 3+ charge states are first summed for each AQUA (P_aq) and analyte (P_v) peptide. The values from the SEE and IRSEE are summed to provide the total counts for peptide ‘Set 1’ and the SVG and SVGR values are summed to provide total counts for peptide ‘Set 2’. The final quantitation is derived by comparing the relative response of the 200 fmol AQUA standard to the endogenous analyte response at 4 ppm and averaging the response between the 2 peptide sets. For absolute eVLP rGP₁ quantitation, only the values derived from peptide set 1 (SEE/IRSEE) were used