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Orbitrap_SciLib
Reputable Mentor II
Reputable Mentor II
Fornelli L, Damoc E, Thomas PM, Kelleher NL, Aizikov K, Denisov E, Makarov A, Tsybin YO.
Mol Cell Proteomics. 2012 Dec;11(12):1758-67.
Primary structure information of proteins employed as bio-therapeutics is essential to understand their structure-function relationship, in the rational design of new therapeutics, and for quality control. Given both the large size (around 150 kDa) and structural complexity of intact immunoglobulins G (IgGs), which include a variable number of disulfide bridges, their extensive fragmentation and subsequent sequence determination by tandem mass spectrometry (MS) are challenging. Here, we applied electron transfer dissociation (ETD) implemented on a hybrid Orbitrap Fourier transform mass spectrometer (FTMS) to analyze a commercial recombinant IgG in a liquid chromatography (LC)-tandem mass spectrometry (MS/MS) top-down experiment. The lack of sensitivity typically observed during top-down MS of large proteins was addressed by averaging time-domain transients recorded in different LC-MS/MS experiments before performing FT signal processing. The results demonstrate that improved signal-to-noise ratio combined with higher resolution and mass accuracy provided by Orbitrap FTMS (compared to previous applications of top-down ETD-based proteomics on IgGs) are essential for their comprehensive analysis. Specifically, ETD on Orbitrap FTMS produced about 33% sequence coverage of an intact IgG, signifying an almost two-fold increase in IgG sequence coverage in comparison with previous ETD-based analysis of intact monoclonal antibodies of a similar subclass. These results suggest the potential application of the developed methodology to other classes of large proteins and biomolecules.

http://www.mcponline.org/content/early/2012/09/10/mcp.M112.019620.full.pdf
Ecole Polytechnique Federale de Lausanne, Switzerland;
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