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J Biomol Tech. 2010 September; 21(3 Suppl): S55–S56.
PMCID: PMC2918076

Increased Sequence Coverage in Complex Protein Digests by Consecutive, Targeted LC-MSMS Runs with Both CID and ETD

A. Schneider,1 M. Lubeck,1 and R. Almeida2
1Bruker Daltonik GmbH, Bremen, Germany;
2Advion, Arnsberg, Germany;
3Bruker Daltonics Inc., Fremont, CA, United States



Highly complex protein digests cannot be analyzed in one single run as not all tryptic peptides will be triggered with an automated MSMS approach. Additionally, many PTMs are low abundant and therefore often not detected in a first run. However, for a second injection the same sample amount is needed. Using the RePlay module a second analysis is possible from the same first injection. When data from the first run are processed in between, the second analysis can be used for a targeted MSMS approach using different fragmentation techniques like CID and ETD. Thus, the ‘replay run’ of the same sample improves protein and peptide coverage, increases identification of post translational modifications, and improves analytical confidence. A standard nanoLC is coupled to the RePlay module (Advion BioSciences). After column separation the flow from a 75 μm column at a typical flow rate of 250 nL/min is split, so that part is directed to the mass spectrometer for analysis whilst the remainder flows to a capillary tube where it is stored. After the direct LCMS run, the flow is switched and the portion stored in the capillary is analyzed (‘replay run’). CID and ETD data are acquired on a Bruker amzon ion trap. A column between the storage capillary and the trap refocuses the stored peaks in the second analyses so that width and intensity is identical to the initial run. When the first run is finished, the data are processed due to their sample origin. Examples of combining exploratory and targeted analysis are shown by targeting PTM's, excluding peptides identified in previous run or using different fragmentation techniques.

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