Concerted Experimental Approach for Sequential ... - ACS Publications

Concerted Experimental Approach for Sequential Mapping of Peptides and Phosphopeptides Using C18-Functionalized Magnetic Nanoparticles He-Hsuan Hsiao,† Hsin-Yu Hsieh,† Chi-Chi Chou,† Shu-Yu Lin,† Andrew H.-J. Wang,†,‡ and Kay-Hooi Khoo*,†,‡ National Core Facilities for Proteomics Research, and Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan Received September 15, 2006

An integrated analytical approach for the enrichment, detection, and sequencing of phosphopeptides using matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS) was developed. On the basis of C18-functionalized Fe3O4 nanoparticles, the enrichment method was designed not only to specifically trap phosphopeptides, but also nonphosphorylated peptides, both of which can be subsequently desorbed selectively and directly for MALDI-MS analysis without an elution step. Peptide binding is afforded by the C18-derivatization, whereas the highly selective capture of phosphopeptides is based on higher binding affinity afforded by additional metal chelating interaction between the Fe3O4 nanoparticles and the phosphate groups. Upon binding, the initial aqueous wash allows desalting, while a second and a third wash with high acetonitrile content coupled with diluted sulfuric acid and ammonia removes most of the bound nonphosphorylated peptides. Selective or sequential mapping of the peptides and phosphopeptides can, thus, be effected by spotting the washed nanoparticles onto the MALDI target plate along with judicious choice of matrices. The inclusion of phosphoric acid in a 2,5-dihydroxybenzoic acid matrix allows the desorption and detection of phosphopeptides, whereas an R-cyano-4-hydroxy-cinnamic acid matrix with formic acid allows only the desorption of nonphosphorylated peptides. The method used to enrich phosphopeptides prior to MS applications is more sensitive and tolerable to sodium dodecyl sulfate than IMAC. We have demonstrated the applicability of C18-functionalized Fe3O4 nanoparticles in the detection of in vitro phosphorylation sites on the myelin basic protein, and at least 17 phosphopeptides were identified, including one previously uncharacterized site. Keywords: Magnetic Nanoparticles • Enrichment • Phosphopeptides • Mass Spectrometry

Introduction Reversible protein phosphorylation is one of the most common post-translational modifications of proteins which plays a critical role in regulating many complex biological processes such as cellular growth, division, and signaling.1,2 Yet, despite considerable interest and recent advances in proteomics, microscale analysis by mass spectrometry (MS) in detecting site-specific phosphorylation remains a formidable technical challenge. Among the most notable problems is the low amount of phosphorylated proteins of interest relative to other nonphosphorylated proteins, and even more so at the peptide level when a phosphopeptide is to be detected in the presence of several orders of magnitude higher amount of nonphosphorylated peptides. Besides the dynamic range of detection issue of an MS instrument, signal corresponding to a phosphopeptide * To whom correspondence should be addressed. Tel, +886-2-27855696; fax, +886-2-27889759; e-mail, [email protected]. † National Core Facilities for Proteomics Research, Academia Sinica. ‡ Institute of Biological Chemistry, Academia Sinica. 10.1021/pr0604817 CCC: $37.00

 2007 American Chemical Society

which is readily detectable in isolation is often suppressed in the company of other more readily ionized nonphosphorylated peptides.3-6 A key to success in detecting phosphopeptides by MS is, thus, the ability to efficiently enrich them from typically a proteolytic digest of phosphoproteins or phosphoproteome. Several approaches have been explored to date for the selective isolation of phosphopeptides. Immobilized metal ion affinity chromatography (IMAC) using iron(III) or gallium(III) prior to MS analysis is the most common method and has been very successful in the studies of individual phosphoproteins as well as in phosphoproteomics investigations.7-16 Enrichment and recovery strongly depend on the type of metal ion, column material, and loading/eluting procedures that are used. However, nonphosphorylated peptides, including multiple acidic residues, are also frequently retained by this procedure. Blocking the carboxylate groups by the formation of a methyl ester has been shown to enhance the specificity of capturing the phosphopeptides.17-20 Nevertheless, the incompleteness of this Journal of Proteome Research 2007, 6, 1313-1324

