title: EJMS protocol: systematic studies on TiO2-based phosphopeptide enrichment procedures upon in-solution and in-gel digestions of proteins. Are there readily applicable protocols suitable for matrix-assisted laser desorption/ionization mass spectrometry-based phosphopeptide stability estimations?
abstract: There have been many successful efforts to enrich phosphopeptides in complex protein mixtures by the use of immobilized metal affinity chromatography (IMAC) and/or metal oxide affinity chromatography (MOAC) with which mass spectrometric analysis of phosphopeptides has become state of the art in specialized laboratories, mostly  applying nanoLC electrospray ionization mass spectrometry-based investigations. However, widespread use of these  powerful techniques is still not achieved. In this study, we present a ready-to-use phosphopeptide enrichment procedure using commercially available TiO(2)-loaded pipette tips in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analyses. Using α-casein as a model protein and citric acid as additive during sample loading, a similar enrichment success can be achieved as compared to applying 2,5- dihydroxy benzoic acid (DHB) for this task. But the DHB-inherited drawbacks are eliminated. In addition, we show that combining DHB and 2,4,6-trihydroxy acetophenone (THAP) as matrix for MALDI-MS measurements retains the sensitivity of DHB for phosphopeptide analysis but adds the homogenous crystallization properties of THAP, enabling  preparation of evenly distributed matrix surfaces on MALDI-MS anchor targets, a prerequisite for automated MALDI- MS analyses. Tripartite motif-containing protein 28 and stathmin are two examples for which successful phosphopeptide enrichment of either sodium dodecyl sulfate polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis-separated proteins is shown. Finally, high resolution MALDI Fourier transform ion cyclotron resonance mass spectrometry after phosphopeptide enrichment suggests that chemical dephosphorylation may occur as a side reaction during basic elution of phosphopeptides bound to MOAC surfaces, suggesting that proteome-wide phosphopeptide analyses ought to be interpreted with caution. In contrast, in-depth analysis of phosphopeptide/non-phosphorylated peptide siblings may be used to estimate stability differences of phosphorylation sites in individual proteins, possibly adding valuable information on biological regulation processes.
authors.affiliation: Proteome Center Rostock, Medical Faculty, University of Rostock, Rostock, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Eickner
authors.forename: Stefan
authors.initials: S
authors.lastname: Mikkat
authors.forename: Peter
authors.initials: P
authors.lastname: Lorenz
authors.forename: Martin
authors.initials: M
authors.lastname: Sklorz
authors.forename: Ralf
authors.initials: R
authors.lastname: Zimmermann
authors.forename: Hans-Jürgen
authors.initials: HJ
authors.lastname: Thiesen
authors.forename: Michael O
authors.initials: MO
authors.lastname: Glocker
mesh_terms: D000098:Acetophenones / Q000737:chemistry; D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D019343:Citric Acid; D003460:Crystallization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D005841:Gentisates / Q000737:chemistry; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis / Q000302:isolation & purification*; D010766:Phosphorylation; D012097:Repressor Proteins / Q000737:chemistry; D012996:Solutions; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D050777:Stathmin / Q000737:chemistry; D014025:Titanium / Q000737:chemistry; D000076183:Tripartite Motif-Containing Protein 28
chemical_list: D000098:Acetophenones; D002364:Caseins; D005782:Gels; D005841:Gentisates; D010748:Phosphopeptides; D012097:Repressor Proteins; D012996:Solutions; D050777:Stathmin; C061082:Stmn1 protein, rat; C009495:titanium dioxide; D019343:Citric Acid; C107067:2,4,6-trihydroxyacetophenone; D014025:Titanium; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28; C010925:2,5-dihydroxybenzoic acid