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Noncovalent interactions in human plasma proteins analyzed by the comparison of nondenaturing and denaturing micro‐2‐D gel electrophoresis patterns after polypeptide assignment using matrix‐assisted laser desorption/ionization‐mass spectrometry and peptide mass fingerprinting
Author(s) -
Manabe Takashi,
Jin Ya
Publication year - 2008
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200700893
Subject(s) - chemistry , gel electrophoresis , mass spectrometry , matrix assisted laser desorption/ionization , spots , chromatography , electrophoresis , two dimensional gel electrophoresis , desorption , proteomics , biochemistry , organic chemistry , adsorption , gene
Previously, we reported the analysis of human plasma proteins by 2‐DE under nondenaturing conditions (Type‐I 2‐DE) followed by the assignment of stained spots using MALDI‐MS and PMF [1]. Here, we employ 2‐DE conditions modified only in the second‐dimensional separation; SDS was added in the gradient slab gel aiming to dissociate noncovalently bound proteins/polypeptides (Type‐II 2‐DE). Totally 169 CBB‐stained spots on a micro‐2‐DE gel were numbered and subjected to polypeptide assignment using MALDI‐MS‐PMF. One hundred sixty spots out of the 169 provided significant match ( p <0.05) with polypeptides in databases. Comparisons of the results of polypeptide assignment on the two 2‐DE patterns indicated that 10 polypeptides in 20 stained spots on the Type‐I 2‐DE pattern [1] shifted toward low‐molecular‐weight positions on the Type‐II 2‐DE pattern, demonstrating the presence of noncovalent interactions. Seventeen polypeptides in 38 stained spots were only assigned on the Type‐II 2‐DE gel, which could mostly be accounted for by the disruption of noncovalent protein–protein interactions in the presence of SDS, i.e ., protein/polypeptide complexes which might form smear bands on the Type‐I 2‐DE gel dissociate to form clear spots on the Type‐II 2‐DE gel. The method employed here, comparisons of nondenaturing and denaturing 2‐DE maps with polypeptide assignment by MALDI‐MS‐PMF, would enable the simultaneous detection of multiple noncovalent interactions in complex protein/polypeptide systems.