title: An optimised version of the secretome protein enrichment with click sugars (SPECS) method leads to enhanced coverage of the secretome.
abstract: The secretome, the entirety of all soluble proteins either being secreted or proteolytically released by a cell, plays a key role in inter-cellular communication of multi-cellular organisms. Pathological alterations contribute to diseases such as hypertension, cancer, autoimmune disorders or neurodegenerative diseases. Hence, studying disease-related perturbations of the secretome and the secretome itself covers an important aspect of cellular physiology. We recently developed the secretome protein enrichment with click sugars (SPECS) method that enables the analysis of secretomes of in vitro cell cultures even in the presence of FCS with MS. So far, SPECS facilitated the identification of protease substrates of BACE1, SPPL3 and ADAM10. Though, the SPECS method has already enabled deep insights into secretome biology, we aimed to improve the SPECS protocol to obtain even more information from MS-based secretome analysis and reduce the amount of input material. Here, we optimised the reaction buffer, the pH and replaced Dibenzocyclooctyne (DBCO) PEG12-biotin with the more water-soluble variant DBCO-sulpho-biotin to finally provide an optimised protocol of the recently published SPECS protocol. Overall, the number of quantified glycoproteins and their average sequence coverage was increased by 1.6- and 2.4-fold, respectively. Thus, the opzimised SPECS protocol allows reducing the input material by half without losing information. These improvements make the SPECS method more sensitive and more universal applicable to cell types with limited availability.
authors.affiliation: Institut für Allgemeine Pathologie und pathologische Anatomie, Technische Universität München, München, Germany.|Institute for Advanced Study, Technische Universität München, Garching, Germany.
authors.forename: Alperen
authors.initials: A
authors.lastname: Serdaroglu
authors.affiliation: Lehrstuhl für Neuroproteomik, Klinikum rechts der Isar, Technische Universität München, München, Germany.|Deutsches Zentrum für Neurodegenerative Erkrankungen e.V., München, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Müller
authors.affiliation: Institut für Toxikologie und Genetik, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Ute
authors.initials: U
authors.lastname: Schepers
authors.affiliation: Institut für Toxikologie und Genetik, Karlsruhe Institute of Technology, Karlsruhe, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bräse
authors.affiliation: Institut für Allgemeine Pathologie und pathologische Anatomie, Technische Universität München, München, Germany.
authors.forename: Wilko
authors.initials: W
authors.lastname: Weichert
authors.affiliation: Institute for Advanced Study, Technische Universität München, Garching, Germany.|Lehrstuhl für Neuroproteomik, Klinikum rechts der Isar, Technische Universität München, München, Germany.|Deutsches Zentrum für Neurodegenerative Erkrankungen e.V., München, Germany.|Munich Cluster for Systems Neurology (SyNergy), Munich, Germany.
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
authors.affiliation: Institut für Allgemeine Pathologie und pathologische Anatomie, Technische Universität München, München, Germany.|Institute for Advanced Study, Technische Universität München, Garching, Germany.
authors.forename: Peer-Hendrik
authors.initials: PH
authors.lastname: Kuhn
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D057930:Click Chemistry / Q000379:methods*; D034242:Cyclooctanes / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism; D051379:Mice; D040901:Proteomics / Q000379:methods*
chemical_list: D034242:Cyclooctanes; D006023:Glycoproteins
keywords: Bioorthogonal chemistry; Glycoprotein enrichment; Mass spectrometry; Secretome; Systems biology