Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis |
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Authors: | Chul Yoo Detlev Suckau Volker Sauerland Michael Ronk Minhui Ma |
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Institution: | 1. Analytical Research and Development, Amgen, Inc., One Amgen Center Drive, Mail stop 25-2-A, 91320, Thousand Oaks, CA, USA 2. Bruker Daltonik GmbH, Bremen, Germany
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Abstract: | A novel matrix assisted laser desorption/ionization (MALDI)-based mass spectrometric approach has been evaluated to rapidly
analyze a custom designed PEGylated peptide that is 31 residues long and conjugated with 20 kDa linear polyethylene glycol
(PEG) at the side chain of Lys. MALDI-TOF MS provided sufficiently high resolution to allow observation of each of the oligomers
of the heterogeneous PEGylated peptide (m/Δm of ca. 500), while a typical ESI-MS spectrum of this molecule was extremely complex
and unresolved. Reflector in-source decay (reISD) analysis using MALDI-TOF MS was attempted to identify the PEGylation site
at intact molecular level without any sample treatment. An reISD spectrum of the free peptide was observed with abundant c-,
y-, and z + 2]-fragment ion series, whereas, in the fragmented PEGylated peptide, the fragment ion series were truncated
at the residue where PEG was attached. Therefore, a direct comparison of these top-down reISD spectra suggested the location
of the PEGylation site. Results from this study demonstrate a clear analytical utility of the ISD technique to characterize
structural aspects of heterogeneous biomolecules. |
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