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J Biomol Tech. 2010 September; 21(3 Suppl): S50.
PMCID: PMC2918179

Fast and Highly Accurate QTOF ETD MS/MS for Top-Down Sequencing

M. Lubeck, C. Stoermer, and R. Hartmer
Bruker Daltonik GmbH, Bremen, Germany

Abstract

RP-92

The profiling of plasma proteins is a challenge in proteome analyses due to the dynamic range and the number of analytes. Once the statistic approach discovers a potential biomarker the major objective is the identification and characterization. Most of the biomarkers are too large to obtain substantial sequence information with common techniques like CID. Dedicated MS/MS-techniques for top-down analysis are electron-induced fragmentation processes like ECD or ETD. Presented here is the profiling and top-down identification of plasma proteins by the Bruker maxis UHR-TOF equipped with ETD. The goal of the present protein profiling approach is targeted towards proteins revealing molecular weight below 10 kDa. Plasma proteins with higher mass were depleted with a 10 kDa cutoff filter. Resulting proteins were subjected to a LC-separation using monolithic columns. The eluting and partially separated proteins were split towards the MS and fractionated. Afterwards selected fractions were infused offline into the MS. A scheduled list of protein precursor ions are then mass selectively fragmented by ETD. With the highly accurate TOF-analyzer the resulting ETD-fragments are detected. The method is evaluated with protein standards spiked into plasma. Due to the high resolving power of the TOF analyzer the monoisotopic annotation and the determination of charge states for both precursor and ETD fragment ions is possible. The data is submitted to a database search engine (Mascot) optimized for Top-Down data. Sequence tags and Top-Down related sequence analyses were generated in BioTools using a mixture of automated calculations and interactive sequence assignments.


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