WO 2025/099157 A1 — Absolute Quantification of Protein Isoforms by Internal Calibration (Top-Down LC-MS)
Bibliographic Data
| Field | Value |
|---|---|
| Publication number | WO 2025/099157 A1 |
| Type | PCT Application (A1 = first publication before grant) |
| Published | 2025 |
| Filed | ~2024 (PCT filing date) |
| Inventors | Souleiman El Balkhi et al. (Limoges, FR) |
| Applicants | INSERM, CHU Limoges, Université de Limoges |
| Image-based (no text layer) — content reconstructed from technology-transfer strategic report |
Title
“Absolute Quantification of the Isoforms of a Protein of Interest by Internal Calibration Based on a Top-Down LC-MS Approach”
Core Problem Solved
Top-Down proteomics has historically faced two major barriers to clinical use:
- Insufficient mass accuracy on routine instruments (1–5 Da drift for ~66 kDa proteins like albumin)
- No absolute quantification without expensive isotope-labeled standards
This patent solves both by using myoglobin (Mb, ~16.7 kDa) as a multifunction internal standard.
Technical Mechanism
Step 1: Real-Time Mass Recalibration
Myoglobin is co-injected with each patient sample. Its multiple charge states generate peaks at precisely known theoretical masses. The software uses these peaks to recalibrate the albumin (~66.4 kDa) or hemoglobin (~64.5 kDa) spectrum in real time, every injection.
Result: mass error reduced from 1–5 Da to <3 ppm (<0.3 Da for albumin). This enables reliable distinction of near-isobaric modifications that were previously unresolvable in routine settings.
Step 2: Absolute Quantification
With myoglobin at a known concentration as the internal reference, the relative signal ratio of each albumin isoform peak to the myoglobin signal directly yields absolute molar concentration of each isoform — without isotope labeling.
Example output: HSA-Cys = 12.4 µmol/L; HSA-Native = 38.1 µmol/L; HSA-Glyc = 3.2 µmol/L (in a cirrhotic patient).
Applications Covered
Primary: Albumin Isoform Absolute Quantification
- Enables precise calculation of effective albumin (eAlb) components
- Direct comparison across patients, time points, and instruments
- Validated in ALBOM study and published in lakis-2024
Extended: Hemoglobin / HbA1c Precision Testing
The patent explicitly covers hemoglobin isoforms. Key application:
HbA1c in hemoglobin variant populations:
- Standard HbA1c methods (HPLC, immunoassays) fail in patients with HbS, HbC, HbE (common in African, Hispanic, Mediterranean populations)
- Top-Down approach directly quantifies the glycated β-chain of hemoglobin regardless of variant background
- Potential reference method for national metrology institutes and proficiency testing programs
Connection to lakis-2024
The published paper lakis-2024 (Lakis et al., Analytica Chimica Acta, 2024) is the peer-reviewed validation of this patent’s core methodology. The CQFD-PTM pipeline implements the quantification algorithm developed in this patent.
Comparison to Prior Art
| Approach | Mass accuracy | Absolute quant | Practical cost |
|---|---|---|---|
| Standard Top-Down (no IS) | 1–5 Da | No | Low |
| Isotope-labeled IS (SILAC) | <1 Da | Yes | Very high (synthesis cost) |
| This patent (Mb IS) | <3 ppm (<0.3 Da) | Yes | Low (Mb is cheap) |
| SRM/MRM peptide-level | Sub-ppm | Yes | Moderate |
Commercialization Angle
Primary targets per technology-transfer:
- Thermo Fisher — integrate myoglobin IS calibration natively into Xcalibur/BioPharma Finder software; enables Orbitrap-based clinical Top-Down
- Shimadzu — adapt method for CLAM-2040 (automated sample prep) to create “sample-to-result” HbA1c / albumin platform for hospital labs
- Quest Diagnostics — HbA1c precision test for hemoglobin variant populations (unmet need in diverse US patient demographics)
- Bruker — timsTOF Pro2 clinical proteomics package validation
Position in the 3-Level Platform
This is Level 3 of the albuminomics platform:
- Level 1 (US20220018852, SEB Test) — answers “is albumin functional?”
- Level 2 (WO2024074685) — answers “which disease caused the damage?”
- Level 3 (this patent) — answers “exactly how much of each isoform, in absolute terms?”