Top-down Characterization of Intact Proteins by Ultra-high-Resolution maXis-ETD

Technical Note

Expression Proteomics

TN-40

UHR-TOF

2010

maXis

Ralf Hartmer, Carsten Stoermer, Laura Main, Dirk Wunderlich, Arnd Ingendoh, Christian Albers, Romano Hebeler, Alexander Harder, Pierre-Olivier Schmit Bruker Daltonik, Bremen, Germany

05-2010, TN-40, #270354

Abstract

The identification of proteins is usually achieved by the well established bottom-up approach where individual or crude mixtures of proteins are enzymatically digested at the whole. MS and MS/MS data information of the resulting peptides is then used for probability searches in sequence databases to reveal the protein identity. This approach typically covers 20-60% of the amino acid sequence for the protein hits. If a full protein characterization is required, like for the analysis of biopharmaceuticals [1], protein homologues or highly conserved proteins with various functional post-translational modifications, alternative or additional methods like top-down sequencing are required. For isolated or a lower number of protein mixtures, top-down can as well be a much faster and direct way to get maximum information on the sequence. Top-down sequencing starts typically by determining the intact protein mass, then fragmentation of the intact proteins is performed, resulting in product ions that allows to pinpoint the protein sequence as well as potential modifications or mutation in the their localization and structure [2].
The maXis is able to determine molecular weights of large intact proteins down to the ppm level, which in many cases is sufficient to get a first confirmation of the correct primary structure including glycosylations patterns. With the introduction of ETD (Electron Transfer Dissociation), direct MS/MS on intact proteins is possible for readout and  confirmation of protein sequences including modifications. The principle and typical data is presented in this technical note. 

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