title: Label-Free Identification and Quantification of SUMO Target Proteins.
abstract: Mass spectrometry-based approaches are utilized with increasing frequency to facilitate identification of novel SUMO target proteins and to elucidate the dynamics of SUMOylation in response to cellular stresses. Here, we describe a robust method for the identification of SUMO target proteins, and the relative quantification of SUMOylation dynamics, using a label-free approach. The method relies on a decahistidine (His10)-tagged SUMO, which is expressed at a low level in a mammalian cell line or model organism. The His10-tag allows for a single-step, high-yield, and high-purity enrichment of SUMOylated proteins, which are then digested and analyzed by high-resolution mass spectrometry. Matching between runs and label-free quantification integrated in the freely available MaxQuant software allow for a high rate and accuracy of quantification, providing a strong alternative to laborious sample or cell labeling techniques. The method described here allows for identification of >1000 SUMO target proteins, and characterization of their SUMOylation dynamics, without requiring sample fractionation. The purification procedure, starting from total lysate, can be performed in ~4 days.
authors.affiliation: Department of Molecular Cell Biology, Leiden University medical Center, Albinusdreef 2, 2300, Leiden, Netherlands.
authors.forename: Ivo A
authors.initials: IA
authors.lastname: Hendriks
authors.affiliation: Department of Molecular Cell Biology, Leiden University medical Center, Albinusdreef 2, 2300, Leiden, Netherlands. vertegaal@lumc.nl.
authors.forename: Alfred C O
authors.initials: AC
authors.lastname: Vertegaal
mesh_terms: D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011499:Protein Processing, Post-Translational; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism*; D012984:Software; D058207:Sumoylation; D053719:Tandem Mass Spectrometry / Q000379:methods; D014162:Transfection
chemical_list: D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D049452:Green Fluorescent Proteins; C033223:polyhistidine; D006639:Histidine
keywords: His10 pulldown; Label-free quantification; Mass spectrometry; Proteomics; SUMO; SUMO target purification