Silica-Lanthanum Oxide: Pioneer Composite of Rare-Earth Metal Oxide in Selective Phosphopeptides Enrichment Fahmida Jabeen, † Dilshad Hussain, † Batool Fatima, † S. Ghulam Musharraf, ‡ Christian W. Huck, § Gű nther K. Bonn, § and Muhammad Najam-ul-Haq* ,†,§ † Division of Analytical Chemistry, Institute of Chemical Sciences, Bahauddin Zakariya University, Multan 60800, Pakistan ‡ H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan § Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 80-82, A-6020 Innsbruck, Austria * S Supporting Information ABSTRACT: Relying on the successful journey of metal oxides in phosphoproteomics, lanthanum oxide is employed for the engineering of an affinity material for phosphopeptide enrichment. The lanthanum oxide is chemically modified on the surface of silica and characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), and Fourier transform infrared spectroscopy (FTIR). The obtained silica-lanthanum oxide composite is applied for the selective enrichment of phosphopeptides from tryptic digest of standard protein (α-casein, β-casein, and commercially available casein mixtures from bovine milk). The enriched entities are analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The mass spectroscopy (MS) results show that the silica-lanthanum oxide composite exhibits enhanced capability for phosphopeptide enrichment with sensitivity assessed to be 50 fmol. Sequence coverage of casein is interpreted showing successful recovery. As a real sample, a protein digest of nonfat milk is applied. Also, the ability of lanthanum in different formats is checked in the selective phosphopeptides enrichment. The composite holds promising future in economic ground as it also possesses the regenerative ability for repetitive use. E nrichment strategies are becoming more and more selective in phosphoproteomics. Phosphorylations are part of the common mechanisms for controlling the behavior of a protein. 1 Phosphorylation takes place mainly on serine residues (86.4%), followed by threonine residues (11.8%) and tyrosine residues (1.8%). 2 With all of these modifications, it is assumed that up to 30% of all proteins may be phosphorylated, some multiple times. Mass spectrometry has been developed for maximum data collection and analysis after enrichment, as it is the predominant analytical tool used in phosphoproteomics. Among the mass instruments, the most widely used for peptide mass analysis is the matrix-assisted laser desorption ionization time-of- flight (MALDI TOF), because it permits the identification of proteins at the smallest levels. 3,4 Immobilized metal ion affinity chromatography (IMAC) has been the most frequently used method for the enrichment of phosphopeptides using different metal ions on various base materials and commercial kits are available from different suppliers. 5-7 Phosphopeptide enrichment has also been done with strong cation exchange (SCX), 8 strong anion exchange, 9 mixed-bed sorbents, 10 and hydrophilic interaction chromatog- raphy (HIC). 11 One of the most powerful and promising approaches that have appeared in recent years is metal oxide affinity chromatography (MOAC), which takes advantage of the particular affinity of metal oxides to phosphate groups. More recently, packed tips 12 and TiO 2 -coated magnetic beads are also commercially available. Research on TiO 2 materials for phosphopeptide enrichment 13-15 and phosphoproteome profil- ing have also been published. 16 ZrO 2 has provided more selective enrichment of singly phosphorylated peptides, compared to the other metal oxides. 17 Different ZrO 2 -coated materials, 18 Al(OH) 3 19 and aluminum oxide nanomaterials, 20 Ga 2 O 3 -coated magnetic particles, 21 bare magnetite (Fe 3 O 4 ) 22 and magnetic microspheres with TiO 2 , 23 Al 2 O 3 , 24 Ga 2 O 3 , 25 and ZrO 2 26 have been used efficiently in phosphoproteomics. In the present work, we report a new composite material for the phosphopeptide enrichment. Silica and lanthanum oxide are selected to synthesize the composite. The lanthanum oxide provides more coordination sites for protein and peptide binding than any other transition-metal oxide used so far as an affinity material. The hydroxyl groups on lanthanum oxide provide hydrophilicity, which makes it compatible with silica and, thus, the composite is beneficial in efficient and specific isolation of phosphopeptides from biological samples. Received: September 23, 2012 Accepted: November 7, 2012 Published: November 7, 2012 Technical Note pubs.acs.org/ac © 2012 American Chemical Society 10180 dx.doi.org/10.1021/ac3023197 | Anal. Chem. 2012, 84, 10180-10185