Top-Down de Novo Protein Sequencing of a 13.6 kDa Camelid Single Heavy Chain Antibody by Matrix-Assisted Laser Desorption Ionization-Time-of-Flight/Time-of-Flight Mass Spectrometry
peer-reviewed · Analytical Chemistry · 2010
| Date | 2010-04-15 |
| Type | peer-reviewed |
| Venue | Analytical Chemistry |
| Publisher | American Chemical Society (ACS) |
| Contribution | downstream-application |
| DOI | 10.1021/ac1000515 |
| Citations (OpenAlex) | 69 |
| Venue 2-year citedness | 7.10 |
Abstract
The primary structure of a 13.6 kDa single heavy chain camelid antibody (V(H)H) was determined by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight (MALDI-TOF/TOF) top-down sequence analysis. The majority of the sequence was obtained by mass spectrometric de novo sequencing, with the N-terminal 14 amino acid residues being determined using T(3)-sequencing and database interrogation. The determined sequence was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of a tryptic digest, which also provided high-energy collisionally induced dissociation (CID) data permitting the clear assignment of 3 of the 14 isobaric Leu/Ile residues. Five of the 11 Leu/Ile ambiguities could be resolved by homology comparisons with known V(H)H sequences. The monoisotopic molecular weight of the V(H)H was determined by ultrahigh-resolution orthogonal electrospray (ESI)-TOF analysis and found to be 13 610.6066 Da, in excellent agreement with the established sequence. To our knowledge, this is the first time that the entire primary structure of a protein with a molecular weight >13 kDa has been established by mass spectrometric top-down sequencing.
Methods and tools
- Camelid VHH top-down sequencing: Determines the primary structure of a 13.6 kDa camelid single heavy chain antibody by MALDI-TOF/TOF top-down analysis, recovering most of the sequence from the intact protein rather than from peptides.