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Updated: May 9, 2026
Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome
Published on: April 2, 2018
Analysis of trypsin inhibition activity in human plasma proteins after separation by non-denaturing two-dimensional
Youji Shimazaki1, Madoka Michhiro
1Graduate School of Science and Engineering (Science Section) and Venture Business Laboratory, Ehime University, Matsuyama, Japan.
Background:
The functions of proteins can be retained following separation by non-denaturing two-dimensional electrophoresis (2-DE). The trypsin inhibition activities can then be examined following the separation and immobilization of the proteins under non-denaturing conditions.
Methods:
Human plasma proteins were separated using 2-DE and transferred onto a polyvinylidene difluoride membrane and stained using Ponceau S. The trypsin inhibition activity of the membrane-bound proteins was qualitatively examined using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The activities were also quantitatively examined by analyzing the release of the azo-chromophore when azocasein was the substrate.
Results:
Trypsin activity was inhibited by the haptoglobin and α2-macroglobulin spots located on the membrane, whereas the protease activity was retained for the spots containing albumin and transferrin. The inhibition activities of the α2-macroglobulin and haptoglobin spots were 4.81- and 4.83-fold higher, respectively, when compared with the inhibition activity of the albumin spot. An axis of the relative activities of trypsin inhibition was added to the 2-DE pattern of human plasma proteins to construct a non-denaturing 3-D map of human plasma proteins.
Conclusion:
This 3-D map should represent a suitable diagnostic tool for the qualitative and quantitative analyses of the trypsin inhibition activities of proteins.
