title: Protein interactomics based on direct molecular fishing on paramagnetic particles: practical realization and further SPR validation.
abstract: 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 (CYB5A), 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 PPIs are especially important.
authors.affiliation: Orechovich Institute of Biomedical Chemistry, Moscow, Russia.
authors.forename: Alexis S
authors.initials: AS
authors.lastname: Ivanov
authors.forename: Alexei
authors.initials: A
authors.lastname: Medvedev
authors.forename: Pavel
authors.initials: P
authors.lastname: Ershov
authors.forename: Andrey
authors.initials: A
authors.lastname: Molnar
authors.forename: Yury
authors.initials: Y
authors.lastname: Mezentsev
authors.forename: Evgeny
authors.initials: E
authors.lastname: Yablokov
authors.forename: Leonid
authors.initials: L
authors.lastname: Kaluzhsky
authors.forename: Oksana
authors.initials: O
authors.lastname: Gnedenko
authors.forename: Olga
authors.initials: O
authors.lastname: Buneeva
authors.forename: Irina
authors.initials: I
authors.lastname: Haidukevich
authors.forename: Gennadiy
authors.initials: G
authors.lastname: Sergeev
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Lushchyk
authors.forename: Alexey
authors.initials: A
authors.lastname: Yantsevich
authors.forename: Marina
authors.initials: M
authors.lastname: Medvedeva
authors.forename: Sergey
authors.initials: S
authors.lastname: Kozin
authors.forename: Igor
authors.initials: I
authors.lastname: Popov
authors.forename: Svetlana
authors.initials: S
authors.lastname: Novikova
authors.forename: Victor
authors.initials: V
authors.lastname: Zgoda
authors.forename: Andrey
authors.initials: A
authors.lastname: Gilep
authors.forename: Sergey
authors.initials: S
authors.lastname: Usanov
authors.forename: Andrey
authors.initials: A
authors.lastname: Lisitsa
authors.forename: Alexander
authors.initials: A
authors.lastname: Archakov
mesh_terms: D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods; D015786:Cytochromes b5 / Q000378:metabolism*; D006801:Humans; D055767:Immobilized Proteins / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000378:metabolism*; D011228:Prealbumin / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000378:metabolism; D020349:Surface Plasmon Resonance / Q000379:methods*; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D055767:Immobilized Proteins; D008024:Ligands; D011228:Prealbumin; D012710:Serum Albumin, Bovine; D015786:Cytochromes b5
keywords: LC-MS/MS; Molecular fishing; Nanoproteomics; Paramagnetic particles; Protein interactomics; SPR biosensor