Structural analysis of intact monoclonal antibodies by electron transfer dissociation mass spectrometry.
Improving qualitative and quantitative characterization of monoclonal antibodies is essential, because of their increasing popularity as therapeutic drug targets. Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth characterization of post-translationally modified large peptides, small- and medium-sized proteins, and noncovalent protein complexes. Here, we describe the performance of ETD-based top-down mass spectrometry for structural analysis of intact 150 kDa monoclonal antibodies, immunoglobulins G (IgGs). Simultaneous mass analysis of intact IgGs as well as a complex mixture of ETD product ions at sufficiently high resolution and mass accuracy in a wide m/z range became possible because of recent advances in state-of-the-art time-of-flight (TOF) mass spectrometry. High-resolution ETD TOF MS performed on IgG1-kappa from murine myeloma cells and human anti-Rhesus D IgG1 resulted in extensive sequence coverage of both light and heavy chains of IgGs and revealed information on their variable domains. Results are superior and complementary to those previously generated by collision-induced dissociation. However, numerous disulfide bonds drastically reduce the efficiency of top-down ETD fragmentation within the protected sequence regions, leaving glycosylation uncharacterized. Further increases in the experiment sensitivity and improvement of ion activation before and after ETD reaction are needed to target S-S bond-protected sequence regions and post-translational modifications.
Biomolecular Mass Spectrometry Laboratory, Ecole Polytechnique Fédérale de Lausanne, 1015 Lausanne, Switzerland. yury.tsybin@epfl.ch
Yury O
YO
Tsybin
Luca
L
Fornelli
Carsten
C
Stoermer
Markus
M
Luebeck
Julien
J
Parra
Sophie
S
Nallet
Florian M
FM
Wurm
Ralf
R
Hartmer
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002460:Cell Line; D004579:Electron Transport; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021241:Spectrometry, Mass, Electrospray Ionization
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G