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.

Atomic Force Microscopy for Protein Detection and Their Physicoсhemical Characterization

“…AFM-based molecular detector combines a method itself and fishing technique (AFM-fishing) [ 127 ]. Fishing is a process of catching target proteins from solution using “bait” [ 128 ]. Molecular fishing is an adaptation of affinity enrichment of target molecules based on the specific interaction between immobilized ligand (bait molecule) and one or several assumed functional partners (captured molecule).…”

The analysis of participation of individual proteins in the protein interactome formation

“…1). Это соответствует данным о взаимодействии цитохрома b 5 c CYP3A4 и CYP3A5 [11] и цитохрома b 5 с глицеральдегид-3-фосфатдегидрогеназой (GAPDH) [6], полученным нами ранее с помощью SPR-биосенсора.…”

“…Однако и данная технология также не лишена ряда недостатков -она охватывает лишь локальные участки интерактома и не обеспечивает его системный (омикс) анализ, а идентифицированные белки зачастую оказываются вторичными и третичными партнерами целевого белка или даже посторонними примесями в составе сложных мицелл и агрегатов, образующихся при приготовлении лизата исследуемого биологического материала [6].…”

Influence of gravity discharge on the content of isatin-binding proteins in mice: results of ground-based and space research under the program Bion-M №1

“…Все процедуры пробоподготовки для масс-спектрометрической идентификации белков выполнены в концентрирующих пробирках с ультрафильтром Vivaspin 500 (MWCO 10 кДа) в соответствии с протоколом, приведённым в [10].…”