Premium
Protein interactomics based on direct molecular fishing on paramagnetic particles: Practical realization and further SPR validation
Author(s) -
Ivanov Alexis S.,
Medvedev Alexei,
Ershov Pavel,
Molnar Andrey,
Mezentsev Yury,
Yablokov Evgeny,
Kaluzhsky Leonid,
Gnedenko Oksana,
Buneeva Olga,
Haidukevich Irina,
Sergeev Gennadiy,
Lushchyk Aliaksandr,
Yantsevich Alexey,
Medvedeva Marina,
Kozin Sergey,
Popov Igor,
Novikova Svetlana,
Zgoda Victor,
Gilep Andrey,
Usanov Sergey,
Lisitsa Andrey,
Archakov Alexander
Publication year - 2014
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.201400117
Subject(s) - proteomics , computational biology , interactome , chemistry , biochemistry , biology , gene
There is increasing evidence that proteins function in the cell as integrated stable or temporally formed protein complexes, interactomes. Previously, using model systems we demonstrated applicability of direct molecular fishing on paramagnetic particles for protein interactomics (Ershov et al. Proteomics , 2012, 12 , 3295). In the present study, we have used a combination of affinity‐based molecular fishing and subsequent MS for investigation of human liver proteins involved in interactions with immobilized microsomal cytochrome b5 ( CYB 5A), and also transthyretin and BSA as alternative affinity ligands (baits). The LC − MS / MS identification of prey proteins fished on these baits revealed three sets of proteins: 98, 120, and 220, respectively. Comparison analysis of these sets revealed only three proteins common for all the baits. In the case of paired analysis, the number of common proteins varied from 2 to 9. The binding capacity of some identified proteins has been validated by a SPR ‐based biosensor. All the investigated proteins effectively interacted with the immobilized CYB5A (Kd values ranged from 0.07 to 1.1 μM). Results of this study suggest that direct molecular fishing is applicable for analysis of protein–protein interactions ( PPI ) under normal and pathological conditions, in which altered PPI s are especially important.