LC-MS (Liquid Chromatography — Mass Spectrometry)
LC-MS is the coupling of liquid chromatographic separation with mass spectrometric detection. It is the core analytical platform for all PTM profiling work in this vault.
Variants used in this research
| Variant | Instrument | Application |
|---|---|---|
| RP-LC-HR-MS (top-down) | Bruker timsTOF Pro2; Sciex TripleTOF 5600+ | HSA isoform profiling (intact protein); el-balkhi-2025 |
| RP-LC-HR-MS (top-down, absolute quant) | Bruker timsTOF Pro2 + equine Mb IS | Absolute quantification; lakis-2024 |
| nanoLC-nanoESI-MS/MS (bottom-up, DDA) | Q-TOF / Orbitrap | SWATH spectral library; rahali-2022 |
| HPLC-ESI-MS (top-down, low-res) | Sciex QStar / Waters Q-TOF Ultima | Early isoform profiling; domenicali-2014, baldassarre-2021-ealb |
| LC-ESI-MS/MS (bottom-up, targeted) | Thermo Orbitrap Velos | Site-level oxidation confirmation; oettl-2013 |
Key parameters for HSA top-down profiling
- Column: C4 reverse-phase (300 Å pore size); 3-min gradient at 1 mL/min
- Sample: serum diluted 1:50; protein precipitation; or direct C4 injection
- Detection: positive ESI mode; multicharged envelope (m/z 1000–1700 for HSA ~66.5 kDa)
- Deconvolution: MaxEnt algorithm → true mass spectrum 61,500–71,500 Da range
- Internal standard: equine myoglobin (16,952 Da) for mass recalibration + absolute quantification
See also
- Top-down proteomics — intact protein LC-MS for isoform profiling
- Bottom-up proteomics — peptide-level LC-MS for site mapping
- SRM — targeted quantification by triple-quad LC-MS
- SWATH — data-independent acquisition for reproducible peptide quantification