De Novo MS/MS Sequencing of Native Human Antibodies

J Proteome Res. 2017 Jan 6;16(1):45-54. doi: 10.1021/acs.jproteome.6b00608. Epub 2016 Nov 2.


One direct route for the discovery of therapeutic human monoclonal antibodies (mAbs) involves the isolation of peripheral B cells from survivors/sero-positive individuals after exposure to an infectious reagent or disease etiology, followed by single-cell sequencing or hybridoma generation. Peripheral B cells, however, are not always easy to obtain and represent only a small percentage of the total B-cell population across all bodily tissues. Although it has been demonstrated that tandem mass spectrometry (MS/MS) techniques can interrogate the full polyclonal antibody (pAb) response to an antigen in vivo, all current approaches identify MS/MS spectra against databases derived from genetic sequencing of B cells from the same patient. In this proof-of-concept study, we demonstrate the feasibility of a novel MS/MS antibody discovery approach in which only serum antibodies are required without the need for sequencing of genetic material. Peripheral pAbs from a cytomegalovirus-exposed individual were purified by glycoprotein B antigen affinity and de novo sequenced from MS/MS data. Purely MS-derived mAbs were then manufactured in mammalian cells to validate potency via antigen-binding ELISA. Interestingly, we found that these mAbs accounted for 1 to 2% of total donor IgG but were not detected in parallel sequencing of memory B cells from the same patient.

Keywords: B cell; antibody discovery; de novo sequencing; mass spectrometry; monoclonal antibody; polyclonal antibody; shotgun protein sequencing.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / biosynthesis
  • Antibodies, Monoclonal / chemistry*
  • Antibodies, Monoclonal / isolation & purification
  • Antibodies, Viral / biosynthesis
  • Antibodies, Viral / chemistry*
  • Antibodies, Viral / isolation & purification
  • Antibody Formation
  • Antibody-Producing Cells / cytology
  • Antibody-Producing Cells / immunology
  • B-Lymphocytes / immunology*
  • B-Lymphocytes / virology
  • Chromatography, Affinity / methods
  • Cytomegalovirus / growth & development
  • Cytomegalovirus Infections / blood
  • Cytomegalovirus Infections / immunology*
  • Cytomegalovirus Infections / virology
  • Enzyme-Linked Immunosorbent Assay
  • HEK293 Cells
  • Humans
  • Immune Sera / chemistry
  • Immunoglobulin G / biosynthesis
  • Immunoglobulin G / chemistry*
  • Immunoglobulin G / isolation & purification
  • Sequence Analysis, Protein*
  • Tandem Mass Spectrometry
  • Viral Envelope Proteins / chemistry
  • Viral Envelope Proteins / immunology


  • Antibodies, Monoclonal
  • Antibodies, Viral
  • Immune Sera
  • Immunoglobulin G
  • Viral Envelope Proteins
  • glycoprotein B, Simplexvirus