title: A periodate-cleavable linker for functional proteomics under slightly acidic conditions: application for the analysis of intracellular aspartic proteases.
abstract: Specific elution of captured proteins greatly improves the quality of proteomic data obtained from pull-downs by avoiding signals from nonspecific proteins, thus allowing more sensitive identification of target proteins. This is important in activity-based proteomics or drug target identification. However, commonly used chemically cleavable linkers can only be cleaved at close to neutral pH, which prevents them from being used for proteins binding only at lower pH when no cross-linking is applied. On the other hand, elution of common acid-cleavable labels can also coelute proteins bound by ionic interactions. Here, we report the synthesis and application of a label readily cleavable by mild oxidation at moderately acidic pH for the noncovalent labeling and pull-down of intracellular aspartic proteases. Using specific release, target proteins cathepsin D and napsin A were identified from human kidney with much higher confidence and without any nontarget hits.
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tuebingen, Ob dem Himmelreich 7, 72074 Tuebingen, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Maurer
authors.forename: Claus
authors.initials: C
authors.lastname: Zeyher
authors.forename: Bushra
authors.initials: B
authors.lastname: Amin
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000302:isolation & purification; D002402:Cathepsin D / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure; D010504:Periodic Acid / Q000737:chemistry*; D040901:Proteomics; D013194:Staining and Labeling
chemical_list: D010504:Periodic Acid; C009288:metaperiodate; D057055:Aspartic Acid Proteases; D016282:Aspartic Acid Endopeptidases; C498549:NAPSA protein, human; C533922:CTSD protein, human; D002402:Cathepsin D