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Scheme 1

derivatization often compromises the sensitivity of this procedure, increases the complexity of the sample, and complicates further data interpretation.21 Therefore, notwithstanding the availability of commercial IMAC products and several notable application successes, the use of IMAC for phosphopeptide analysis is far from a routine running. Recently, a promising phosphopeptide enrichment strategy was introduced by Sano et al. where titanium dioxide (TiO2) was used as an alternative to IMAC.22-25 Further coupling of TiO2 with magnetic nanoparticles (Fe3O4/TiO2 core shell) has increased its ease of use and has been shown to selectively concentrate phosphopeptides from protein digest products.26 Notably, in most applications using either IMAC or TiO2-based resins or microparticles, only the phosphopeptides were targeted for analysis. Nonphosphorylated peptides, except those retained nonspecifically and coeluting with the phosphopeptides, were usually not further investigated. A commonly held view is that these would be recovered in the initial wash due to nonbinding, although further analysis often revealed that a significant portion of these would not be recovered.27 MSmapping of the proteolytic digests of a phosphoprotein or phosphoproteome is, thus, normally a two-stage process in which an initial mapping without enrichment would precede the phosphopeptide analysis step. In practice, this initial peptide mapping will itself benefit from desalting and concentrating, usually based on a reversed-phase C18 resin and, hence, the popularity of devices such as C18 ZipTip. Others have recently introduced C18-functionalized magnetic beads for similar applications in sample precleaning.28-31 Searching for a more versatile and efficient method to facilitate MS analysis of both peptides and phosphopeptides, we first explored the performance of C18-functionalized magnetic nanoparticles which can be applied directly to the MALDI target plate along with matrix solution. Unexpectedly, we found that phosphopeptides were retained more tightly than nonphosphorylated peptides, most likely through additional metal chelating interactions afforded by the functionalized Fe3O4 nanoparticles. This enables us to formulate a concerted experimental approach to sequentially map the peptides and 1314

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phosphopeptides based on an optimized, differential desorption strategy. The efficacy of this method is demonstrated against standard phosphoproteins and further applied to successfully identify the phosphopeptides of an in vitro phosphorylated myelin basic protein.

Experimental Section Materials. Iron(II, III) oxide nanopowder (Fe3O4, 20-30 nm), trimethoxypropylsilane, octadecyltrimethoxysilane, bovine serum albumin (BSA), R-casein from bovine milk, β-casein from bovine milk, myelin basic protein (MBP) from bovine brain, 2,5-dihydroxybenzoic acid (DHB), R-cyano-4-hydroxy-cinnamic acid (CHCA), phosphoric acid, and ammonium bicarbonate were obtained from Sigma-Aldrich (St. Louis, MO). Protein kinase A was obtained from New England BioLabs, Inc. (Ipswich, MA). Sequencing grade, modified trypsin was obtained from Promega (Madison, WI). Sequencing grade endoproteinase Lys-C and alkaline phosphatase were obtained from Roche (Indianapolis, IN). Formic acid, ammonia solution, sulfuric acid, acetonitrile (ACN), and ethanol were obtained from Merck (Darmstadt, Germany). ZipTipMC were obtained from Millipore (Billerica, MA). Preparation of C3 and C18 Magnetic Nanoparticles. Fe3O4 nanoparticles were coated with C3 and C18 as described.26,32,33 In brief, 10 mg of Fe3O4 nanoparticles were added to a freshly prepared 2% (v/v) solution of trimethoxypropylsilane and octadecyltrimethoxysilane (20 µL in 970 µL of ethanol and 10 µL of water), followed by sonication and then vigorous stirring at 45 °C for 8 h. Upon completion of the reaction, the nanoparticles were aggregated onto the wall of the tube by positioning a magnet adjacent to the edge of the tube which allowed the remaining solution to be readily removed by pipet. The isolated nanoparticles were then washed repeatedly with ethanol, 0.1% formic acid, and 95% ACN/0.1% formic acid for five times in order to remove any unreacted impurities. Finally, the derivatized nanoparticles were resuspended in 1 mL of 0.1% formic acid and stored at room temperature until needed. Proteolytic Digestions. In vitro phosphorylated MBP was prepared by treating the protein with protein kinase A in 50

Phosphopeptides Mapping with C18-Functionalized Magnetic Nanoparticles

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Figure 1. MALDI mass spectra of BSA tryptic peptides: (a) 10 fmol, extracted with C3-functionalized Fe3O4 nanoparticles; (b) 10 fmol, extracted with C18-functionalized Fe3O4 nanoparticles; (c) 1 fmol, before extraction; (d) 1 fmol, extracted with C3-functionalized Fe3O4 nanoparticles; (e) 1 fmol, extracted with C18-functionalized Fe3O4 nanoparticles. The insets show the Mascot search results. The matrix peaks are marked with M.

mM Tris-HCl buffer containing 10 mM MgCl2 and 0.2 M ATP for 4 h at room temperature, followed by trichloroacetic acid precipitation. Solutions of proteins, trypsin, and Lys-C were prepared in aqueous ammonium bicarbonate buffer (25 mM, pH 8.5). Stock solutions of proteins (approximately 10 µg) were reduced with dithiothreitol at 37 °C for 1 h, alkylated with iodoacetamide at 37 °C for 1 h, and then treated overnight with trypsin or Lys-C at an enzyme-to-substrate ratio of 1:50 at 37 °C as described previously.34 The digested products were then

diluted with 0.1% formic acid to the proper concentration for further experiments. Immobilized Metal Affinity Chromatography. The method was carried out according to the Millipore User Guide for ZipTipMC Pipettes. Briefly, the tip was equilibrated by repeatedly aspirating and dispensing 10 µL of freshly prepared 0.1% acetic acid in 50% ACN. Charging of the tips was accomplished by performing 10 cycles of aspiration and dispensing of 10 µL of 200 mM ferric chloride (FeCl3) in 10 mM HCl, followed by two Journal of Proteome Research • Vol. 6, No. 4, 2007 1315

