title: Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies.
abstract: Site-specific solvent accessibility of the primary amines (mainly lysine or the N-termini) on proteins is of great interest in many research areas because amines are an important functional group for protein conjugation. In this study, we coupled dimethyl labeling via reductive amination with liquid chromatography-mass spectrometry (LC-MS) to fully characterize the solvent accessibility of lysine residues and the N-termini on human immunoglobulin G (IgG). Circular dichroism (CD) and fluorescence spectroscopy revealed that dimethyl labeling did not alter the conformation of the native IgG molecule. Based on intact protein measurements, up to 28 (light chain) and 66 (heavy chain) dimethyl tags, covering all lysine residues and the N-termini, were sequentially incorporated into IgG molecules in 1000 s. All labeled sites were identified and quantified by a bottom-up proteomics approach. Some highly exposed hot-spots (for example, the N-termini of both the heavy and the light chains) and some buried sites (for example, K415 in the heavy chain and K39 in the light chain) were unambiguously revealed. This method was also used to characterize aggregation-induced structural changes in IgGs by increasing the temperature. Substantial changes in the labeling percentage of many lysine sites were observed, indicating a non-native aggregation triggered by thermal stress. Due to high labeling yields and the van der Waals surface of the labeling reagents being comparable to that of water, dimethyl labeling is a highly promising technique for probing the amine's surface topography of proteins. It can also be used as a complementary approach to other methods for resolving the higher-order structure of proteins by LC-MS.
authors.affiliation: Department of Chemistry, National Cheng Kung University , No. 1 College Road, Tainan 701, Taiwan, Republic of China.
authors.forename: Sin-Yi
authors.initials: SY
authors.lastname: Jhan
authors.affiliation: Department of Chemistry, National Cheng Kung University , No. 1 College Road, Tainan 701, Taiwan, Republic of China.
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Huang
authors.affiliation: Department of Chemistry, National Cheng Kung University , No. 1 College Road, Tainan 701, Taiwan, Republic of China.
authors.forename: Tzu-Fan
authors.initials: TF
authors.lastname: Wang
authors.affiliation: Department of Chemistry, National Cheng Kung University , No. 1 College Road, Tainan 701, Taiwan, Republic of China.
authors.forename: Ho-Hsuan
authors.initials: HH
authors.lastname: Chou
authors.affiliation: Department of Chemistry, National Cheng Kung University , No. 1 College Road, Tainan 701, Taiwan, Republic of China.
authors.forename: Shu-Hui
authors.initials: SH
authors.lastname: Chen
mesh_terms: D000588:Amines / Q000737:chemistry*; D002853:Chromatography, Liquid; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D040901:Proteomics
chemical_list: D000588:Amines; D007074:Immunoglobulin G; D008239:Lysine