Analytica Chimica Acta 761 (2013) 92–101 Contents lists available at SciVerse ScienceDirect Analytica Chimica Acta jou rn al hom epa ge: www.elsevier.com/locate/aca C 60 -fullerene bound silica for the preconcentration and the fractionation of multiphosphorylated peptides Martin Fischnaller a , Rania Bakry a,∗ , Rainer M. Vallant a , Lukas A. Huber b , Günther K. Bonn a a Institute of Analytical Chemistry and Radiochemistry, Leopold Franzens University, Innrain 80-82, 6020 Innsbruck, Austria b Biocenter, Division of Cell Biology, Innsbruck Medical University, Innrain 80-82, 6020 Innsbruck, Austria h i g h l i g h t s ◮ Phosphopeptides can be selec- tively fractionated using C 60 -aminopropylsilica. ◮ Selective isolation of mono- and multiphosphorylated peptides was performed according to their pI val- ues. ◮ Elution was carried out using pH gradient in presence of different con- centration of acetonitrile. ◮ The binding and fractionation can be attributed to amino groups together with the hydrophobic fullerene moi- eties. g r a p h i c a l a b s t r a c t a r t i c l e i n f o Article history: Received 27 September 2012 Received in revised form 5 November 2012 Accepted 10 November 2012 Available online 5 December 2012 Keywords: C60-fullerene Phosphopeptides Grade of phosphorylation MALDI-MS Enrichment Sample preparation a b s t r a c t Phosphorylation of proteins is an important cellular regulatory process. The analysis of protein phos- phorylation is challenging due to the high dynamic range and low abundance natures of phosphorylated species. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests is the method- of-choice for characterization of phosphorylated proteins. However, determination of phosphopeptides by MS represents a major challenge, especially in the presence of unmodified peptides. Due to lower ionization efficiency of phosphopeptides, as well as the fact that the stoichiometry of phosphorylation is often present at low relative abundance, efficient enrichment of the phosphorylated peptides prior to MS analysis is therefore of high demand. In addition, successful identification of peptides with different phosphorylation grades still remains challenging. This work presents a new strategy for enrichment and subsequent selective elution of multi-, mono- and nonphosphorylated peptides based on their difference in pI by using pH gradient elution in presence of different concentration of acetonitrile prior to matrix-assisted laser desorption/ionization time-of- flight mass spectrometric analysis (MALDI-MS). The developed protocol was successfully applied for - casein tryptic digest and bovine serum albumin digest spiked with 9 synthetic phosphopeptides. Further selectivity for phosphopeptides was demonstrated by fractionation of peptides from a milk digest. © 2012 Elsevier B.V. All rights reserved. ∗ Corresponding author. Tel.: +43 512507 57308; fax: +43 512507 57399. E-mail address: rania.bakry@uibk.ac.at (R. Bakry). 1. Introduction Protein phosphorylation is one of the most common mech- anisms for covalent modification of proteins and is found in as many as one-third of eukaryotic gene products [1,2]. Reversible 0003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2012.11.019