Mass spectrometric analysis of proteasomes affinity puirified from the human myelogenous leukemia cells K562

• Published in: Bioorganicheskaia khimiia Vol. 40; no. 6; p. 720
• Main Authors: Artamonova, T O, Khodorkovskiĭ, M A, Tsimokha, A S
• Format: Journal Article
• Language: Russian
• Published: Russia (Federation) 01.11.2014
• Subjects: Gene Expression Regulation, Leukemic; Heat-Shock Proteins - biosynthesis; Humans; K562 Cells; Leukemia, Myeloid - genetics; Leukemia, Myeloid - pathology; Mass Spectrometry; Proteasome Endopeptidase Complex - chemistry; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - isolation & purification; Proteasome Endopeptidase Complex - metabolism; Proteomics
• ISSN: 0132-3423

Proteasomes carry out regulated proteolysis of most proteins and thereby play a crucial role in the regulation of different cellular processes. Dissecting subunit composition and post-translational modifications of proteasome is one of the important milestones in understanding their functions and mechanisms of regulation in the cell. To this end a strategy we followed a strategy for affinity purification of proteasomes from human myeloid leukemia cells with subsequent mass spectrometric analysis. Proteasomes were purified from the stable cell line K562 expressing HTBH tag-labeled 20S proteasome subunit β7 (PSMB4) by non-covalent affinity purification on biotin-avidin beads, followed by elution with TEV protease. We identified all known subunits of the 26S proteasome, as well as PA200 and regulators PA28γ amongst the eluted proteins, using MALDI-ICR mass spectrometry. We have shown that the proteasomes are associated with heat shock proteins, components of the ubiquitin-proteasome system of some cytoskeleton proteins. A number of novel phosphorylation, ubiquitination and N-terminal modification of proteasome subunits were found for 16 proteasome subunits. Our results might be useful for further proteomic studies of proteasomes.