PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jbtJBT IndexAssociation Homepage
 
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

Abstract

RP-90

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.


Articles from Journal of Biomolecular Techniques : JBT are provided here courtesy of The Association of Biomolecular Resource Facilities