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Reputable Mentor II
Reputable Mentor II
Hao Z, Jiang L, Mohtashemi I, Hühmer A, Ding Y, Dong M.
Application Note 484
N-terminal sequencing is widely accepted as a reliable tool for protein characterization throughout all stages of drug discovery and biopharmaceutical manufacturing. The two major direct methods of protein N-terminal sequencing are Edman degradation and mass spectrometry. Although the traditional Edman technique is very robust and provides de novo capabilities, the method suffers from several limitations, namely throughput, sensitivity, cost, and the need for specific data interpretation expertise. Mass spectrometry-based methods use the bottom-up approach, which is not only labor intensive but also has limitations in terms of digestion efficiency and efficient capture of the N-terminal peptides. In contrast, emerging top-down approaches involve direct analysis of intact proteins. Direct analysis can preserve the post-translationally modified forms of proteins, resolve protein-level variations, and determine expression ratios of intact protein forms. Mass spectrometry using electron transfer dissociation (ETD) is particularly advantageous for top-down sequencing applications because ETD is relatively unaffected by protein size, amino acid composition, and post-translational modifications. ETD randomly cleaves protein backbone bonds while preserving post-translational modifications.1 ETD technology has been implemented in hybrid linear ion trap – Thermo Scientific Orbitrap mass spectrometers whose high mass resolution and mass accuracy facilitate top-down analysis of intact proteins.2 The ability of a hybrid Orbitrap™ mass spectrometer to perform multiple stages of MS/MS with a variety of activation types extends the rapid N-, and C-terminal sequencing capability to complete sequencing of the terminus – even for proteins with modifications. In this study, the utility of ETD mass spectrometry for N- and C-terminal sequencing of intact proteins is presented. The high-resolution, high-mass accuracy capabilities of Orbitrap ETD hybrid mass spectrometry coupled with multiple fragmentation techniques enabled complete characterization of N- and C-termini of a truncated protein.

Thermo Fisher Scientific
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Last update:
‎10-15-2021 07:06 AM
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