Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies

Sin Yi Jhan, Li Juan Huang, Tzu Fan Wang, Ho-Hsuan Chou, Shu-Hui Chen

Research output: Contribution to journalArticle

7 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)4255-4263
Number of pages9
JournalAnalytical Chemistry
Volume89
Issue number7
DOIs
Publication statusPublished - 2017 Apr 4

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Labeling
Amines
Mass spectrometry
Monoclonal Antibodies
Lysine
Immunoglobulin G
Liquid chromatography
Proteins
Agglomeration
Circular dichroism spectroscopy
Amination
Molecules
Fluorescence spectroscopy
Surface topography
Thermal stress
Functional groups
Conformations
Membrane Proteins
Water
Temperature

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

Cite this

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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.",
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Dimethyl Labeling Coupled with Mass Spectrometry for Topographical Characterization of Primary Amines on Monoclonal Antibodies. / Jhan, Sin Yi; Huang, Li Juan; Wang, Tzu Fan; Chou, Ho-Hsuan; Chen, Shu-Hui.

In: Analytical Chemistry, Vol. 89, No. 7, 04.04.2017, p. 4255-4263.

Research output: Contribution to journalArticle

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AU - Jhan, Sin Yi

AU - Huang, Li Juan

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AU - Chen, Shu-Hui

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