title: [Mass spectrometric analysis of proteasomes affinity puirified from the human myelogenous leukemia cells K562].
abstract: 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.
authors.forename: T O
authors.initials: TO
authors.lastname: Artamonova
authors.forename: M A
authors.initials: MA
authors.lastname: Khodorkovskiĭ
authors.forename: A S
authors.initials: AS
authors.lastname: Tsimokha
mesh_terms: D015973:Gene Expression Regulation, Leukemic; D006360:Heat-Shock Proteins / Q000096:biosynthesis; D006801:Humans; D020014:K562 Cells; D007951:Leukemia, Myeloid / Q000235:genetics* / Q000473:pathology; D013058:Mass Spectrometry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D040901:Proteomics
chemical_list: D006360:Heat-Shock Proteins; C108898:PSMB4 protein, human; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease