title: Enrichment and sequencing of phosphopeptides on indium tin oxide coated glass slides.
abstract: Unambiguous identification of phosphorylation sites is of premier importance to biologists, who seek to understand the role of phosphorylation from the perspective of site-specific control of biological phenomena. Despite this widely asked and highly specific information, many methods developed are aimed at analysis of complete proteomes, indeed even phospho-proteomes, surpassing the basic requests of many biologists. We have therefore further developed a simple method that specifically deals with the analysis of multiple phosphorylation sites on singular proteins or small collections of proteins. With this method, the whole purification process, from sample application to MALDI-MS analysis, can be performed on commercially available indium tin oxide (ITO) coated glass slides. We show that fifteen (15) samples can be purified within one hour, and that low femtomole sensitivity can be achieved. This limit of identification is demonstrated by the successful MS/MS-based identification of 6 fmol of monophosphopeptide from β-casein. We demonstrate that the method can be applied for identifying phosphorylation sites from recombinant and cell-derived biological protein samples. Since ITO-coated glass slides are inexpensive and available from several suppliers the method is readily and inexpensively available to other researchers. Taken together, the presented protocols and materials render this method as an extremely fast and sensitive phosphopeptide identification protocol that should aid biologists in discovery and validation of phosphorylation sites.
authors.affiliation: University of Turku, Centre for Biotechnology, Turku, Finland.
authors.forename: Petri
authors.initials: P
authors.lastname: Kouvonen
authors.forename: Eeva-Marja
authors.initials: EM
authors.lastname: Rainio
authors.forename: Veronika
authors.initials: V
authors.lastname: Suni
authors.forename: Päivi
authors.initials: P
authors.lastname: Koskinen
authors.forename: Garry L
authors.initials: GL
authors.lastname: Corthals
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002364:Caseins / Q000737:chemistry; D002522:Chlorocebus aethiops; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry; D005898:Glass; D057230:Limit of Detection; D050778:NFATC Transcription Factors / Q000096:biosynthesis / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry* / Q000302:isolation & purification; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D053719:Tandem Mass Spectrometry; D017971:Tin Compounds / Q000737:chemistry*
chemical_list: D002364:Caseins; D050778:NFATC Transcription Factors; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D017971:Tin Compounds; C109984:indium tin oxide; D017868:Cyclic AMP-Dependent Protein Kinases