Semi-synthetic human albumin isoforms: Production, structure, binding capacities and influence on a routine laboratory test

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.…”

“…Sample Preparation. Plasma samples and HSAc five-point calibration curves (10,20,30,40, and 50 g/L) were prepared with water (0.9% NaCl). Myoglobin (4 g/L) was prepared with Milli-Q-water.…”

“…This solution was prepared according to a protocol developed previously as follows: HSAc (100 g/L) was reduced by incubating it with DL-dithiothreitol (DTT) (concentration ratio DTT/albumin = 1/10), a reducing agent, for 5 min at 37 °C. 20 The second calibration curve (10−50 g/L) was prepared by using the HSAc standard solution (HSAc; 200 g/L). Samples used to create the calibration curves, and the actual calibration curve points prepared by simple dilution from the initially concentrated solutions were determined by immunoturbidimetry, a widely applied quantification method in clinical settings for protein quantification, for more accuracy.…”

“…Conventional antigen–antibody-based methods, such as immunoturbidimetry, can directly quantify a protein by measuring the light scattered by the immune complexes formed but are unable to distinguish isoforms. , Proteomic approaches based on mass spectrometry were investigated as a possible solution to this limitation. Bottom-up strategies, a peptide-centric proteomic approach, offer deep proteome coverage and reliable quantification of isoforms, but it is unsuitable for clinical settings due to time-consuming sample preparation procedures, data processing, and loss of information related to the 3D structure caused by proteolytic digestion. ,,, Since the introduction of nebulization–ionization technologies in top-down strategies, liquid chromatography–high-resolution mass spectrometry (LC-HR-MS) techniques gained popularity for native-like folded-state analyses of molecules, ,,− specifically for isoform characterization. , In this way, we recently reported the detection and quantification of several HSA isoforms with minimal sample preparation and relatively short acquisition time. , …”

“…17,18 Conventional antigen−antibody-based methods, such as immunoturbidimetry, can directly quantify a protein by measuring the light scattered by the immune complexes formed but are unable to distinguish isoforms. 19,20 Proteomic approaches based on mass spectrometry were investigated as a possible solution to this limitation. Bottom-up strategies, a peptide-centric proteomic approach, offer deep proteome coverage and reliable quantification of isoforms, but it is unsuitable for clinical settings due to time-consuming sample preparation procedures, data processing, and loss of information related to the 3D structure caused by proteolytic digestion.…”