Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach

“…After deconvolution of the multicharged spectra to the zero-charged mass spectra using the intervals m / z : 1250–1650 for albumin and 925–1450 for myoglobin, the deconvoluted mass spectrum of Mb yielded one intense peak at 16951.16 Da (Figure E), while HSA presented several peaks corresponding to different isoforms between 66,000 and 67,500 Da (Figure C). These isoforms correspond to truncated, native, oxidated, cysteinylated, and glycated forms of albumin, , which will be then used to create calibration curves for their proper absolute quantification.…”

“…On this basis, we have attempted to develop an absolute quantification method utilizing the direct flow injection technique that we previously applied. 15 Initially, our strategy involved separating HSA from potential IS candidates in the deconvolution spectrum. For this purpose, we set four criteria for our IS candidates: proximity to HSA in terms of physiological properties, nonoverlapping deconvoluted mass spectra within the mass range of potential HSA isoforms (66,000−69,000 Da), good solubility in water, and a comparable response in MS. We evaluated various animal albumins, such as ovalbumin and horse, rat, and mouse serum albumin as well as other protein standards such as lysozyme and myoglobin.…”

“…15,31 These factors might arise from the presence of salt adducts, small ligands that are bound noncovalently to HSA, or post-translational modifications (PTM) 48 in general and more specifically those with closely matching masses such as acetylation (+42.0 Da), carbamylation (+43.0 Da), or trioxidation (+48.0 Da). 15 Part of the present study was dedicated to the validation of the method using three distinct plasma samples with different concentrations representing a control patient as well as various stages of liver pathologies. Albumin levels quantified using deconvoluted spectra and extracted ion chromatogram (XIC) approaches closely matched those obtained through immunoturbidimetry (Tables 2 and 3), which validated our method for the absolute quantification of albumin.…”

“…It is assumed that an IS can play its part in reducing the impact of those fluctuations provided that it is well separated from the analyte and well ionized in the same m / z range as the analyte. On this basis, we have attempted to develop an absolute quantification method utilizing the direct flow injection technique that we previously applied …”

“…However, a high risk of misidentification is possible when identifying isoforms based on their characteristic mass shifts after deconvolution of the charged mass spectra of HSA. Low-intensity peaks, poor peak resolution, and unsatisfactory mass accuracy associated with deconvolution are all factors that can contribute to deviations equivalent to 1–5 Da and therefore to identification errors. , These factors might arise from the presence of salt adducts, small ligands that are bound noncovalently to HSA, or post-translational modifications (PTM) in general and more specifically those with closely matching masses such as acetylation (+42.0 Da), carbamylation (+43.0 Da), or trioxidation (+48.0 Da) …”