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J Biomol Tech. 2007 February; 18(1): 52.
PMCID: PMC2292042

P150-T Utilization of ABRF sPRG Protein Standard for Developing Optimized Experimental Strategies for ETD Based Protein Identification

Abstract

Conventional methodology of protein mass spectrometry using collision induced dissociation (CID) has been widely adopted for confident identification and characterization of proteins in recent years. However, CID-based protein analysis works best with relatively short peptides and many important post translational modifications are not preserved during CID-based analyses. A new fragmentation method, electron transfer dissociation (ETD), has been developed recently and introduced to linear ion trap mass spectrometer. In ETD, peptide backbone is fragmented along pathways that are analogous to those observed in ECD. ETD has been reported to not only favor multiply charged, relatively large peptides, but also preserve post translational modifications. Thus, ETD is drawing more and more attention as mass spectrometry based methods for protein identification and analysis of post-translational modifications. However, current experimental strategies, which perfectly match CID-based analysis, are not optimized for ETD. This lack of efficient experimental strategies that enhance ETD-based analysis could significantly limit the benefits which ETD can provide for the analysis of proteins.

The purpose of this study is to optimize experimental strategies for the comprehensive analysis of the ABRF sPRG Protein Standard and to compare sensitivity of CID and ETD activation methods using various concentrations of this standard. Protease digested ABRF sPRG 49 protein standard mixture was analyzed on a Finnigan LTQXL with ETD employing the combination of CID and ETD activation types. Comparison of data generated by ETD and CID indicates complementarity of amino acid sequence coverages in the resultant datasets. Many peptides only show strong fragmentation patterns by either one or the other method. However, improved protein sequence coverage was enabled by alternating between CID and ETD activation types. Increased protein sequence coverage not only allowed a more thorough characterization of the 49 proteins in the standard, but also reduced the number of false positive protein hits.


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