Posttranslational-Modifications of Human-Serum-Albumin Analysis by a Top-Down Approach Validated by a Comprehensive Bottom-Up Analysis
Bibliographic info
- Authors: Mohamad-Ali Rahali, Roy Lakis, François-Ludovic Sauvage, Emilie Pinault, Pierre Marquet, Franck Saint-Marcoux, Souleiman El Balkhi
- Journal: Journal of Chromatography B (submitted 2022/2023; manuscript CHROMB-S-23-00284)
- Institution: P&T UMR1248 INSERM / CHU Limoges
Key question
Do top-down (TD) LC-MS isoform identifications accurately reflect the true PTM status of HSA, and can a comprehensive bottom-up (BU) approach — including SWATH-MS library — confirm TD assignments and reveal additional modifications?
Methods
- Sample type: Human serum — controls + liver dysfunction patients
- Approach 1 (Top-down): LC-TOF-MS; multicharged spectrum → deconvolution → isoform identification by MW; trapezoidal integration for relative quantification
- Approach 2 (Bottom-up):
- Phase 1: Reduction ± alkylation → trypsin digestion → Q-TOF DDA → SWATH-MS spectral library (127 HSA peptides)
- Phase 2: Same samples analyzed in DIA mode → confirms isoforms and PTM sites using the library
- Cross-validation: compare TD isoform list against BU-confirmed modification sites
Main findings
- Top-down detected 15 isoforms: glycation, cysteinylation, nitrosylation, oxidation (di- and tri-)
- BU spectral library: 127 peptides; enabled site-level confirmation of isoforms and identification of additional PTMs
- BU-only PTMs: carbamylation, deamidation, amino acid substitution — not detected by TD due to resolution limitations at intact protein MW
- Critical validation result: all CLD-relevant HSA isoforms detected by TD were confirmed by BU — TD is not generating false positives for the clinically significant isoforms
- TD risks: some isoforms can be missed or misidentified by TD (overlapping masses), particularly low-abundance or closely-spaced modifications
- Reproducibility: intra- and inter-assay CV < 15% for all isoforms present at relative abundance > 2%
- Speed: 3-minute analysis runtime
PTMs characterized
| PTM | Method | Modified site(s) | Clinical relevance |
|---|---|---|---|
| Cysteinylation (+119 Da) | TD + BU | Cys34 | Oxidative stress |
| Glycation (+162 Da) | TD + BU | Lys (multiple) | Metabolic stress |
| Oxidation (di/tri, +32/+48 Da) | TD + BU | Cys34 | Severe oxidation |
| Nitrosylation | TD + BU | Cys34 / Tyr | Inflammation |
| Carbamylation | BU only | Lys | Uremia, renal disease |
| Deamidation | BU only | Asn/Gln | Spontaneous modification |
| AA substitution | BU only | Various | Genetic variant |
| N-term truncation (-Asp) | TD + BU | N-terminus | CLD marker |
| C-term truncation (-Leu) | TD + BU | C-terminus | — |
Clinical context
- Disease: Liver dysfunction patients vs controls
- Implication: Establishes the TD approach as valid for CLD-relevant isoforms, while flagging its limitations for less abundant or novel PTMs
- TD vs BU trade-off: TD = fast (3 min), clinically deployable, good for known isoforms; BU = comprehensive, slow, requires digestion, but can find novel PTMs
Limitations
- BU library required substantial upfront effort (DDA library generation)
- Carbamylation and deamidation found by BU not quantifiable by TD — missing important PTMs in renal/inflammatory disease contexts
- Relative quantification only (TD) — absolute quantification requires lakis-2024 method
Connections
- Top-down proteomics — primary approach validated here
- Bottom-up proteomics — validation approach; SWATH/DIA library
- HSA — sole protein; 15 isoforms by TD, 127 peptides by BU
- CQFD-PTM pipeline — pipeline is built on TD approach validated in this paper
- lakis-2024 — subsequent absolute quantification method (same group, same platform)
- el-balkhi-2025 — ALBOM clinical application using the validated TD approach
My notes
- This paper is the methodological foundation justifying the TD-only approach used in el-balkhi-2025 and lakis-2024. Its key message: TD is not perfect, but for the specific set of CLD-relevant isoforms, it’s valid and fast enough for clinical use.
- Nitrosylation detected by TD — not quantified in ALBOM. Potential biomarker of inflammation in CLD?
- Carbamylation by BU only: relevant for patients with comorbid renal disease (common in decompensated cirrhosis). This is a gap in the current ALBOM panel.
- 127-peptide BU library is a valuable resource for future site-level resolution studies.