Structural characterization of intact antibodies by high-resolution LTQ Orbitrap mass spectrometry

Journal of Mass Spectrometry : JMS
Jennifer ZhangViswanatham Katta

Abstract

Recombinant monoclonal antibodies (MAbs) can be heterogeneous due to modifications that can occur during expression, purification or during storage. These large multichain proteins (approximately 150 kDa) are structurally challenging for detailed characterization to identify the sites of modifications. We report the use of LTQ Orbitrap mass spectrometry to accurately measure the average masses of individual glycoforms by direct infusion of an intact antibody. To identify the site-specific modification of methionines in the antibody caused by forced oxidation, we used a 'middle-down' approach. The antibody was subjected to limited digestion using the endoproteinase Lys-C and reduced to generate Fab heavy chain, single chain Fc and light chain fragments (approximately 25 kDa each). These species were subjected to on-line liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) analysis using an LTQ Orbitrap, where these large precursors were dissociated by higher-energy collisions in the C-trap. High resolution and accuracy achieved for resulting fragments allowed us to show in a site-specific manner that only the methionines in the Fc heavy chain were oxidized under the studied conditions.

References

Mar 1, 1995·Analytical Chemistry·D S AshtonM A Smith
Jan 1, 1995·Free Radical Biology & Medicine·W Vogt
Dec 31, 1997·Journal of Pharmaceutical Sciences·X M LamJ L Cleland
Dec 10, 2003·Oncogene·Dale L LudwigPeter Bohlen
Jun 5, 2004·Biotechnology Progress·A Cecília A RoqueM Angela Taipa
Jun 12, 2004·Analytical Chemistry·Neil L Kelleher
Sep 25, 2004·Mass Spectrometry Reviews·Fanyu MengNeil L Kelleher
Mar 1, 2005·Journal of the American Society for Mass Spectrometry·John C Le, Pavel V Bondarenko
Apr 19, 2005·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Alain BeckJean-François Haeuw
Oct 27, 2005·Molecular & Cellular Proteomics : MCP·Jesper V OlsenMatthias Mann
Feb 16, 2006·Molecular & Cellular Proteomics : MCP·Boris MacekMatthias Mann
Apr 25, 2006·Journal of the American Society for Mass Spectrometry·Himanshu S GadgilPavel V Bondarenko
Jun 6, 2006·Journal of the American Society for Mass Spectrometry·Alexander MakarovStevan Horning
Jul 11, 2006·Advanced Drug Delivery Reviews·John R Birch, Andrew J Racher
Jul 11, 2006·Advanced Drug Delivery Reviews·Steven Kozlowski, Patrick Swann
Sep 26, 2006·Journal of Pharmaceutical Sciences·Wei WangSandeep Nema
Oct 19, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Abhinav A ShuklaDuncan Low
Dec 22, 2006·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Chris ChumsaeHongcheng Liu
Jan 6, 2007·Journal of Proteome Research·Ari M FrankPavel A Pevzner
Jun 27, 2007·Analytical Chemistry·Zhongqi Zhang, Bhavana Shah
Jul 11, 2007·Journal of Pharmaceutical Sciences·Himanshu S GadgilMichael Treuheit
Aug 28, 2007·Nature Methods·Jesper V OlsenMatthias Mann
Sep 11, 2007·Journal of Pharmaceutical Sciences·Hongcheng LiuJoanne Sun
Sep 29, 2007·Nature Methods·Nertila Siuti, Neil L Kelleher
Jan 19, 2008·Journal of Pharmaceutical and Biomedical Analysis·Gang Xiao, Pavel V Bondarenko
Aug 23, 2008·Mass Spectrometry Reviews·Zhongqi ZhangXiaoyu Chen
May 5, 2009·Journal of the American Society for Mass Spectrometry·Pavel V BondarenkoZhongqi Zhang
Feb 1, 1994·Journal of the American Society for Mass Spectrometry·S Guan, A G Marshall

❮ Previous
Next ❯

Citations

Jul 9, 2013·Analytical and Bioanalytical Chemistry·Johann HolzmannHansjoerg Toll
May 5, 2009·Journal of the American Society for Mass Spectrometry·Pavel V BondarenkoZhongqi Zhang
Jun 3, 2011·The Journal of Biological Chemistry·Jeremiah D TiptonNeil L Kelleher
Apr 23, 2021·Journal of the American Society for Mass Spectrometry·Iain D G Campuzano, Wendy Sandoval

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.