title: Ultraviolet matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for phosphopeptide analysis with a solidified ionic liquid matrix.
abstract: A solidified ionic liquid matrix (SILM) consisting of 3-aminoquinoline, α-cyano-4- hydroxycinnamic acid and ammonium dihydrogen phosphate combines the benefits of liquid and solid MALDI matrices and proves to be well suitable for phosphopeptide analysis using MALDI-MS in the low femtomole range. Desalting and buffer exchange that typically follow after phosphopeptide elution from metal oxide affinity chromatography (MOAC) materials can be omitted. Shifting the pH from acidic to basic during target preparation causes slow matrix crystallization and homogeneous embedding of the analyte molecules, forming a uniform preparation from which (phospho)peptides can be ionized in high yields over long periods of time. The novel combination of MOAC-based phosphopeptide enrichment with SILM preparation has been developed with commercially available standard phosphopeptides and with α-casein as phosphorylated standard protein. The applicability of the streamlined phosphopeptide analysis procedure to cell biological and clinical samples has been tested (i) using affinity-enriched endogenous TRIM28 from cell cultures and (ii) by analysis of a two-dimensional gel-separated protein spot from a bladder cancer sample.
authors.affiliation: Proteome Center Rostock, University of Rostock, Germany.. gargee.mukherjee@uni-rostock.de.
authors.forename: Gargee
authors.initials: G
authors.lastname: Mukherjee
authors.affiliation: Proteome Center Rostock, University of Rostock, Germany.. claudia.roewer@uni-rostock.de.
authors.forename: Claudia
authors.initials: C
authors.lastname: Claudia Röwer
authors.affiliation: Proteome Center Rostock, University of Rostock, Germany.. cornelia.koy@uni-rostock.de.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Koy
authors.affiliation: Urology Clinic and Polyclinic, University Medicine Rostock, Germany.. chris.protzel@med.uni-rostock.de.
authors.forename: Chris
authors.initials: C
authors.lastname: Protzel
authors.affiliation: Institute of Immunology, University Medicine Rostock, Germany.. peter.lorenz@med.uni-rostock.de.
authors.forename: Peter
authors.initials: P
authors.lastname: Lorenz
authors.affiliation: Institute of Immunology, University Medicine Rostock, Germany.. hans-juergen.thiesen@med.uni-rostock.de.
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Thiesen
authors.affiliation: Urology Clinic and Polyclinic, University Medicine Rostock, Germany. oliver.hakenberg@med.uni- rostock.de.
authors.forename: Oliver W
authors.initials: OW
authors.lastname: Hakenberg
authors.affiliation: Proteome Center Rostock, University of Rostock, Germany.. michael.glocker@med.uni-rostock.de.
authors.forename: Michael O
authors.initials: MO
authors.lastname: Glocker
mesh_terms: D000465:Algorithms; D006367:HeLa Cells; D006801:Humans; D052578:Ionic Liquids / Q000737:chemistry*; D044367:Phase Transition; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D052578:Ionic Liquids; D010748:Phosphopeptides