Protein Sequencing Research Group (PSRG): Results of the PSRG 2013 Study Year 2: Terminal Sequencing of Standard Proteins in a Mixture

摘要

Establishing the N- and C-terminal sequences of intact proteins plays a critical role in biochemistry and drug development. Terminal sequence analysis is necessary for quality control of protein biologics, for determining sites of signal peptide cleavage events, for characterizing monoclonal antibodies, and in elucidating sequences of genes from uncommon species. N-terminal sequencing is in the midst of a technology transition from classical Edman sequencing to mass spectrometry (MS) based terminal sequencing, with C-terminal sequencing largely accessible only through top-down MS. Protein homogeneity (absence of interfering protein, non-protein contaminants and buffer components) is critical to the success of analysis. While it has been straightforward to isolate the protein of interest out of protein mixtures in preparation for Edman sequencing, core facilities now need to adopt novel sample preparation techniques to isolate proteins in high purity and make them amenable for terminal sequencing by MS. There is a lack of easy-to-use, field proven methods for sample clean-up.In order to address the upcoming change in technology platforms, the PSRG is conducting a two-year study with the goal of sample preparation and terminal sequencing of a protein mixture. The 2013 study is the second phase towards this goal and entails terminal sequencing and identification of fusion proteins, which were provided in mixture. Participants used Edman and/or MS techniques, along with bioinformatics tools, to derive the termini of the sample proteins. Study participants were directed to a website to anonymously upload sequences and supporting data.Our analysis focused on comparison of results obtained by different technology platforms. The results obtained by Edman sequencing and MS as well as information on instrumentation and protocols will be presented. This data and information will be provided to the community in conjunction with year one of the study, which entailed the same proteins supplied as isolated, homogeneous proteins.