Journal of Proteomics & Bioinformatics
Open Access

Abstract

Targeted SCX Based Peptide Fractionation for Optimal Sequencing by Collision Induced, and Electron Transfer Dissociation

Henk W.P. van den Toorn, Shabaz Mohammed, Joost W. Gouw, Bas van Breukelen and Albert J.R. Heck

Electron transfer dissociation (ETD) of peptide ions has been introduced as a tool for mass spectrometry based peptide sequencing, complementary to the commonly used collision induced dissociation (CID). It has been proposed that ETD may have better performance than CID for more highly charged and/or larger peptides. Here, we compare the performance of ETD and CID on data generated in a large-scale proteomics experiment. First, tryptic proteolytic peptides of Drosophila melanogaster oocytes were off-line separated based on their insolution net charge state using strong cation exchange chromatography (SCX), followed by an on-line reversephase (RP) liquid chromatography separation coupled to an ion trap mass spectrometer with ETD capabilities. The mass spectrometer selected MS peaks were subjected to both ETD and CID thus allowing a fair comparison. Around 2300 peptides were exclusively identified by CID and similarly more than 3000 by ETD with approximately 1400 by both ETD and CID. In total nearly 7,000 peptides were identified with a very conservative Mascot peptide cut-off score of 60 clearly verifying that ETD and CID are complementary techniques. In the early SCX fractions, which contain peptides with a ‘low’ net charge, more than 90% of the peptides could be successfully identified by CID whereas in the later SCX fractions more than 90% of the identified peptides could be successfully identified by using ETD only. The chosen strategy, with a combination of SCX and RP-LC-MS/MS, allows the user to make targeted decisions on whether to optimally use CID and/or ETD. Analysis of the sequence and amino acid contents of all identified peptides clearly revealed that the impressive performance of ETD for peptides possessing charge states above three do not require CID based sequencing which, at best, would be solely confirmatory.