Selective enrichment of phosphopeptides by a metal–organic framework† Christoph B. Messner, a Munazza R. Mirza, a Matthias Rainer, * a Oliver M. D. Lutz, a Y¨ uksel G¨ uzel, a Thomas S. Hofer, b Christian W. Huck, a Bernd M. Rode b and G¨ unther K. Bonn a A metal–organic framework, consisting of Er(III) linked together by 1,4-phenylenediacetate, was synthesised by a one-pot reaction and successfully used as an affinity material for the selective capturing of phosphopeptides. An optimised protocol for loading, washing and elution was developed and the eluents were analysed via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A standard protein digest (a-casein, b-casein and ovalbumin) as well as digested egg white proteins were used to test the efficiency and selectivity of the presented approach. 14 phosphopeptides could be recovered from the peptide mixture and in the case of digested egg-white, four phosphorylated peptides which could be assigned to ovalbumin were isolated. Ab initio calculations based on the affinities of various ligands to the material have provided reasonable explanations of the observed experimental properties. 1 Introduction Metal–organic frameworks (MOFs) are a class of intriguing crystalline compounds, consisting of metal ions linked together by organic bridging ligands. Due to their high porosity and their chemical and structural diversity, MOFs received great attention in recent years and are widely regarded as promising materials for catalysis, separation and gas storage. 1–4 The possibility to rationally design MOFs with specic adsorption properties (tunable pore size and chemical functionalities) makes these materials highly attractive for applications in analytical and bioanalytical chemistry. Several studies dealing with MOFs in analytical chemistry, including liquid chromatography and gas chromatography separations, can be found in the literature. 5 However, the only bioanalytical application was reported by Gu et al., 6 who used MOFs for the extraction of peptides out of complex samples. The present study uses MOFs for the rst time as an affinity material for the selective enrichment of phosphopeptides. Protein phosphorylation is a key mechanism in a number of cellular processes and abnormal phosphorylation reactions are related to many diseases (e.g. cancer). 7 Thus, the mechanistic understanding of cellular activities at the molecular level is fundamental and requires accurate and reliable methods for characterisation and identication of phosphorylation states. Mass spectrometric methods, such as matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) 8,9 and electrospray ionisation mass spectrometry (ESI MS), 10 have become the most powerful tools for the analysis of protein digests. Nevertheless, the analysis of phosphopeptides in complex samples remains challenging due to the higher abundance of non-phosphorylated peptides, resulting in signal suppression. Therefore, approaches to enrich phosphopeptides prior to MS analysis are required, with immobilised metal-ion affinity chromatography (IMAC) 11 and metal oxide affinity chromatography (MOAC) 12,13 being the most prominent tech- niques. Nevertheless, these methods still suffer from unspecic binding and the development of innovative strategies and novel materials is highly desirable. 2 Experimental 2.1 Chemicals and reagents Acetonitrile (ACN; for HPLC, $99.9%), 2,5-dihydroxybenzoic acid (DHB; 99.0%), dithiothreitol (DTT, 99.0%), erbium nitrate pentahydrate (99.9%), erbium trichloride (ErCl 3 99.9%), p-phenylenediacetic acid (PDA; 97.0%), iodoacetamide ($98.0%), ammonium bicarbonate ($99.9%) acetone ($99%), ethanol absolute (for HPLC $99.8%), a-casein from bovine milk ($70%), b-casein from bovine milk ($90%) and ovalbumin from chicken egg white ($98%) were purchased from Sigma- Aldrich (St. Louis, MO). Triuoroacetic acid (TFA; $99.5%), ammonium hydroxide solution and n-octylglucopyranoside a Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria. E-mail: m.rainer@uibk.ac.at; Fax: +43-512- 507-57399; Tel: +43-512-507-57307 b Theoretical Chemistry Division, Institute of General, Inorganic and Theoretical Chemistry, Leopold-Franzens University, Innrain 80-82, 6020 Innsbruck, Austria † Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ay40308d Cite this: Anal. Methods, 2013, 5, 2379 Received 24th February 2013 Accepted 20th March 2013 DOI: 10.1039/c3ay40308d www.rsc.org/methods This journal is ª The Royal Society of Chemistry 2013 Anal. Methods, 2013, 5, 2379–2383 | 2379 Analytical Methods PAPER