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J Biomol Tech. 2010 September; 21(3 Suppl): S63.
PMCID: PMC2918049

Towards 100% Sequence Coverage in Protein QC: In-depth Sequence Characterization of Monoclonal Antibodies

U. Schweiger-Hufnagel, P. Hufnagel, R. Hebeler, M. Witt, P.O. Schmit, and M. Macht
Bruker Daltonik GmbH, Bremen, Germany



Quality control of biotherapeutic proteins requires access to the full protein sequence to account for every single aminoacid and its modification status. This is typically not achieved in a single LC-MS/MS analysis, but requires combined analytical strategies. However, this complicates the downstream data analysis due to distribution of relevant information in multiple datasets. A new bioinformatics platform, ProteinScape, is described here, which facilitates in-depth protein characterization tasks at an unparalleled level of efficiency. The analysis of a monoclonal antibody (mAB) described here utilized digestion by multiple enzymes (trypsin, chymotrypsin, LysC, GluC), with and without upfront SDS-PAGE separation, followed by LC-MALDI-TOF/TOF. LC-MALDI analysis of these digests yielded individual sequence coverages between 64 and 88%. A significant increase in sequence coverage was achieved when compiling the four individual digest analyses into one overall sequencing result. This rather demanding compilation step is easily performed using ProteinExtractor, a unique software algorithm implemented in ProteinScape. ProteinExtractor compilation of the LC-MALDI data yielded 100% coverage for the mAB light chain, and 98% for the heavy chain (HC). Additional electrospray measurements were performed to obtain information on the mAB HĆs sequence range [177-184] which was not accessible by MALDI. Again, ProteinExtractor was used to compile ESI and MALDI data resulting in a further increased sequence coverage of 99% for the HC. ESI measurements added 2 unique MS/MS spectra covering 3 aminoacid residues [range 182-184] that had not been detected by MALDI. In addition to the aminoacid sequence, the HĆs N-terminal modification status was characterized. Evaluation of the respective MS/MS spectra, which are directly accessible in ProteinScape, confirmed N-terminal conversion Glu to pyroGlu based on consistent ESI and MALDI data.

Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities