Serum Proteomics
Overview
Serum proteomics (in the antibody context) uses nano-flow liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to identify individual antibody heavy-chain protein sequences circulating in serum. Affinity-purified serum antibody fractions are digested with proteases (chymotrypsin, elastase, trypsin), and the resulting peptide fragments — particularly those spanning the highly diverse CDR3 region — are matched against a reference database of antibody sequences from the same patient. When the reference database is built from next-generation sequencing (NGS) of sorted B cell and ASC populations from the same blood draw, this approach achieves “perfect identity” linkage between serum protein sequences and B/ASC clonotype sequences. This is the most direct available method for proving that a specific B cell clone is the source of a specific serum antibody.
Key Points from Literature
- Direct serum–ASC linkage: In Tipton2015, 9G4⁺ antibody fractions were affinity-purified from serum using monoclonal 9G4, then proteolytically digested and analysed by LC-MS/MS (SEQUEST against NGS databases). Perfect identity was established for 39 serum 9G4⁺ antibodies representing 20 distinct clonotypes, directly confirming which ASC clones produced the dominant serum autoantibodies (see Tipton2015 - ASC Diversity and Origin in SLE).
- Persistence confirmation: Two clonally related serum 9G4 antibodies with shared HCDR3 (ARAPGLDRDYYYYYYMDV) were identical matches to ASC sequences from the largest VH4-34⁺ clone in patient SLE-3, and both serum clonotype and the corresponding ASC clones persisted for over 8 weeks (see Tipton2015 - ASC Diversity and Origin in SLE).
- Unmutated serum autoantibodies: The serum 9G4 antibodies linked to acN-derived ASC clones were almost completely devoid of VH mutation — directly proving that germline-encoded (unmutated) antibodies from naive-derived ASCs contribute substantially to the circulating serum autoantibody repertoire in SLE (see Tipton2015 - ASC Diversity and Origin in SLE).
- Limitation: Serum proteomics is technically demanding, requires both deep NGS coverage of B cell populations and high-confidence LC-MS/MS peptide spectral matching. Coverage is incomplete — the method identifies sequences with high-confidence peptide spectral matches but will miss low-abundance clones or sequences with poor proteolytic coverage of CDR3 peptides.
Contradictions & Debates
None documented in current wiki sources.
Related Pages
BCR Sequencing, Plasmablast, Activated Naive B Cell, ELISpot, Somatic Hypermutation