⚖️ Proteomics: Mass Spectrometry & Peptide Sequencing
Biomolecular mass spectrometry measures the mass-to-charge ratio ($m/z$) of ionized macromolecules, enabling high-throughput proteome identification, post-translational modification mapping, and structural crosslinking.
1. Soft Ionization: MALDI vs ESI
| Ionization Technique | Matrix-Assisted Laser Desorption/Ionization (MALDI) | Electrospray Ionization (ESI) |
|---|---|---|
| Sample State | Solid co-crystallized with organic UV matrix ($alpha$-CHCA, sinapinic acid) | Liquid phase in volatile acidic solvent spray |
| Charge States | Predominantly singly charged ions ($[M+H]^+$) | Multiple charge states ($[M+zH]^{z+}$), bringing large proteins into readable $m/z$ ranges |
| Coupling | Typically coupled to Time-of-Flight (TOF) analyzers | Directly interfaced online with reverse-phase nano-LC (LC-MS/MS) |
2. Tandem MS (MS/MS) Peptide Fragmentation
In collision-induced dissociation (CID), precursor peptide ions collide with inert gas (Ar, N2), breaking peptide amide bonds to generate sequence-informative fragment ions:
- b-ions: Fragments retaining the positive charge on the N-terminal fragment.
- y-ions: Fragments retaining the positive charge on the C-terminal fragment.
- The mass difference between consecutive b-ions or y-ions equals the exact residue mass of the corresponding amino acid, enabling de novo sequence assignment.