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Anal Chem. 2018 Sep 18;90(18):10670-10675. doi: 10.1021/acs.analchem.8b02563. Epub 2018 Sep 6.

Heat-Induced Rearrangement of the Disulfide Bond of Lactoglobulin Characterized by Multiply Charged MALDI-TOF/TOF Mass Spectrometry.

Zhan L1,2, Liu Y2, Xie X1,2, Xiong C1, Nie Z1,3.

Author information

1
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry , Chinese Academy of Sciences , Beijing 100190 , China.
2
University of Chinese Academy of Sciences , Beijing 100049 , China.
3
National Center for Mass Spectrometry in Beijing , Beijing 100190 , China.

Abstract

Disulfide bonds are an important post-translational modification of proteins and play a significant role in stabilizing protein structure. While both mass spectrometry-based bottom-up and top-down proteomics are widely used in the identification of disulfide linkages, the top-down approach can avoid potential information loss of disulfide linkage occurring in the bottom-up analysis. In the present work, we applied matrix-assisted laser desorption/ionization tandem Time-of-Flight (MALDI-TOF/TOF) mass spectrometry to investigate the heat-induced disulfide rearrangement of β-lactoglobulin (β-LG). Since β-LG (18 kDa) is too large for common TOF/TOF analysis, we use 2-nitrophloroglucinol (2-NPG) as a matrix to generate multiply charged proteins by MALDI. Fragmentation of doubly charged protein ions yields characteristic triplet peaks of disulfide bonds. We found that the characteristic fragments derived from the heterolytic cleavage of disulfide bonds decreased sharply when the incubation temperature of β-LG solution reached the critical point of 75 °C. These results indicate that the disulfide linkage between C160 and C66 has been broken during the heating process and, probably, new disulfide formed. In conclusion, our work highlights the analytical value of the multiply charged MALDI-TOF/TOF method in the identification of larger proteins (>12 kDa) and disulfide-containing proteins.

PMID:
30139250
DOI:
10.1021/acs.analchem.8b02563
[Indexed for MEDLINE]

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