title: Identification of ADP-ribosylation sites of CD38 mutants by precursor ion scanning mass spectrometry.
abstract: Protein ADP-ribosylation, including mono- and poly-ADP-ribosylation, is increasingly recognized to play important roles in various biological pathways. Molecular understanding of the functions of ADP-ribosylation requires the identification of the sites of modification. Although tandem mass spectrometry (MS/MS) is widely recognized as an effective means for determining protein modifications, identification of ADP-ribosylation sites has been challenging due to the labile and hydrophilic nature of the modification. Here we applied precursor ion scanning-triggered MS/MS analysis on a hybrid quadrupole linear ion trap mass spectrometer for selectively detecting ADP-ribosylated peptides and determining the auto-ADP-ribosylation sites of CD38 (cluster of differentiation 38) E226D and E226Q mutants. CD38 is an enzyme that catalyzes the hydrolysis of nicotinamide adenine dinucleotide (NAD) to ADP-ribose. Here we show that NAD can covalently label CD38 E226D and E226Q mutants but not wild-type CD38. In this study, we have successfully identified the D226/Q226 and K129 residues of the two CD38 mutants being the ADP-ribosylation sites using precursor ion scanning hybrid quadrupole linear ion trap mass spectrometry. The results offer insights about the CD38 enzymatic reaction mechanism. The precursor ion scanning method should be useful for identifying the modification sites of other ADP-ribosyltransferases such as poly(ADP-ribose) polymerases.
authors.affiliation: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Robert
authors.initials: R
authors.lastname: Sherwood
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Xuling
authors.initials: X
authors.lastname: Zhu
authors.forename: Qun
authors.initials: Q
authors.lastname: Liu
authors.forename: Richard
authors.initials: R
authors.lastname: Graeff
authors.forename: Irina A
authors.initials: IA
authors.lastname: Kriksunov
authors.forename: Hon Cheung
authors.initials: HC
authors.lastname: Lee
authors.forename: Quan
authors.initials: Q
authors.lastname: Hao
authors.forename: Hening
authors.initials: H
authors.lastname: Lin
mesh_terms: D051997:ADP-ribosyl Cyclase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D009243:NAD / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011064:Poly Adenosine Diphosphate Ribose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D011506:Proteins; C024857:poly(ADP)-ribosylated proteins; D009243:NAD; D011064:Poly Adenosine Diphosphate Ribose; C498154:CD38 protein, human; D051997:ADP-ribosyl Cyclase 1