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J Biomol Tech. 2010 September; 21(3 Suppl): S49.
PMCID: PMC2918146

Optimizing In-source Decay as a High-Throughput Alternative to Edman Degradation for the Determination of Protein Termini

P. Liu, C. Bakalarski, and W. Sandoval
Genentech, Inc., South San Francisco, CA, United States



For years, Edman degradation has been the method of choice for obtaining N-terminal sequence information from a protein. Though Edman sequencing is slow and costly with other limitations it has been an invaluable tool for protein characterization and is often used as a core facility function to verify correct processing of a recombinant purified protein. Matrix assisted laser desorption ionization in-source decay (MALDI-ISD) is a top-down method by which an intact protein is fragmented in the presence of a proton-donating matrix after excitation by a laser beam. In recent years, MALDI-ISD has been shown to be a rapid method for obtaining N- and C- terminal sequence information of an intact protein. Our laboratory has utilized MALDI-ISD in a high-throughput manner to replace a significant percentage of our routine Edman service samples, and to reduce our sample analysis and turn-around time from hours to minutes. We have studied the effects of matrix composition on terminal specificity with a variety of purified proteins. In addition, we have developed methods for obtaining N- and C- terminal information from complex mixtures such as antibodies, clipped termini or proteolytic cleavage sites through the incorporation of chromatography prior to MALDI-ISD, thereby further reducing our Edman workload. Though sample prep and data acquisition can be done in mere minutes, manual interpretation of the spectra and assignment of the correct N-terminus from a protein sequence could take an hour or more. To alleviate this burden, we have developed a semi-de novo bioinformatic tool for rapid data processing named “ISDetect.” This software accepts a given protein sequence and probabilistically determines the most likely N- or C- termini based on the ISD spectrum.

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