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

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.