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Table 1. Overview of Observed Peptides Derived by Tryptic Digestion of R-Casein and β-Casein sequence

number of phosphoryl groups

[M + H]+ (theoretical)

after alkaline phosphatase

R-Casein Nonphosphorylated peptides FALPQYLK (Rs2 189-196) YLGYLEQLLR (Rs1 106-115) FFVAPFPEVFGK (Rs1 38-49) HQGLPQEVLNENLLR (Rs1 23-37) IGVNQELAYFYPELFR (Rs1 151-166) EPMIGVNQELAYFYPELFR (Rs1 148-166) FPQYLQYLYQGPIVLNPWDQVK (Rs2 153-164) Phosphopeptides TVDMEpSTEVFTK (Rs2 153-164) TVDMEpSTEVFTKK (Rs2 153-165) VPQLEIVPNpSAEER (Rs1 121-134) DIGpSEpSTEDQAMEDIK (Rs1 58-73) YKVPQLEIVPNpSAEER (Rs1 119-134) VNELpSKDIGpSEpSTEDQAMEDIK (Rs1 52-73) QMEAEpSIpSpSpSEEIVPNpSVEQK (Rs1 74-94) NTMEHVpSpSpSEESIIpSQETYKQ (Rs2 17-37) EKVNELpSKDIGpSEpSTEDQAMEDIK (Rs1 50-73) NANEEEYSIGpSpSpSEEpSAEVATEEVK (Rs2 61-85) NANEEEYpSIGpSpSpSEEpSAEVATEEVK(Rs2 61-85)

979.6 1267.7 1384.7 1760.0 1959.0 2316.1 2709.4 1 1 1 2 1 3 5 4 3 4 5 β-Casein

Nonphosphorylated peptides LLYQEPVLGPVR (β206-217) DMPIQAFLLYQEPVLGPVR (β199-217) IHPFAQTQSLVYPFPGPIPN (β64-83) SLPQNIPPLTQTPVVVPPFLQPEVMGVSK (β64-83) Phosphopeptides FQpSEEQQQTEDELQDK (β48-63) RELEELNVPGEIVEpSLpSpSpSEESITR (β16-40)

washing steps with water and 1.0% acetic acid in 10% ACN. For maximum binding, sample was aspirated and dispensed with 10 µL of binding buffer (0.1% acetic acid) in 10 cycles. The tip was washed once with 0.1% acetic acid, followed by 0.1% acetic acid in 10% acetonitrile, and finally with pure water. The phosphopeptides were then eluted from the tip with 10 µL of freshly prepared 0.3 N ammonium hydroxide solution and dried under vacuum for MALDI measurements. Application of Functionalized Nanoparticles in MALDI-MS Analysis. Typically, 1 µL of peptide digest solution was added to 10 µL of the nanoparticles solution (10 mg/mL in 0.1% formic acid), and the mixture was sonicated for 10 s to thoroughly suspend the nanoparticles. Subsequently, the nanoparticles, with peptides adsorbed, were isolated by positioning a magnet to the outside of the tube, and the solution was then removed using a pipet. For MALDI-MS mapping of nonphosphorylated peptides, the isolated nanoparticles were washed with 0.1% formic acid solution (2 × 10 µL) to remove salt and other impurities. A total of 1 µL of CHCA solution (5 mg/mL in 75% ACN/0.1% formic acid) was then added to resuspend the nanoparticles, and the mixture was spotted directly onto the MALDI target plate. For phosphopeptide enrichment, two additional washes with 75% ACN/0.25% sulfuric acid and 75% ACN/1% ammonia were included to remove nonphosphorylated peptides. Subsequently, 1 µL of DHB solution (10 mg/ mL in 75% ACN/1% phosphoric acid,) was added to resuspend the nanoparticles, and the mixture was spotted directly onto the target plate for MALDI-MS and -MS/MS analysis (Scheme 1). Mass Spectrometry Analysis. MALDI-MS detection and MS/ MS sequencing of peptides in reflectron mode were performed on an Applied Biosystems 4700 Proteomics Analyzer mass spectrometer (Applied Biosystems, Framingham, MA) equipped with an Nd:YAG laser (355 nm wavelength, < 500-ps pulse, and 200 Hz repetition rate in both MS and MS/MS modes). In total, 1316


