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

Abstract

It becomes increasingly clear that most proteins of living systems exist as components of various protein complexes rather than individual molecules. The use of various proteomic techniques significantly extended our knowledge not only about functioning of individual complexes but also formed a basis for systemic analysis of protein-protein interactions. In this study gel-filtration chromatography accompanied by mass-spectrometry was used for the interactome analysis of human liver proteins. In six fractions (with average molecular masses of 45 kDa, 60 kDa, 85 kDa, 150 kDa, 250 kDa, and 440 kDa) 797 proteins were identified. In dependence of their distribution profiles in the fractions, these proteins could be subdivided into four groups: (1) single monomeric proteins that are not involved in formation of stable protein complexes; (2) proteins existing as homodimers or heterodimers with comparable partners; (3) proteins that are partially exist as monomers and partially as components of protein complexes; (4) proteins that do not exist in the monomolecular state, but also exist within protein complexes containing three or more subunits. Application of this approach to known isatin-binding proteins resulted in identification of proteins involved in formation of the homo- and heterodimers and mixed protein complexes.

Affinity Isolation and Mass Spectrometry Identification of Prostacyclin Synthase (PTGIS) Subinteractome

“…SPR biosensor makes it possible to perform the preliminary semi-quantitative characterization of tissue lysates according to either the presence or absence of protein partners for a bait protein [22,23]. Comparison of different rat tissue lysates (testis, lung, liver, heart, aorta, brain) revealed that the highest binding levels and moderate relative dissociation of the protein material bound from the lysates with immobilized PTGIS were in case of the testis lysate (Table 2).…”

“…Tissue lysates are experimental models containing all types of cells that can form a tissue, providing important information about the pattern of participation of a particular cell protein in the formation of stable protein complexes, and SEC profiling of tissue lysates is another tool for the disclosure of such information [12,22,23,25]. SEC profiling of lysate made it possible to expand molecular fishing information about identified proteins.…”

Quality Control Of FITC-labeled Proteins for Interactomics Investigations Using SDS Capillary Gel Electrophoresis and SPR Biosensor

“…Приготовление лизата ткани печени крысы осуществляли при помощи буферного раствора для экстракции белков CellLytic Mammalian Tissue Lysis/ Extraction Reagent («Sigma Aldrich») с добавлением коктейля ингибиторов протеаз («Sigma Aldrich») по методике, описанной в работе [1]. Содержание общего белка в образцах тканевого лизата определяли по методу Бредфорда.…”

“…Ключевые слова: поверхностный плазмонный резонанс (SPR); капиллярный гель-электрофорез; интерактомика; цитохром с; флуоресцентный краситель; FITC DOI: 10.18097/BMCRM00112 ВВЕДЕНИЕ В нашей предыдущей работе [1] были выполнены эксперименты по SEC (Size Exclusion Chromatography)профилированию тканевого лизата и, таким образом, стало возможным составить интерактомный «паспорт» для каждого идентифицированного белка в лизате. Было выявлено, что почти три четверти всех идентифицированных во фракциях белков находятся в виде гомо-и гетеродимерных комплексов и белковых комплексов более высокого порядка.…”