使用LTQ Orbitrap ETD自上而下的对蛋白质进行N端和C端测序
简介：
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.
仪器：
Thermo Scientific LTQ Orbitrap XL ETD mass spectrometer
总结：
• Confident and complete characterization of the N-terminus and C-terminus of a truncated protein can be achieved using a top-down approach on an LTQ Orbitrap XL ETD hybrid mass spectrometer with ProSightPC software.
• In many cases, a simple top-down ETD MS2 approach is sufficient to obtain intact protein N- and C-terminal sequence.
• Top-down CID followed by ETD MS3 of product ions from CID MS2 can be used to locate truncation as well as to sequence the terminus of the protein. This approach can provide complete terminal sequence coverage and is therefore particularly useful when the first several amino acids from the terminus were missed using top-down ETD MS2.
• For a truncated protein with unknown terminus, ProSightPC is the software of choice for terminus identification as well as for N- and C-terminal sequencing. Extremely confident identifications can be obtained by analyzing high-resolution ETD data using the delta M mode of ProSightPC search under the BioMarker category.