1466.6 1594.7 1660.8 1927.7 1952.0 2678.0 2720.9 2747.0 2935.2 3008.0 3088.0

1580.8 1872.0 2438.1 2427.0 2688.2 2688.2

1383.8 2186.2 2223.2 3112.7 1 4

2061.8 3122.3

1981.9 2802.4

1000 and 5000 shots were accumulated in positive ion mode MS and MS/MS modes, respectively. For collision-induced dissociation (CID) MS/MS operation, the indicated collision cell pressure was increased from 3.0 × 10-8 Torr (no collision gas) to 5.0 × 10-7 Torr, with the potential difference between the source acceleration voltage and the collision cell set at 1 kV. The resolution of timed ion selector for precursor ion was set at 200. For peptide mass fingerprinting scoring, the MALDIMS data were searched against Swiss-Prot database using the MASCOT program with the following parameters: peptide mass tolerance, 50 ppm; allow up to one missed cleavage; variable modifications considered were methionine oxidation and cysteine carboxyamidomethylation. All MALDI-MS/MS data were manually acquired for each detected peak and manually assigned to verify the sequences for both nonphophopeptides and phosphopeptides alike.

Results and Discussion C3- and C18-Functionalized Magnetic Fe3O4 Nanoparticles. C18-based solid-phase extraction methods have been widely used in proteomics and other analyses for concentrating and desalting peptides. Among the numerous variations, miniaturized packing of C18-coated microparticles or resins into spincolumns or pipet microtips is the most popular application format for microscale handling of peptides.7,35,36 On the other hand, C3, C4, or C8 coating is widely used for larger polypeptides or proteins. Our C3- and C18-functionalized magnetic Fe3O4 nanoparticles compare favorably with these devices for ease of use in extracting, desalting, and concentrating proteins and peptides. Importantly, after removing hydrophilic contaminants and salts with washes, the peptides could be selectively eluted off the nanoparticles or desorbed directly from a spotted MALDI target plate for MS and MS/MS analysis. The latter is particularly useful when handling samples at the low femtomole level, as it would minimize loss incurred by sample


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Figure 2. MALDI mass spectra of the tryptic peptides from 50 fmol of R-casein (a-c) and β-casein (d-f). (a and d) Without extraction; using CHCA as matrix; (b and e) extracted with C18-functionalized Fe3O4 nanoparticles, washed with 0.1% formic acid, using CHCA as matrix; (c and f) treated with alkaline phosphatase and then extracted with C18-functionalized Fe3O4 nanoparticles, washed with 0.1% formic acid, using CHCA as matrix. The phosphorylated peptides are marked with *.

elution and subsequent spotting onto plate. Although the C3functionalized magnetic nanoparticles were derived and used mainly for protein work, its efficacy against peptides and hence functional versatility was initially evaluated along with the C18functionalized nanoparticles. As demonstrated with tryptic peptides of BSA, our C3- and C18-derivatized Fe3O4 nanoparticles afforded excellent trapping efficiency (Figure 1). A peptide mass fingerprinting (PMF) search against the Swiss-Prot database using the Mascot search engine returned a high score and sequence coverage (52%) for MALDI-MS analysis of the tryptic peptides from 10 fmol of BSA, desorbed directly from the spotted nanoparticles (Figure 1a,b). Sufficiently high quality MALDI-MS data were still obtained with as low as 1 fmol of BSA digests. At this level, although the improvement in the actual number of relevant BSA peptides detected and hence the Mascot score is marginal, the additional purification afforded by the application of C3- or C18-functionalized nanoparticles has clearly improved the quality of the resulting spectra (Figure 1d,e). Several of the CHCA matrix peaks, which have been attributed to a multiplicity of sodium

and potassium adducts37,38 (e.g., m/z 1044.1, 1050.1, 1060.1, 1066.1, 1082.1, 1098.0), were suppressed due to the desalting function, and the signal-to-noise ratios of the peptide signals were generally enhanced. However, the C3-functionalized nanoparticles apparently failed to retain some of the more hydrophilic peptides, thus, affording a lower sequence coverage (16%) and Mascot score than that given by the nontreated sample. In contrast, a combination of extremely small size and optimal hydrophobicity endow the C18-functionalized nanoparticles with high capturing efficiency for tryptic peptides, allowing them to be desalted and desorbed directly for MALDIMS analysis at lower detection limit. Identification of Phosphopeptides with C18 Functionalized Fe3O4 Nanoparticles. To evaluate if the C18-functionalized Fe3O4 nanoparticles could equally enhance the detection of phosphopeptides, tryptic peptides from bovine milk R- and β-caseins were subjected to MALDI-MS mapping, either directly or after adsorption to the nanoparticles (see Table 1 for the observed amino acid sequences of peptides and phosphopeptides). As shown in Figure 2a,d, a direct analysis of the tryptic digests of Journal of Proteome Research • Vol. 6, No. 4, 2007 1317

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Figure 3. MALDI mass spectra of tryptic peptides extracted from 50 fmol of R-casein digests (a-d) and β-casein digests (e-h). (a and e) Extracted with C18-functionalized Fe3O4 nanoparticles, washed with 0.1% formic acid, using DHB and 1% phosphoric acid as matrix; (b and f) extracted with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile and 0.1% formic acid, using DHB and 1% phosphoric acid as matrix; (c and g) extracted with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile/0.25% sulfuric acid and then 75% acetonitrile/1% ammonia, using DHB and 1% phosphoric acid as matrix; (d and h) the combined wash solution from panels c and g, respectively, using DHB and 1% phosphoric acid as matrix. The phosphorylated peptides are marked with *. The double charge of phosphopeptides are marked with **. Metastable ions of phosphopeptides are marked with #.

50 fmol of R- and β-caseins, mixed with CHCA matrix solution and spotted onto the MALDI target plate using a dried droplet method, resulted in the detection of weak signals corresponding to their respective phosphorylated peptides at m/z 1660.8 (Rs1 121-134), 1952.0 (Rs1 119-134), and 2061.8 (β 48-63), among other nonphosphorylated peptides and sodium adduct ions. In contrast, if the tryptic peptides were analyzed directly from the C18-functionalized Fe3O4 nanoparticles, all sodium adduct ions were removed and these phosphopeptides were conspicuously absent from the MALDI-mass spectra (Figure 2b,e). This is somewhat unexpected since similar treatment with other C18 resins would normally retain these phosphopeptides from Rand β-caseins. It implies that the C18-functionalized nanoparticles used were either not retaining the phosphopeptides sufficiently in the first place or, retained too strongly for them to be subsequently eluted or desorbed directly for MALDI-MS analysis. 1318


To distinguish between the two possibilities, the phosphopeptides were first dephosphorylated by alkaline phosphatase. When their negatively charged functional group was removed, the dephosphorylated peptides of R- and β-caseins became readily detectable using the same experimental approach (Figure 2c,f), giving signals at m/z 1580.8 (Rs1 121-134), 1872.0 (Rs1 119-134), 2427.0 (Rs2 17-37), 2438.1 (Rs1 52-73), 2688.2 (Rs2 61-85), 1981.9 (β48-63), and 2802.4 (β16-40). The tryptic peptide chains which carry the phosphate groups were therefore of sufficient hydrophobicity to be retained efficiently by the C18-functionalized nanoparticles. Since the phosphopeptides were not found in the nonbinding or washed fractions, it would seem most likely that they were retained too strongly instead, possibly through metal chelating interactions between the phosphate and the Fe3O4 nanoparticles, in a manner similar to that commonly used in immobilized metal affinity chromatography (IMAC).


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Figure 4. MALDI-MS/MS spectra of a tryptic phosphopeptide FQpSEEQQQTEDELQDK and RELEELNVPGEIVEpSLpSpSpSEESITR from 50 fmol of β-casein extracted with C18-functionalized Fe3O4 nanoparticles, (a and c) Without or (b and d) after treatment with alkaline phosphatase. Product ions designated as * contain the phosphorylated residue, whereas those marked with + resulted from further β-elimination of a phosphoric acid moiety from phosphoserine.

Taking cues from previous findings which showed that phosphoric acid not only acts as an excellent eluant for IMAC but also leads to an enhanced response of phosphopeptide signals in MALDI-MS,10 we next employed 2,5-DHB and 1% phosphoric acid as MALDI matrix for the analysis of the C18functionalized Fe3O4 nanoparticles. As shown in Figures 3a,e, the respective phosphopeptides for R- and β-caseins, including the signals at m/z 1466.6 (Rs2 153-164), 1927.7 (Rs1 58-73), 2678.0 (Rs1 52-73), 2720.9 (Rs1 74-94), 2747.0 (Rs2 17-37), 2935.2 (Rs1 50-73), 3008.0 (Rs2 61-85), 3088.0 (Rs2 61-85), and 3122 (β16-40), which were not observed without using the nanoparticles (Figure 2a,d) were now detected, along with other nonphosphorylated tryptic peptides. Furthermore, their sequence identities could be readily confirmed by direct MALDI-MS/MS analysis (Figure 4). Our results thus demonstrate that phosphopeptides could be strongly trapped by the C18-functionalized Fe3O4 nanoparticles. The additional interaction afforded by the phosphate group with the Fe3O4 nanoparticles necessitates the inclusion of phosphoric acid in either the elution or MALDI desorption conditions to allow the detection of phosphopeptides. This suggests a possibility in designing a novel strategy to sequentially map and sequence the adsorbed peptides and phosphopeptides, respectively, at enhanced sensitivity and selectivity. Specific Enrichment of Phosphopeptides with C18 Functionalized Fe3O4 Nanoparticles. If the aim is simply to enrich for phosphopeptides, additional C18 derivatization is apparently not needed, and the Fe3O4 nanoparticles would behave just like TiO2 nanoparticles26 albeit with a higher tendency to retain more of the nonphosphorylated peptides. This ‘nonspecific’ interactions apparently draw from additional ionic chelation of acidic peptides through their carboxylic groups, especially those containing Glu and Asp, in common with other

IMAC-type of applications.17-20 In this context, higher selectivity in retaining only the phosphopeptide would necessitate incorporation of not only high percentage of organic solvent but also a wash solution that is of sufficient strength to compete off all other nonphosphorylated, acidic peptides. This would leave behind mostly the phosphopeptides on the Fe3O4 nanoparticles which could then be directly applied to the MALDI target plate for MS and MS/MS analysis. Comparative analyses showed that a sequential wash with 75% ACN/0.25% sulfuric acid and then 75% ACN/1% ammonia solution (Figure 3c,g) performed better than a wash of 75% ACN alone (Figure 3b,f) to selectively wash off the nonphosphorylated peptides. No phosphopeptide could be detected in the combined washed fraction (Figure 3d,h) which is indicative of its minimal loss from the nanoparticles at this stage. Thus, an additional functionalization with C18 would offer the advantage of binding most peptides in the first place and therefore serve the initial need in desalting and enhancing signal-to-noise ratio by enabling direct desorption from the nanoparticles for PMF. A concerted PMF strategy would then be to first obtain a MALDI-MS profile of the nonphosphorylated peptides from the tryptic digests adsorbed on the nanoparticles using a CHCA matrix. The phosphopeptides could then be specifically detected and sequenced by reapplying a DHB/phosphoric acid matrix, after washing away the remaining nonphosphorylated peptides on plate. Alternatively, the nonphosphorylated peptides could first be washed from the nanoparticles for separate analysis, while the phosphopeptides retained on the nanoparticles could be spotted onto the plate for direct MALDI desorption. The resulting spectra with either of the two Journal of Proteome Research • Vol. 6, No. 4, 2007 1319

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Figure 5. MALDI mass spectra of the tryptic peptides from 50 fmol of R-casein and β-casein digests containing 1% SDS. (a) Without extraction, using DHB and 1% phosphoric acid as matrix; (b) extracted with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile/0.25% sulfuric acid and 75% acetonitrile/1% ammonia, using DHB and 1% phosphoric acid as matrix; (c) extracted with IMAC, using DHB and 1% phosphoric acid as matrix. The phosphorylated peptides are marked with *. The double charge of phosphopeptides are marked with **.

approaches were similar (data not shown). Scheme 1 summarizes the various possible workflows for the approach developed. High Tolerance of Sodium Dodecyl Sulfate. Apart from being highly sensitive and specific in capturing phosphopeptides, an advantage of our C18-functionalized Fe3O4 nanoparticles over the highly popular IMAC is a higher tolerance to SDS interference in MALDI-MS analysis. As shown in Figure 5a, phosphopeptides were hardly detectable in the spectrum of R-casein and β-casein digests containing 1% SDS. However, an additional enrichment step with the C18-functionalized Fe3O4 nanoparticles resulted in the positive identification of 12 phosphopeptides (Figure 5b) with only the one at m/z 3088.0 (Rs2 61-85) missing. In comparison, a similar approach using IMAC returned only 5 phosphopeptides at much lower signalto-noise ratio (Figure 5c). Detection Sensitivity of Phosphopeptides. Taken together, we have shown that the advantage of using the C18-functionalized Fe3O4 nanoparticles is the ability to capture and preconcentrate the phosphopeptides based on dual binding functions, as well as minimizing loss incurred by sample transfer between elution and spotting. The lower limit of 1320


detection using this approach is explored using different starting amounts of R- and β-casein digests, with the affinity capture step performed after serial dilutions of the respective tryptic peptides to 10 and 1 fmol. As shown in Figure 6, eight phosphopeptide signals could be detected if starting from 10 fmol of digests, but only four of the stronger signals, namely, m/z 1660.8 (Rs1 121-134), 1952.0 (Rs1 119-134), 2061.8 (β48-63), and 3122.3 (β16-40), were detectable at the 1 fmol level. This subfemtomole level detection limit for the R- and β-casein phosphopeptides is lower than other current approaches that use IMAC or TiO2 beads in conjunction with MALDI-MS analysis.26,27,39 Identification of Phosphopeptides from Myelin Basic Protein. To further demonstrate the strengths of the developed strategy in mapping phosphopeptides, the C18-functionalized Fe3O4 nanoparticles were applied to proteolytic digests of in vitro phosphorylated myelin basic protein (MBP) from bovine brain. Table 2 lists the observed amino acid sequences of phosphopeptides from MBP. In fact, the distribution of tryptic cleavage sites in MBP is such that many small peptides would be produced by trypsin digestion. Therefore, a 1 h tryptic digestion was done to generate large peptides for a direct

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Figure 6. MALDI mass spectra of the tryptic peptides extracted from (a) 10 fmol and (b) 1 fmol of R-casein and β-casein digestion. The phosphorylated peptides are marked with *. Table 2. Observed Phosphopeptides from Tryptic or Lys-C Digestion of Bovine Brain Myelin Basic Proteina enzyme

Trypsin

phosphopeptide sequence 113FpSWGAEGQK121 64TpTHYGSLPQK73 30HRDpTGILDSLGR41 91NIVpTPRTPPPSQGK104 or

number of phosphoryl groups

[M + H]+

1 1 1 1

1089.5 1211.6 1419.7 1571.8

1 1 1 2 1 1

1880.8 2445.1 2494.2 2574.1 925.5 938.5

1 1

1419.7 1571.8

1 1 1 1 1

1682.8 1702.8 1915.9 1929.9 2441.2

91NIVTPRpTPPPSQGK104 or 91NIVTPRTPPPpSQGK104 113FpSWGAEGQKPGFGYGGR129 113FpSWGAEGQKPGFGYGGRASDYK134 107GLSLSRFpSWGAEGQKPGFGYGGR129 107GLpSLSRFpSWGAEGQKPGFGYGGR129

Lys-C

105GRGLpSLSR112 6PpSQRSK11 or 6PSQRpSK11 30HRDpTGILDSLGR41 91NIVpTPRTPPPSQGK104 or 91NIVTPRpTPPPSQGK104 or 91NIVTPRTPPPpSQGK104 155LGGRDpSRSGSPMARR169 107GLSLSRFSWGAEGQK121 105GRGLSLSRFSWGAEGQK121 6PSQRSKYLASASTMDHA22 52RGSGKDGHHAARTTHYGSLPQK73

a The phosphopeptides were detected by MALDI-MS after enrichment with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile/0.25% sulfuric acid and 75% acetonitrile/1% ammonia, and desorbed using DHB and 1% phosphoric acid as matrix. All phosphopeptides detected carried one single phosphate group except the phosphopeptide 107GLpSLSRFpSWGAEGQKPGFGYGGR129, which was phosphorylated at 2 sites. The phosphorylation sites are inferred from those reported by Kishimoto et al.40 except for the phosphopeptide 91NIVTPRTPPPSQGK104, which was not identified previously.

MALDI-MS analysis of MBP. The tryptic peptides using CHCA as matrix afforded mostly signals corresponding to nonphosphorylated peptides (Figure 7a). Two barely detectable signals

at m/z 1419.7 and 1880.8 could be attributed to phosphopeptides M*30-41 and M*113-129, respectively, but were too weak to allow informative MS/MS to confirm their identities. In comJournal of Proteome Research • Vol. 6, No. 4, 2007 1321

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Figure 7. MALDI mass spectra of the tryptic peptides of myelin basic protein from 1 h (a-c) or (d) overnight tryptic digestion. (a) Before extraction, using CHCA as matrix; (b) Extracted with C18-functionalized nanoparticles, washed with 0.1% formic acid, using CHCA as matrix; (c and d) extracted with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile/0.25% sulfuric acid and 75% acetonitrile/1% ammonia, using DHB and 1% phosphoric acid as matrix. The phosphorylated peptides are marked with *. The double charge of phosphopeptides are marked with double asterisks **. Metastable ions of phosphopeptides are marked with #. The observed sequences of phosphopeptides from MBP are listed in Table 2.

parison, these two phosphopeptide signals were not detected if the tryptic peptides derived from the same amount of MBP were adsorbed on the nanoparticles and analyzed directly using the same CHCA matrix (Figure 7b). Thus, the disappearance of signals in this instance provided the first clue that they indeed represent phosphopeptides. More importantly, if the nanoparticles were first washed with 75% ACN/0.25% sulfuric acid and then 75% ACN/1% ammonia before subjecting to MALDI-MS analysis using DHB/1% phosphoric acid as matrix, not only were the two phosphopeptide signals significantly enhanced in relative intensities, but also other five phosphopeptide signals were additionally detected at m/z 1089.5, 1571.8, 2445.1, 2494.2 and 2574.1 (Figure 7c), and the greater part of nonphosphorylated peptides were removed. It was noted that if the tryptic digestion was performed overnight, an eighth phosphopeptide could be detected at m/z 1211.6 (Figure 7d). The molecular ions corresponding to each of these putative phosphopeptides could be readily selected for MS/MS analysis which afforded unambiguous confirmation of their identity based on facile neutral loss of the phosphate moiety (-98 u) in positive ion mode (Figure 8). If endo-Lys-C was used instead to avoid cleavages at arginine, additional phosphopeptides could be identified from the same amount of MBP using this strategy, and each could be confirmed by MS/MS analysis (Table 2). Taken together, 1322


phosphopeptides carrying all reported phosphorylation sites of MBP40 could be detected by this simple approach, namely, an enrichment with the functionalized nanoparticles for direct MALDI-MS analysis.

