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Establishing the N-terminal sequence of intact proteins plays a critical role in biochemistry and drug development. N-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. Protein homogeneity (absence of interfering protein, non-protein contaminants and buffer components) is absolutely critical to the success of analysis. While it has been very 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 in 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 ultimate goal of sample preparation and terminal sequencing of a protein mixture. The 2012 study is the first phase towards the overall goal and entails terminal sequencing and identification of fusion proteins, which were provided as separated, homogeneous proteins. 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 critical comparison of results obtained by different technology platforms. The results obtained by Edman sequencing and MS as well as information on the type of instruments and protocols will be presented. This data and information will be provided to the community in conjunction with year two of the study, which will be the same proteins supplied in mixture.

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 an 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 o 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.

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.

r9-1

The most common form of dementia is Alzheimer’s disease. According to the amyloid hypothesis, the disease is preceded by an accumulation of the amyloid-β (Aβ) protein, which leads to downstream events including activation of microglia, inflammation, synaptic dysfunction, and neuronal loss. The objective of this research is to address the physiology of Aβ in humans by measuring its in vivo metabolic rates.