Analysis of the ABRF-PSRG 2010 Protein Sequencing Sample by Mass Spectrometric Top-Down de Novo Sequencing
abstract · PubMed Central · 2010
| Date | 2010-09-01 |
| Type | abstract |
| Venue | PubMed Central |
| Contribution | downstream-application |
| Link | https://www.ncbi.nlm.nih.gov/pmc/articles/2918021 |
| Citations (OpenAlex) | 0 |
Abstract
A monoclonal antibody was provided to participants in this study with the goal to provide as much terminal sequences of both the light and heavy chains (LC and HC) as possible. Two vials with 50 μg of proteins each were received from PSRG. The samples were reduced with TCEP and HC and LC separated by HPLC on a Zorbax 300SB-C8 column and collected according to their UV signal. Sinapinic acid and 1,5-diaminonaphthalene were used for molecular weight determination on a MALDI-TOF/TOF instrument as well as for top-down sequencing [1]. All MALDI-ISD spectra were de novo sequenced using dedicated and semiautomatic software (BioTools 3.2). The terminal calls (approx. residues 1-20) were assigned using Tз-sequencing and standard MASCOT based protein identification. MALDI top-down sequencing, i.e., the combined use of In-Source Decay (ISD) and Tз-sequencing, provided approx. 70 N- and C-terminal sequence calls each from both antibody chains from a total of 5 Spectra. The acquisition of MALDI spectra after the chromatographic chain separation took a few minutes and the data analysis a few hours. Included in these analysis is the confirmation of the pyroglutamylated N-terminus of the HC and the C-terminus from which the terminal K was excised. Simple MALDI-MS spectra of the HC allowed to monitor the glycoprofile from the same preparations. Obviously, MALDI top-down sequencing is a very economic approach to characterize antibodies with regard to their terminal status and glycocomposition and can accelerate the relevant QC of therapeutic proteins including antibodies.
Methods and tools
- ABRF-PSRG 2010 antibody top-down de novo sequencing: Sequenced the N- and C-terminal regions of both chains of the ABRF-PSRG 2010 study antibody by MALDI in-source decay top-down de novo sequencing.