N-terminal Top-Down Protein Sequencing of the ABRF-PSRG2012 Study Samples by ETD-UHR-TOF Mass Spectrometry

• Published in: Journal of biomolecular techniques Vol. 23; no. Suppl; p. S51
• Main Authors: Albers, Christian, Hartmer, Ralf, Jabs, Wolfgang, Baessmann, Carsten, Kaspar, Stephanie
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Association of Biomolecular Resource Facilities 01.01.2012
• Subjects: Scientific Poster Abstracts
• ISSN: 1524-0215; 1943-4731

MS based top-down protein sequencing (TDS) techniques are becoming increasingly important mainly because relevant sequence information such as N-terminal sequence assignment is obtained very quickly and accurately. This meets the demand of many growing areas like identity confirmation of biopharmaceuticals. In this study, we applied a general analysis strategy for top-down sequencing (TDS) based on Electron Transfer Dissociation (ETD) mass spectrometry. Samples were obtained for the participation in the ABRF-PSRG research study from the PSRG and the identities were provided: BSA, Endostatin and protein A. Samples were directly subjected to offline nano-spray. Using a Bruker maXis ETD mass spectrometer the molecular weight of the intact proteins as well as the charge states for the ETD fragmentation were determined and the ETD fragment spectra acquired. Identification of N-terminal sequences was readily obtained using BioTools software applying a two-step approach: The first step consists of a TDS based Mascot database search. If the protein terminus cannot be obtained, most likely because the protein sequence is not included in the database, a BLAST protein homology search is performed as second step. For the BLAST search, different sequence tags of the proteins top-down spectrum are automatically generated. The retrieved sequences were aligned with the ETD fragment sequence tags. Misalignments indicate sequence variation such as methylation as in protein A. The three proteins were identified via Mascot database search. N-Terminal sequences could be determined directly for BSA and two Collagen alpha-1-(XVIII) fragments (Endostatin) up to the position of the first Cysteine-Cysteine bridge. As protein A could not be identified via Mascot database search, alternative BLAST searches were performed. ETD-TDS in combination with dedicated TDS software provided N-terminal sequences for all PSRG samples – even in cases of a ragged or modified N-term. Precision intact MW determination provided also information about C-term Lys-excision in Endostatin.