N- and C-terminal Protein Sequencing

by MALDI ISD

Full characterization of recombinant proteins requires confirmation of the expected N- and the C-terminals. Alphalyse offers N- and C-terminal protein and peptide sequencing by mass spectrometry using top-down sequencing by MALDI ISD. The analysis can be used for characterisation of degradation in therapeutic protein products. And for analysis of enzymatic cleavage specificity.

The top-down sequence analysis confirms 20-50 residues from both the N-terminal and the C-terminal of the intact protein. Truncated proteins and modified terminals can also be sequenced, including acetylation, pyroglutamic acids, and signal sequences.

C-terminal and N-terminal sequences are obtained
N-terminal blocked proteins can be sequenced
Long sequence reads of 20-50 residues from both termini in a single mass spectrum

The application note "N- and C-terminal sequencing by MALDI ISD (PDF File), demonstrates the technique on a variety of recombinant proteins and monoclonal antibodies.

Sample preparation
The top-down sequencing analysis requires 20-50 ug HPLC purified protein and the expected amino acid sequence.

For N-terminal sequencing using Edman degradation chemistry see N-terminal Edman sequencing.

: MALDI ISD spectrum of recombinant vaccine protein. The observed N- and C-terminal protein fragment masses are used for a Mascot database search to correlate the MS data with the expected protein sequence. The Mascot search (insert) confirms the C-terminal sequence, and shows that the N-terminal sequence has been truncated. The analysis verifies 80 amino acid residues in one single mass spectrum.