Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

A. S. Ivanov; P. V. Ershov; A. A. Molnar; Yu. V. Mezentsev; L. A. Kaluzhskiy; E. O. Yablokov; A. V. Florinskaya; O. V. Gnedenko; A. E. Medvedev; S. A. Kozin; V. A. Mitkevich; A. A. Makarov; A. A. Gilep; A. Ya. Luschik; I. V. Gaidukevich; S. A. Usanov

doi:10.1134/S1068162016010052

Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

Авторы: Ivanov A.S.¹, Ershov P.V.¹, Molnar A.A.¹, Mezentsev Y.V.¹, Kaluzhskiy L.A.¹, Yablokov E.O.¹, Florinskaya A.V.¹, Gnedenko O.V.¹, Medvedev A.E.¹, Kozin S.A.², Mitkevich V.A.², Makarov A.A.², Gilep A.A.³, Luschik A.Y.³, Gaidukevich I.V.³, Usanov S.A.³
Учреждения:
1. Institute of Biomedical Chemistry
2. Engelhardt Institute of Molecular Biology
3. Institute of Bioorganic Chemistry
Выпуск: Том 42, № 1 (2016)
Страницы: 14-21
Раздел: Article
URL: https://bakhtiniada.ru/1068-1620/article/view/227822
DOI: https://doi.org/10.1134/S1068162016010052
ID: 227822

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An original experimental method of direct molecular fishing has been developed for identification of potential partners of protein–protein and protein–peptide interactions. It is based on combination of surface plasmon resonance technology (SPR), size exclusion and affinity chromatography and mass spectrometric identification of proteins (LC-MS/MS). Previously, we demonstrated applicability of this method for protein interactomics using experimental model system, as well as in the pilot study in the frame of the Human Proteome Project (HPP). In the present paper, this method was successfully applied to identify possible molecular partners of 7 target proteins encoded by genes of 18 chromosome (also in the frame of the HPP). Fishing on the affinity sorbents with immobilized target proteins as ligands was carried out using total lysate of human liver tissue as well as pooled sets of fractions (individual for each bait-protein) obtained by means of a combination of size exclusion chromatography and SPR analysis for the presence of potential prey-proteins in each fraction. As a result we obtained lists of possible molecular partners of all 7 proteins and performed a comparative evaluation of direct fishing specificity for these target proteins. Direct molecular fishing was also successfully used for search of potential protein partners interacting with different isoforms of amyloid-beta peptide, playing a key role in the development of Alzheimer’s disease. The synthetic peptides that are analogues of the metal-binding domain isoforms of beta-amyloid were used as molecular baits and the fishing was performed in various fractions of immortalized human neural cells. As a result, 13 potential partner proteins were identified in the cytosol fraction of the cells by fishing on amyloid-beta peptide (1-16).

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intermolecular interactions, optical biosensor, proteins’ mass-spectrometry, molecular fishing, human proteome, Alzheimer’s disease

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Direct molecular fishing in molecular partners investigation in protein–protein and protein–peptide interactions

Полный текст

Аннотация

Ключевые слова

Об авторах

A. Ivanov

P. Ershov

A. Molnar

Yu. Mezentsev

L. Kaluzhskiy

E. Yablokov

A. Florinskaya

O. Gnedenko

A. Medvedev

S. Kozin

V. Mitkevich

A. Makarov

A. Gilep

A. Luschik

I. Gaidukevich

S. Usanov

Дополнительные файлы