PSRG 2011 Study Results: Sensitivity Assessment of Terminal Sequencing Techniques Using an Unknown Protein

Establishing the N-terminal sequence of intact proteins plays a critical role in biochemistry and potential drug development. N-terminal sequence analysis is necessary for quality control of protein biologics, for determining sites of signal peptide cleavage events, as a first step in elucidating the sequences of genes from uncommon species and for the characterization of monoclonal antibodies. Automated Edman degradation has been the method of choice for these analyses. However, alternate methods for N-terminal sequence analysis have emerged. The recent PSRG studies have established that Edman sequencing and mass spectrometry based techniques have varied strengths and weaknesses depending on several experimental factors and both play an important role in terminal sequencing. With this complimentary role realized, the 2011 PSRG study attempts to evaluate the sensitivity limits of the various sequencing techniques. The PSRG distributed three sample sets of 3 tubes each, varying by sample format (lyophilized, gel slice or membrane piece). Each set of three samples contains the same recombinant protein with increasing amounts of material. The sequence of this protein is not listed in any database. Participants could request any one, two, or all three sample sets. Including PSRG committee, a total of 38 participants requested 74 sample sets. The participants were asked to determine as many amino acids from both termini by their method of choice, and were encouraged to try multiple methods for sequence elucidation. Study participants were directed to a website to anonymously upload sequences and supporting data. Our analysis focuses on the length and accuracy of the sequence calls reported by the participants, and how that compares with decreasing amounts of protein and the type of sample format analyzed. A comparison of the results obtained by Edman chemistry and by alternative technologies as well as information on the type of instruments and protocols is reported.