Toward top-down determination of PEGylation site using MALDI in-source decay MS analysis.
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/Deltam 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.
Yoo
Chul
C
Analytical Research and Development, Amgen Inc., Thousand Oaks, California 91320, USA. cyoo@amgen.com
Suckau
Detlev
D
Sauerland
Volker
V
Ronk
Michael
M
Ma
Minhui
M
D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D010455:Peptides; D011092:Polyethylene Glycols