Silica–Lanthanum Oxide: Pioneer Composite of Rare-Earth Metal

Nov 7, 2012 - At a molar ratio of 1:1:50, after enrichment, the phosphopeptides from ..... is supported by the Higher Education Commission (HEC) of Pa...
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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,§ Gűnther 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

More recently, packed tips12 and TiO2-coated magnetic beads are also commercially available. Research on TiO2 materials for phosphopeptide enrichment13−15 and phosphoproteome profiling have also been published.16 ZrO2 has provided more selective enrichment of singly phosphorylated peptides, compared to the other metal oxides.17 Different ZrO2-coated materials,18 Al(OH)319 and aluminum oxide nanomaterials,20 Ga2O3-coated magnetic particles,21 bare magnetite (Fe3O4)22 and magnetic microspheres with TiO2,23 Al2O3,24 Ga2O3,25 and ZrO226 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.

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 chromatography (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. © 2012 American Chemical Society

Received: September 23, 2012 Accepted: November 7, 2012 Published: November 7, 2012 10180

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Table 1. Identified Phosphopeptides from Tryptic β-Casein Digest by Native and End-Capped Silica-Lanthanum Oxide (SiO2− La2O3) Compositea MS Spectrac b

peak label

m/z

sequence

β-1 β-2 β-3 β-4 β-5 β-6 β-7 β-8 β-9 β-10 α-1 α-2 Nppα

1157.45 1383.87 1881.12 1994.46 2061.74 2352.22 2556.04 2965.13 3054.32 3477.81 1660.92 1847.01 1267.92

LGPVRGPFPIIV LLYQEPVLGPVR LYQEPVLGPVRGPFPIIV LLYQEPVLGPVRGPFPIIV FQS*EEQQQTEDELQDK NVPGEIVES*LS*S*S*EESITR FQS*EEQQQTEDELQDKIHPF RELEELNVPGEIVES*LS8S*S*EESITR KIEK FQS*EEQQQTEDELQDKIHPF RELEELNVPGEIVES*LS*S*S*EESITRINK VPQLEIVPNS*AEER (αS1) DIGES*ESTEDQAMEDIK (αS1) YLGYLEQLLR ((αS1)

phosphorylation sites

raw digest

silica−La2O3

end-capped Si−La2O3

212−224 206−217 208−224 206−224 47−63 (1P) 7−24 (1P) 47−67(1P) 1−24 (4P) 43−67(1P) 1−28 (4P) 121−134(1P) 58−73 (2P) 106−115

− − − ● − − − − − − − − ●

● ● ● ● ● ● ● ● ● − ● ● ●

− − − − ● ● ● ● ● ● ● ● −

Each material is loaded with 1 pmol (2 μL) of the β-casein digest with 1% α-casein as an impurity and eluted with 0.01M NH4OH. bAn asterisk (*) indicates the phosphorylation site at the serine residue to its left. cA solid circle (●) indicates the presence of peak mass loaded by the material. a

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particles were washed with 200 μL of 80% ACN in 0.1% (v/v) TFA, followed by several washings with deionized water. The supernatant containing nonphosphorylated peptides was collected for sequence coverage analysis. Solid material was collected for material-enhanced laser desorption/ionization (MELDI) analysis29−32 of loaded phosphopeptides. Bound phosphopeptides were eluted by 20 μL ammonium hydroxide solution (pH 10). The eluted phosphopeptides were acidified with 2 μL of TFA 10% (v/v) for MALDI-MS analysis. MALDI-MS Analysis of Phosphopeptides. Mass spectra were recorded on Ultraflex II MALDI-TOF/TOF-MS, (Bruker Daltonics), with CHCA in 50% acetonitrile:0.1% (v/v) TFA (1:1) as matrix. The Flex Analysis Version 3.0 (Bruker, Bremen, Germany) software was used for data processing. Database search was performed by the Mascot program (www. matrixscience.com) to query the SWISS-PROT database, which correctly identified the phosphorylated peptides for αcasein (αS1 and αS2), β-casein, and κ-casein (SWISS-PROT Accession Nos. P02662, P02663, P02666, and P02668, respectively). Regeneration of the Silica−Lanthanum Oxide Composite. To regenerate the already used silica−lanthanum oxide composite, particles were washed with an activation buffer (100 μL 80% ACN in 0.1% (v/v) TFA) at room temperature. The activated composite was then incubated with equilibration buffer to restore all the surface functionalities. The regenerated composite was loaded with tryptic casein digest and phosphopeptide analysis was carried out as described previously.

EXPERIMENTAL SECTION Synthesis of the Silica−Lanthanum Oxide Composite. The synthesis of the silica−lanthanum oxide composite involved four steps: (a) hydroxyl functionalization of silica gel, increased by immersing 2.0 g of silica gel in a 1:1 mixture of 50% aqueous sulfuric acid:30% hydrogen peroxide for 30 min;27 (b) chlorination of hydroxyl functionalized silica, using thionyl chloride;28 (c) amino modification of lanthanum oxide (0.4 g) by aminopropylsilane (0.2 g), with anhydrous toluene as a solvent (30 mL); and, finally, (d) synthesis of the composite by using product from steps (b) and (c) in the presence of toluene (30 mL) with magnetic stirring at 80 °C for 24 h. The particles were washed with anhydrous toluene, dried overnight under vacuum at 140 °C, and labeled as native SiO2− La2O3. Half of the synthesized composite was end-capped by treating with 5 mL of hexamethyldisilazane solution in toluene (1:4, v/v) at 60 °C for half an hour, after thorough washing with 80% ACN and dry toluene (20 mL). Finally, they were washed with 80% ACN, dried under vacuum, and labeled as end-capped SiO2−La2O3. Sample Preparation: Tryptic Digestion of Standard Proteins/Nonfat Milk. Tryptic digestion of phosphoproteins standards (α and β-casein), commercially available casein, and nonfat milk was carried out by the reported methodology. Detailed digestion protocol is given in the Supporting Information. Sample Preparation for Spiked Serum. Four different concentrations of spiked serum were prepared using 40 μL of serum sample spiked with tryptic digest of β-casein in concentrations of 1 μg (50 pmol), 100 ng (5 pmol), 10 ng (500 fmol), and 1 ng (50 fmol). The composite was activated, washed and eluted in the same way as in the case of phosphopeptide enrichment using standard proteins (α- or βcasein). Phosphopeptide Enrichment. Enrichment experiments were performed by using peptide samples (1 mg/mL) with silica−lanthanum oxide particles. The peptide sample (tryptic digests of standard proteins and nonfat milk) was loaded after activation of the particles by 100 μL 80% ACN in 0.1% (v/v) TFA and incubated for 30 min at room temperature. The

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RESULTS AND DISCUSSION Choice of Materials. The ideology behind the selection of silica and lanthanum oxide for the synthesis of the composite in this research is supported by various facts. Keeping in view the priority of research interest, the discussion about the choice of materials and the characterization of the composite by EDAX and SEM is given in the Supporting Information (Figure S1). Feasibility of Composite in Phosphopeptide Enrichment. Bovine β-casein is used to evaluate the performance of the silica−lanthanum oxide composite for selective phosphopeptides enrichment. Tryptic digest of β-casein (1 pmol, 2 μL) diluted with 0.1% TFA is incubated with both native and end-

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13, 5, and 1−2 phosphate groups, respectively. Because of the low abundance of κ-casein (∼1.2%−2%), the phosphopeptides present are difficult to enrich; however, the silica−lanthanum oxide composite successfully enriches the phosphopeptides of κ-casein (the residue Ser-170),33 which is fully phosphorylated. This proves its working efficiency for low abundance phosphopeptides in complex mixtures. Amino modifications for casein variants are given as follows: for αS1-CNpSer56, 61, 63, 79, 81, 82, 83, 90, 130;34 for αS2-CNpSer23, 24, 25, 31, 71, 72, 73, 76, 144, 146, 158;35 for β-CNpSer30, 32, 33, 34, 50;36 and for κ-CNpSer 148, 170.37 Therefore, tryptic digest of commercially available casein is taken as a complex mixture containing all three variants of casein (αS1, αS2, β as a maximum, with a trace amount of κ-casein) in the ratio 4:1:4:1.38 After loading, the SiO2−La2O3 composite is investigated regarding its applicability as a MELDI carrier material, followed by MS analysis (see Figure S-4A in the Supporting Information). MELDI analysis provides a complete profile for tryptic casein digest; however, for identification purposes, the elution of adsorbed peptides is necessary. All the phosphopeptides shown by MELDI MS spectra have been successfully eluted (see Figure S-4B in the Supporting Information) which confirms that the adsorption of peptides on composite is reversible. It is important to mention that the direct MALDI analysis of loaded sample on material (MELDI), as well as the analysis of eluted phosphopeptides, has resulted in the same peak pattern. The MS spectrum shows characteristic peaks of casein variants. A complete list of identified peptide signals, along with amino acid sequences and phosphorylation sites, is given in Table S-5 in the Supporting Information. MS spectra for nonphosphorylated peptides are also recorded for the sequence coverage. Sequence Coverage. Earlier sequence coverage for tryptic casein digest (αS1-CN (52.8%), αS2-CN (18.9%), β-CN (39.7%), and κ-CN (11.0%) has been reported by using ZrO2.39 The commercially available casein is selected as the sample, because it contains many nonphosphorylated counterparts (κ-casein as glycoprotein),40 which complicate the selective enrichment of phosphopeptides from the tryptic digest. The databases are available for casein variants which help to score the data with better confidence level and easier to compare and report. It strengthens the in-solution proteomic approach, giving a relatively good characterization of protein mixture of moderate complexity. The sequence coverage after enrichment of casein digest by SiO2−La2O3 composite has shown compatible results (see Table S6 in the Supporting Information), which helps to move onto the real sample application using the same enrichment protocol. Regeneration Studies. The regeneration of adsorbents is a crucial step in all affinity separation techniques. The most economic aspect of SiO2−La2O3 composite is its regenerative property. The MS spectra of eluted fractions show that all of the enriched phosphopeptides have been successfully eluted. Therefore, the solid material is collected after the elution and again spotted onto the MALDI target plate. Surprisingly, there is not a single peak in MS spectrum (see Figure S-7A in the Supporting Information). The idea of regeneration popped up instantly and the used composite is again activated by applying previously described conditions. The used composite is thoroughly washed with deionized water and then with activation buffer, to restore all the surface chemistry of the composite. The entire enrichment procedure is performed

capped composite. Keeping in view the role of buffer conditions to minimize the nonspecific bindings, the activation and loading buffer systems are comprised of TFA, for pH control, and acetonitrile (at levels of 50%−80%), to prevent the hydrophobic interactions. Using harsh washing conditions with high concentrations of additives may improve specificity, but can also result in the loss of weakly bound (mono) phosphopeptides. Therefore, an activation buffer (80% ACN in 0.1% (v/v) TFA) and Milli-Q water are used for washings. A pH shift to alkaline conditions using ammonium hydroxide (pH 10) is employed for elution to have better recovery of enriched phosphopeptides. For comparison, direct analysis of the βcasein digest is performed; the result is presented in Figure S1A in the Supporting Information. None of the characteristic phosphopeptides derived from β-casein are present in the mass spectroscopy (MS) spectrum of raw digest. Some abundant nonphosphopeptides can be observed, which result in low signal-to-noise ratio (S/N) for phosphopeptides, if present. Therefore, enrichment is done using native and end-capped SiO2−La2O3 composites, and the corresponding MALDI-TOF MS spectra are illustrated in Figure S-2 in the Supporting Information. The identified peptides from β-casein digest are labeled as β-1 to β-10, with detailed descriptions given in Table 1. The comparison is helpful to strengthen the use of endcapping in order to avoid the nonspecific bindings. The MS spectra demonstrate the enrichment of phosphopeptides to the composite surface by showing major peaks of phosphopeptides from β-casein (β-5, β-6, β-7, β-8, β-9, and β-10). Since the original protein sample contains a small amount of α-casein, phosphopeptide residues (α-1 and α-2) derived from this impurity have also been enriched and detected. In the case of the SiO2−La2O3 composite, nonphosphopeptides peaks also are observed after enrichment, indicating that there is nonspecific adsorption of peptides onto the SiO2 surface, whereas the end-capped SiO 2−La2O3 composite yields significantly higher signal intensity, allowing unambiguous detection of less-abundant phosphopeptides, such as β-10, and displays a clean background. Selective Enrichment of Phosphopeptides. To evaluate the selectiveness of the synthesized composite, a semicomplex mixture analysis is done by employing BSA for increasing complexity of digested casein mixture. To mimic a complex biological sample, BSA is added to the tryptic digest of α-casein and β-casein at different molar ratios (α-casein:β-casein:BSA = 1:1:50 and 1:1:100). At a molar ratio of 1:1:50, after enrichment, the phosphopeptides from α-casein (α-1 at 1237.56, α-2 at 1253.32, α-3 at 1411.99, α-4 at 1660.34 (PO43− adduct), α-5 at 1832.67, α-6 at 1927.90, and α-7 at 2247.98), as well as those from β-casein (β at m/z 2061.19) are detected (see Figure S3 in the Supporting Information). When the molar ratio decreases to 1:1:100, after enrichment, still the phosphopeptides from α-casein (α-4, α-5, and α-6), as well as from β-casein (β at 2061.46) can be distinguished, even in the presence of an exceedingly high concentration of BSA. These results prove that the trace amount of phosphopeptides can be enriched by the SiO2−La2O3 composite, even in the presence of a large amount of interfering protein. Complex Mixture Analysis. Most common type of casein phosphorylation involves the formation of phosphate ester bonds with the hydroxyl side chains of serine and, much less frequently, threonine. Thus, bovine caseins are phosphorylated at different levels, and the common genetic variants of αS1-CN, αS2-CN, β-CN, and κ-CN caseins normally contain 8−9, 11− 10182

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using casein digest and MS spectra are recorded for MELDI and eluted phosphopeptides (see Figures S-7B and 7C in the Supporting Information). It can be observed that most of the phosphopeptides has been enriched by the regenerated composite, showing its successful regeneration. It provides the possibility of using the composite several times, however, the reuse over a longer period of time depends substantially on achieving and maintaining the chemical functionalities of the composite. The comparison shows that the regenerated SiO2− La2O3 composite has enriched phosphopeptides as efficiently as the original composite. Real Sample Application. Sensitivity Assessment for Serum. In the case of advanced cancer treatment with antiangiogenesis agents and protein kinase inhibitors,41 the phospho-modified proteins are secreted from cells into the circulation system and are available in the serum. However, direct determination of these modified proteins in a biological system has been difficult, because of the serum complexity and lower concentrations. There is no method available to study this aspect in detail with consistency.42 In order to estimate the detection limit for phosphopeptides enriched from serum, a sensitivity assessment is done using spiked serum with tryptic digest of β-casein in different ratios (40 μL serum sample spiked with 1 μg (50 pmol), 100 ng (5 pmol), 10 ng (500 fmol), and/or 1 ng (50 fmol) β-casein). The identified phosphopeptides from β-casein are at m/z 1103 (KFQS*EEQQQT), 1473 (KKIEKFQS*EEQQQT), and 2061 (FQS*EEQQQTEDELQDK), with relatively good MS peak intensity. However, a 1-ng β-casein-spiked sample yielded low MS peak intensity (see Figures 1A−D). These results indicate that developed method allows the detection of serum

phosphoproteins/phosphopeptides with amounts greater than ∼10−100 fmol. The identification also shows that method has huge potential to enrich the phospho- content from complex real samples such as serum. Phosphopeptide Enrichment from Nonfat Milk. The tryptic digest of nonfat milk has been selected for real sample application. Nonfat milk, obtained from a local market, contains abundant phosphorylated proteins such as α- and β-casein, and its application helps to further examine the effectiveness and selectivity of the composite in the enrichment of phosphopeptides from complex sample. The direct analysis of tryptic digest of nonfat milk is not shown, because of the significant suppression of ionization in MALDI due to the presence of high concentrations of salts in the digest. After phosphopeptide enrichment using the silica−lanthanum oxide composite in its both native and end-capped forms, the salt concentration is low enough for MALDI processing and several phosphopeptide peaks appear in the mass spectrum. Most of these signals are derived from α- and β-casein, as shown in Figures 2A and 2B

Figure 2. MALDI-TOF mass spectra of phosphopeptides enriched form tryptic digest of nonfat milk by (A) native SiO2−La2O3 composite and (B) end-capped SiO2−La2O3 composite.

for native and end-capped SiO2−La2O3 composite, respectively. The detail of identified peaks is given in Table 2. The results demonstrate the potential of SiO2−La2O3 composite for the selective enrichment of phosphopeptides in real biological samples. It is worth noting that the current process does not involve the desalting step (e.g., with C18 Zip Tips), which is typically required for the enrichment of natural samples, because of their high salt content. The inherent desalting capability of the composite and sample preparation protocol is advantageous, because, otherwise, some phosphopeptides may be lost, because of their hydrophilic nature. Use of Lanthanum in Different Formats. For the first time, lanthanum oxide is being used in phosphopeptide enrichment. The study was based on the idea of the silica− lanthanum oxide composite. After marvelous results, the use of

Figure 1. Sensitivity assessment of phosphopeptides enrichment from human serum spiked with β-casein in different concentrations: (A) 1 μg (50 pmol), (B)100 ng (5 pmol), (C) 10 ng (500 fmol), and (D) 1 ng (50 fmol). 10183

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Table 2. Identified Tryptic Peptide Fragments of Nonfat Milk Digest Enriched by the Silica−Lanthanum Oxide (SiO2−La2O3) Composite peak label

[M+H]+

α-1 α-2 α-3 α-4 α-5 α-6 α-7 α-8 α-9 α-10

1197.009 1253.301 1330.388 1660.510 1954.668 2202.873 2247.190 2362.623 2453.609 2616.678

β-1 ß-2 β-3 β-4 β-5 β-6 β-7 β-8 β-9

847.054 975.768 1103.906 1473.009 1688.459 1881.107 2186.740 2910.255 3054.779

amino acid sequencea

sequence No.

