Studies of interaction between insulin and glutathione using electrospray ionization mass spectrometry.
BACKGROUND
The interaction of glutathione (GSH) with insulin plays an important role in the degradation or regulation of insulin. The characterization of the reaction products of GSH and insulin is very important for a proper understanding of the mechanism of insulin regulation of GSH.


METHODS
Solutions of insulin and glutathione were incubated under different experimental conditions in vitro. The reaction products were determined by electrospray ionization (ESI) ion trap mass spectrometry combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry.


RESULTS
The multi-reaction products were identified, including insulin A chain with two intrachain disulfides, insulin B chain with one intrachain disulfides, GSH-modified insulin, GSH-modified A chain, GSH-modified B chain, aggregates of A chain and B chain, and reduced A chain and B chain. The binding site of the B chain with insulin was determined directly by tandem mass spectrometry (MS/MS) without enzyme digestion. It was found that the reaction between GSH and insulin was pH-, O(2)- and temperature-dependent.


CONCLUSIONS
The results provide insight into the interaction between GSH and insulin. It has also been demonstrated that ESI-MS combined with high-resolution FTICRMS and MS/MS provides a powerful tool for screening the reactions of proteins and small molecules.
Changchun Center of Mass Spectrometry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, PR China.
Yonggang
Y
Du
Ningbo
N
Zhang
Meng
M
Cui
Zhiqiang
Z
Liu
Shuying
S
Liu
D000595:Amino Acid Sequence; D001665:Binding Sites; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013696:Temperature
D004220:Disulfides; D007328:Insulin; D006861:Hydrogen Peroxide; D005978:Glutathione; D010100:Oxygen