Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach
Bibliographic info
- Authors: Roy Lakis§, François-Ludovic Sauvage§, Emilie Pinault, Pierre Marquet, Franck Saint-Marcoux, Souleiman El Balkhi (§ equal contribution)
- Journal: Analytical Chemistry, 2024, vol. 96, pp. 746–755
- DOI: 10.1021/acs.analchem.3c03933
- Institution: P&T UMR1248 INSERM / CHU Limoges
- File:
raw/my_work/lakis-et-al-2024-...pdf
Key question
Can a top-down LC-MS method with an equine myoglobin internal standard provide reliable absolute quantification of HSA isoforms in clinical plasma samples, with sufficient accuracy and precision for use as a clinical biomarker assay?
Methods
- Sample type: Human plasma / serum (clinical samples + spiked standards)
- Technique: Top-down LC-HR-MS; Top-down proteomics
- Internal standard: Equine myoglobin (Mb) — dual purpose: (1) mass recalibration per sample, (2) absolute quantification anchor
- Sample prep: 1:50 dilution in 0.9% NaCl + Mb (4 g/L final in diluted sample); C4 column
- Two quantification approaches tested:
- Deconvoluted peak integration (DPI) — from the deconvoluted mass spectrum
- Extracted Ion Chromatogram (XIC) — from raw m/z data
Main findings
- Myoglobin IS solves the mass accuracy problem: conventional top-down deconvolution has 1–5 Da mass errors; Mb IS enables systematic per-sample mass recalibration → extremely low mass deviations
- 8 HSA isoforms quantified with specific calibration curves showing good linearity (deconvoluted peak approach)
- Isoform-dependent ionization: HSA isoforms do not ionize equally → using a single generic HSA standard underestimates some isoforms. An enriched isoform calibration solution is required for accurate absolute quantification
- Good repeatability, reproducibility, accuracy — CV acceptable for clinical use
- Better sensitivity than routine biochemical assays for low albumin samples (relevant in advanced liver disease where albumin is severely depleted)
- Workflow: relatively simple; high-throughput compatible; aimed at clinical translation
PTMs reported
| Protein | Isoform | Modification | Δmass (Da) | Notes |
|---|---|---|---|---|
| HSA | Native | None | 0 | Reference isoform |
| HSA | HSA+CYS | Cysteinylation Cys34 | +119 | — |
| HSA | HSA+GLYC | Glycation (Lys) | +162 | — |
| HSA | HSA+CYS+GLYC | CYS + GLYC | +281 | — |
| HSA | HSA+2GLYC | Double glycation | +324 | — |
| HSA | HSA+CYS+2GLYC | CYS + 2×GLYC | +443 | — |
| HSA | HSA-DA | N-term truncation | −115 | — |
| HSA | HSA-L | C-term truncation | variable | — |
Clinical context
HSA isoforms as biomarkers of diabetes, kidney disease, and liver disease. This method enables quantitative (g/L) isoform profiling — prior methods only gave relative abundances (%). Absolute quantification is critical for clinical cut-off definition and multicenter deployment.
Limitations
- Enriched isoform calibration solution required (adds complexity)
- XIC approach not superior to DPI; DPI preferred
- Validation focused on methodological performance; clinical cohort data not in this paper (done in el-balkhi-2025)
Connections
- ALBOM study / el-balkhi-2025 — this method (Ref 22 in ALBOM) is the foundation of all absolute HSA quantification in ALBOM
- CQFD-PTM pipeline — this is the quantification engine underlying the pipeline
- Top-down proteomics — methodological context
- HSA — protein studied
My notes
This is the foundational methods paper for ALBOM. Everything in el-balkhi-2025 rests on this validated method. Key innovation: isoform-dependent ionization is a genuine calibration challenge that was properly solved here by using enriched isoform standards rather than a single commercial HSA calibrator. This paper should be cited in any clinical or regulatory context for the ALBOM method.