α-Casein KNMAINPS*KENL (αS2) 39−50 (1P) TVDMMES*TEVF (αS2) 153−162 (1P) EQLS*TS*EENSK (αS2) 141−151 (2P) VPQLEIVPNS*AEER (αS1) 121−134 (1P) YKVPQLEIVPNS*AEER (αS1) 119−134 (1P) YKVPQLEIVPNS*AEERLHS (αS1) 119−137 (1P) KEKVNELS*KDIGS*ES*TEDQA (αS1) 49−68 (3P) PNS*VEQKHIQKEDVPSERY (αS1) 88−106 (1P) NTMEHVS*S*S*EES*IISQETY (αS2) 17−35 (4P) NTMEHVS*S*S*EES*IISQETYK (αS2) 17−36 (4P) β-Casein KFQS*EEQQ 47−54 (1P) KFQS*EEQQQ 47−55 (1P) KFQS*EEQQQT 47−56 (1P) KKIEKFQS*EEQQQT 43−56 (1P) LTDVENLHLPLPLLQSW 142−157 LLYQEPVLGPVRGPFPIIV 206−224 DMPIQAFLLQEPVLGPVR 199−217 DMPIQAFLLQEPVLGPVRGPFPIIV 199−224 KKIEKFQS*EEQQQTEDELQDKIHPFA 43−67 (1P)

native SiO2−La2O3b

end-capped SiO2−La2O3b,c

● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ● ●

● ● ● ● ● ● ● ● ●

● ○ ● ● ○ ○ ● ○ ●

a

Asterisk (*) indicates the phosphorylation site at the serine residue to its left. bSolid circle indicates the presence of peak mass loaded by the material. cOpen circle indicates the absence of peak mass not enriched by material.

lanthanum in different formats was also investigated to compare the enrichment efficiency from digested protein samples. The lanthanum is loaded on IMAC material as La3+, as the counterpart in a composite (SiO2−La2O3 composite), and as an oxide itself (La2O3). Tryptic β-casein digest is employed to investigate the use of lanthanum and to study the differences in the enrichment of phosphopeptides, if any. The MS results (see Figures 3A−C) provide the evidence that, in any format, lanthanum ensures maximum enrichment without any prominent difference. The efficient enrichment of phosphopeptides in the case of La3+ ions loaded on IMAC material might be due to the presence of an f-orbital, which offers coordination bonds to phosphopeptides, as in the case of transition metals (Fe3+, Zr4+, Ti4+) used in IMAC loadings so far. As a composite, its working efficiency has been checked in detail as the SiO2− La2O3 composite (see Table S8 in the Supporting Information). The presence of nonspecific bindings in the case of composite or loaded IMAC material can be attributed to the base material. Therefore, end-capping of the IMAC or composite base material is necessary to reduce the nonspecific bindings. As a pure oxide (La2O3), there is neat spectrum and maximum phosphopeptide enrichment. So, this comparison provides choices in the research field, showing that lanthanum can be used in solution, as well as particle form, without compromising on the loss of phosphopeptides.

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Figure 3. MALDI-TOF mass spectra of phosphopeptides enriched from mixture of tryptic digest of α-casein and β-casein (1 pmol, 2 μL) in ratio of 1:1 by (A) IMAC material loaded with La3+ (0.01 M LaCl3 solution for loading); (B) SiO2−La2O3 composite; and (C) activated La2O3 particles (commercially available).

CONCLUSION

In nature, protein modifications, such as phosphorylation, contribute to the complex signaling pathways. There is always room for new materials to enrich them in order to study the protein phosphorylation. Silica−lanthanum oxide composite synthesized from chemical derivatization of silica and lanthanum oxide provides an efficient media for selective enrichment of phosphopeptides from standard protein digests and real samples such as milk and serum. The elution

conditions are compatible for balanced recovery of singly and multiply phosphorylated peptides. The regeneration phenomenon enhances the commercial value of the silica−lanthanum oxide composite. The material also has been proven to be an efficient material-enhanced laser desorption/ionization 10184

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(MELDI) carrier material. In addiiton, sufficient data are gained to cover all the major steps of protein phosphorylation: from enrichment of phosphopeptides to the identification of phosphorylation sites and sequence coverage.

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +92 61 9210085. Fax: +92 61 9210138. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by the Higher Education Commission (HEC) of Pakistan. REFERENCES

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dx.doi.org/10.1021/ac3023197 | Anal. Chem. 2012, 84, 10180−10185