title: Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP).
abstract: Efficient protein solubilization using detergents is required for in-depth proteome analysis, but successful LC-MS/MS analysis greatly depends on proper detergents removal. A commonly used sample processing method is the filter-aided sample preparation (FASP), which allows protein digestion and detergent removal on the same filtration device. Many optimizations of the FASP protocol have been published, but there is no information on the influence of the filtration unit typology on the detergents removal. The aim of this study was to compare the performance of conic and flat bottom filtration units in terms of number of proteins identified by LC-MS/MS. We have analyzed 1, 10 and 100 μg of total cell lysate prepared using lysis buffer with different SDS concentrations. We compared the FASP protocol using conic and flat bottom filtration units to ethanol precipitation method. Subsequently, we applied our most performant protocol to single murine pancreatic islet, and identified up to 2463 protein using FASP versus 1169 proteins using ethanol precipitation. We conclude that FASP performance depends strongly on the filter shape: flat bottom devices are better suited for low-protein samples, as they allow better SDS removal leading to the identification of greater number of proteins.
authors.affiliation: The CPN Proteomics Facility - 3P5, Center of Psychiatry and Neuroscience, UMR INSERM 894, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Joanna
authors.initials: J
authors.lastname: Lipecka
authors.affiliation: Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Cerina
authors.initials: C
authors.lastname: Chhuon
authors.affiliation: Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Bourderioux
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité, 1151, Paris, France.|Centre National de la Recherche Scientifique, Unité, 8253, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Marie-Andrée
authors.initials: MA
authors.lastname: Bessard
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité, 1151, Paris, France.|Centre National de la Recherche Scientifique, Unité, 8253, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Peter
authors.initials: P
authors.lastname: van Endert
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité, 1151, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Edelman
authors.affiliation: Proteomic Platform Necker, PPN-3P5, Structure Fédérative de Recherche SFR Necker US24, Paris, France.|Université Paris Descartes, Sorbonne Paris Cité, Paris, France.
authors.forename: Ida Chiara
authors.initials: IC
authors.lastname: Guerrera
mesh_terms: D000818:Animals; D002460:Cell Line; D005591:Chemical Fractionation / Q000295:instrumentation / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003902:Detergents / Q000302:isolation & purification*; D004867:Equipment Design; D005374:Filtration / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007515:Islets of Langerhans / Q000737:chemistry*; D051379:Mice; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D012995:Solubility; D053719:Tandem Mass Spectrometry / Q000295:instrumentation* / Q000379:methods
chemical_list: D003902:Detergents; D020543:Proteome
keywords: FASP; SDS removal; Sample preparation; Sensitivity; Single pancreatic islet; Technology