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.

Early detection of liver injuries by the Serum enhanced binding test sensitive to albumin post-transcriptional modifications.

Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.

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

Human Serum Albumin (HSA) undergoes Post-Translational-Modifications (PTMs) leading to isoforms affecting its oncotic and non-oncotic properties. HSA is comprised of several isoforms whose abundance may vary with pathologies such as diabetes, kidney and liver diseases. Studying their impact separately may help to understand their sources and potential pathogenicity and further their evaluation as biomarkers. The present study examined semi-synthetic HSA isoforms to investigate independently their structure by means of advanced mass spectrometry techniques (LC-TOF-MS and ICP-MS), influence on the HSA binding/antioxidant activities using a binding capacity test, and potential impact on albumin quantification by a routine immunoturbidimetric assay. Applying different chemical reactions to a commercial HSA solution, we obtained different solutions enriched up to 53 % of native HSA, 78 % of acetylated HSA, 71 % of cysteinylated HSA, 94 % of oxidized HSA, 58 % of nitrosylated HSA and 96 % of glycated HSA, respectively. Moreover, the semi-synthetic isoforms showed differently altered binding capacities for a panel of ligands (Cu, Cd, Au, Ds and L-T4). Furthermore, immunoturbidimetry was found to be insensitive to the presence and abundance of the different isoforms. The fully characterized semi synthetic HSA isoforms obtained should be useful to further investigate their pathogenicity and potential roles as biomarkers.

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

The posttranslational modifications (PTM) of human serum albumin (HSA) can result in the development of isoforms that have been identified as potential biomarkers for advanced hepatic diseases. However, previous approaches using top-down (TD) analysis to identify isoforms based on molecular weight may have resulted in misidentifications. The nature of the identified isoforms has never been confirmed in previous works. Here, we aimed to critically evaluate TD for the characterization and determination of HSA isoforms in patients and make an inventory of HSA-PTM. Serum samples from control subjects and patients with liver dysfunctions were analyzed using both top-down (TD) and bottom-up (BU) approaches. TD analysis involved using a LC-TOF-MS system to obtain a multicharged spectrum of HSA, which was deconvoluted to identify isoforms. Spectra were then used for relative quantitation analysis of albumin isoform abundances based on trapezoidal integration. For BU analysis, serums were reduced +/- alkylated, digested with trypsin and analyzed in the Q-TOF, data-dependent acquisition (DDA) mode to generate a SWATH-MS high-resolution mass spectral library of all HSA peptides. Tryptic digests of another set of serum samples were then analyzed using data-independent acquisition (DIA) mode to confirm the presence of HSA isoforms and their modification sites. TD detected 15 isoforms corresponding to various modifications, including glycation, cysteinylation, nitrosylation, and oxidation (di- and tri-). In BU, the spectral library containing 127 peptides allowed for the characterization of the important isoforms with their modified sites, including some modifications that were only characterized in BU (carbamylation, deamidation, and amino-acid substitution). The method used for determining isoforms offered acceptable reproducibility (intra-/inter-assay CVs < 15%) for all isoforms present at relative abundances higher than 2%. Overall, the study found that several isoforms could be missed or misidentified by TD. However, all HSA isoforms identified by TD and reported to be relevant in liver dysfunctions were confirmed by BU. This critical evaluation of TD approach helped design an adequate and reliable method for the characterization of HSA isoforms in patients and offers the possibility to estimate isoform abundances within 3 min. These findings have significant implications for the diagnosis and treatment of liver dysfunctions.