title: Identifying Unknown Enzyme-Substrate Pairs from the Cellular Milieu with Native Mass Spectrometry.
abstract: The enzyme-substrate complex is inherently transient, rendering its detection difficult. In our framework designed for bisubstrate systems-isotope-labeled, activity-based identification and tracking (IsoLAIT)-the common substrate, such as S-adenosyl-l-methionine (AdoMet) for methyltransferases, is replaced by an analogue (e.g., S-adenosyl-l-vinthionine) that, as a probe, creates a tightly bound [enzyme⋅substrate⋅probe] complex upon catalysis by thiopurine-S-methyltransferase (TPMT, EC 2.1.1.67). This persistent complex is then identified by native mass spectrometry from the cellular milieu without separation. Furthermore, the probe's isotope pattern flags even unknown substrates and enzymes. IsoLAIT is broadly applicable for other enzyme systems, particularly those catalyzing group transfer and with multiple substrates, such as glycosyltransferases and kinases.
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
authors.forename: Kalli C
authors.identifier: 0000-0003-1941-0678
authors.initials: KC
authors.lastname: Catcott
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH, 43210, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Yan
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
authors.forename: Wanlu
authors.initials: W
authors.lastname: Qu
authors.affiliation: Department of Chemistry and Biochemistry, The Ohio State University, 100 West 18th Avenue, Columbus, OH, 43210, USA.
authors.forename: Vicki H
authors.initials: VH
authors.lastname: Wysocki
authors.affiliation: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.|Barnett Institute of Chemical and Biological Analysis, Northeastern University, 360 Huntington Avenue, Boston, MA, 02115, USA.
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D002250:Carbon Radioisotopes; D004926:Escherichia coli / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism; D015335:Molecular Probes / Q000032:analysis* / Q000737:chemistry; D009590:Nitrogen Radioisotopes; D012435:S-Adenosylhomocysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D002250:Carbon Radioisotopes; D015335:Molecular Probes; D009590:Nitrogen Radioisotopes; C000622610:S-adenosylvinthionine; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C022745:thiopurine methyltransferase
keywords: bioorganic chemistry; enzymes; mass spectrometry; substrate identification; transferases