title: The parallel reaction monitoring method contributes to a highly sensitive polyubiquitin chain quantification.
abstract: Ubiquitylation is an essential posttranslational protein modification that is implicated in a diverse array of cellular functions. Although cells contain eight structurally distinct types of polyubiquitin chains, detailed function of several chain types including K29-linked chains has remained largely unclear. Current mass spectrometry (MS)-based quantification methods are highly inefficient for low abundant atypical chains, such as K29- and M1-linked chains, in complex mixtures that typically contain highly abundant proteins. In this study, we applied parallel reaction monitoring (PRM), a quantitative, high-resolution MS method, to quantify ubiquitin chains. The ubiquitin PRM method allows us to quantify 100attomole amounts of all possible ubiquitin chains in cell extracts. Furthermore, we quantified ubiquitylation levels of ubiquitin-proline-β-galactosidase (Ub-P-βgal), a historically known model substrate of the ubiquitin fusion degradation (UFD) pathway. In wild-type cells, Ub-P-βgal is modified with ubiquitin chains consisting of 21% K29- and 78% K48-linked chains. In contrast, K29-linked chains are not detected in UFD4 knockout cells, suggesting that Ufd4 assembles the K29-linked ubiquitin chain(s) on Ub-P-βgal in vivo. Thus, the ubiquitin PRM is a novel, useful, quantitative method for analyzing the highly complicated ubiquitin system.
authors.affiliation: Laboratory of Protein Metabolism, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Tsuchiya
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Saeki
mesh_terms: D015153:Blotting, Western; D008239:Lysine / Q000235:genetics / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009154:Mutation; D025821:Polyubiquitin / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; C509339:Ufd4 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D001616:beta-Galactosidase; D008239:Lysine