In-situ enrichment of phosphopeptides on MALDI plates modified by ambient ion landing Luká  s Krásný, a,b Petr Pompach, a,c Martin Strohalm, a Veronika Obsilova, d Marcela Strnadová, a Petr Novák a,c and Michael Volný a,e * We report substantial in-situ enrichment of phosphopeptides in peptide mixtures using titanium and zirconium dioxide-coated matrix assisted laser desorption-ionization (MALDI) plates prepared by recently reported ambient ion landing deposition technique. The technique was able to modify four common materials currently used for MALDI targets (stainless steel, aluminum, indium-tin oxide glass and polymeric anchor chip). The structure of the deposited dioxide was investigated by electron micros- copy, and different surfaces were compared and discussed in this study. Two standard proteins were used to test the enrichment capabilities of modified MALDI plates: casein and in-vitro phosphorylated trehalase. The enrichment of casein tryptic digest resulted in identification of 20 phosphopeptides (including miscleavages). Trehalase was used as a suitable model of larger protein that provided more complex peptide mixture after the trypsin digestion. All four possible phosphorylation sites in trehalase were identified and up to seven phosphopetides were found (including methionine oxidations and miscleavages). Two different mass spectrometers, MALDI-Fourier transform ion cyclotron resonance (FTICR) and MALDI-time of flight, were used to detect the phosphopeptides from modified MALDI plates after the enrichment procedure. It was observed that the desorption- ionization phenomena on the modified surfaces are not critically influenced by the parameters of the different MALDI ion sources (e.g. different pressure, different extraction voltages), and thus the presence of dioxide layer on the standard MALDI plate does not significantly interfere with the main MALDI processes. The detection of phosphopeptides after the enrichment could be done by both instruments. Desorption electrospray ionization coupled to the FTICR was also tested, but, unlike MALDI, it did not provide satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd. Supporting Information can be found in an online version of this article. Keywords: phosphopetides; MALDI; FTICR; enrichment; casein; trehalase Introduction Post-translational modifications of proteins are a late step in the protein biosynthesis and a key tool in the regulation of their func- tionality. [1] Protein phosphorylation, a covalent attachment of a phosphate group, was described more than 50 years ago [2] , and currently it is considered to be one of the most important signaling mechanisms in cells. Many cellular processes such as metabolism, transcription and differentiation are controlled by phosphorylation of serine, threonine, tyrosine (O-linked) or histidine (N-linked) residues. It is recognized that protein kinases and phosphatases mediate most of the signal transduction in eukaryotic cells [3] , and the misregulation of enzymes that control cellular phos- phorylation activities has been implicated in a wide range of diseases. [4–6] There are many, often complementary techniques, used in phosphoproteomics. [7–10] Mass spectrometry (MS), which is capable of fast protein sequencing, has also become a key method for investigation of protein phosphorylation. [8,9,11–14] Theoretical prediction of eukaryotic phosphorylation is still extremely difficult [15] , and its elucidation relies on experimental approaches. The detection of phosphorylated peptides is hin- dered by the fact that phosphorylation is a very dynamic event, and thus only a fraction of isolated protein molecules might be phosphorylated. [11] This results in low occurrence of phospho- peptides and in their low stoichiometry with respect to the corresponding non-modified analogs. Moreover, addition of a phosphate group, which increases the peptide acidity, depresses the ionization efficiency in positive ion mode due to the difficulty to form positive ions by protonation. This leads to phosphopeptides signal suppression in MS. [11] Harder ionization approaches can result in cleavage of the phosphate group bond from the peptide backbone and in the loss of the information about phosphorylation status. Isolation, extra chromatographic separation or prior enrichment of phosphopeptides from a pep- tide mixture after enzymatic digest of a protein are often used to overcome the above described difficulties. [8,11,16,17] Many techniques for phosphopeptide enrichment compatible with MS were reported and used. [16] They can be roughly divided into several groups, although the classification serves mainly a * Correspondence to: Michael Volny, Department of Chemistry, University of Washington, Seattle, Washington, 98195-1700, USA. E-mail: volny@uw.edu The first two authors, LK and PP, contributed equally to this work. a Institute of Microbiology of the ASCR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic b Institute of Chemical Technology, Technická 5, Prague, 16628, Czech Republic c Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, 128 40 Prague 2, Czech Republic d Institute of Physiology of the ASCR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic e Department of Chemistry, University of Washington, Seattle, Washington, 98195-1700, USA J. Mass Spectrom. 2012, 47, 1294–1302 Copyright © 2012 John Wiley & Sons, Ltd. Research article Received: 15 June 2012 Revised: 25 July 2012 Accepted: 3 August 2012 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jms.3081 1294