Conclusions We have demonstrated that the C18-functionalized Fe3O4 nanoparticles not only can be used to effectively isolate, desalt, and concentrate the peptide mixtures in one step, but can also be further employed to enrich the phosphopeptides for MALDIMS analysis. Tryptic peptides adsorbed onto the C18-functionalized nanoparticles can be directly analyzed by MALDI-MS and -MS/MS without an additional elution step. This feature significantly reduces the analysis time and sample loss, and allows extracting tryptic peptides for protein identification at low femtomole levels. Furthermore, we have conclusively shown that these C18-functionalized Fe3O4 nanoparticles have a good selectivity to trap traces of phosphopeptides which can be subsequently desorbed after additional washing steps. We found that small amount of sulfuric acid and ammonia in washing solution can efficiently remove nonphosphorylated peptides from C18 functionalized Fe3O4 nanoparticles, and phosphoric acid in matrix solution can enhance the detection


research articles

Figure 8. MALDI-MS/MS spectra of the tryptic phosphopeptides of bovine brain myelin basic protein at m/z 1089.5 (a), 1880.8 (b), 1419.7 (c), and 2574.1 (d), as detected after extraction with C18-functionalized Fe3O4 nanoparticles, washed with 75% acetonitrile/0.25% sulfuric acid and 75% acetonitrile/1% ammonia, desorbed using DHB and 1% phosphoric acid as matrix.

of phosphorylated peptides in MALDI-MS. Together, these favorable properties afford a low detection limit in the femtomole range. In summary, the C18-functionalized Fe3O4 nanoparticles can be strategically adapted for concerted extraction and preconcentration of both nonphosphorylated and phosphorylated tryptic peptides prior to MS analysis in proteomic applications. However, when applied specifically to larger scale phosphoproteomics, this method is not without its limitation, but in our hands, its performance compared favorably against other commonly adopted enrichment methods including IMAC and TiO2. In the presence of highly abundant proteins and nonphosphorylated peptides, as would typically be found in unfractionated total cell lysates, all enrichment methods resulted in an incomplete removal of nonphosphorylated peptides, whereas the extent of false negative are difficult to assess. By careful and extensive washing using the optimized conditions developed in this study, we have demonstrated that most nonphosphorylated peptides can indeed be removed from the C18-functionalized Fe3O4 nanoparticles without additional derivatization, such as methyl esterification, to improve its specificity. We propose that our method is a viable alternative for phosphoproteomic applications, although its true unique

strength resides in facile and highly sensitive analysis of defined phosphoprotein sample when coupled with direct MALDI-MS and MS/MS desorption. Abbreviations: ACN, acetonitrile; ATP, adenosine triphosphate; BSA, bovine serum albumin; CHCA, R-cyano-4-hydroxycinnamic acid; CID, collision-induced dissociation; DHB, 2,5dihydroxybenzoic acid; FA, formic acid; IMAC, immobilized metal ion affinity chromatography; MALDI, matrix-assisted laser desorption/ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; MBP, myelin basic protein; PMF, peptide mass fingerprinting; TOF, time-of-flight.

Acknowledgment. This work was supported by Taiwan NSC Grant 94-3112-B-001-009-Y and 95-3112-B-001-014 to the National Core Facilities for Proteomics located at the Institute of Biological Chemistry, Academia Sinica. We gratefully acknowledge Dr. Tzu-Ching Meng and Ms. Han Lee for the preparation of in vitro phosphorylated myelin basic protein. References (1) Mann, M.; Ong, S. E.; Gronborg, M.; Steen, H.; Jensen, O. N.; Pandey, A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome. Trends Biotechnol. 2002, 20 (6), 261-8.

Journal of Proteome Research • Vol. 6, No. 4, 2007 1323

research articles (2) Hunter, T. Signaling-2000 and beyond. Cell 2000, 100 (1), 11327. (3) Conrads, T. P.; Issaq, H. J.; Veenstra, T. D. New tools for quantitative phosphoproteome analysis. Biochem. Biophys. Res. Commun. 2002, 290 (3), 885-90. (4) Mann, M.; Jensen, O. N. Proteomic analysis of post-translational modifications. Nat. Biotechnol. 2003, 21 (3), 255-61. (5) Kalume, D. E.; Molina, H.; Pandey, A. Tackling the phosphoproteome: tools and strategies. Curr. Opin. Chem. Biol. 2003, 7 (1), 64-9. (6) Peters, E. C.; Brock, A.; Ficarro, S. B. Exploring the phosphoproteome with mass spectrometry. Mini-Rev. Med. Chem. 2004, 4 (3), 313-24. (7) Kokubu, M.; Ishihama, Y.; Sato, T.; Nagasu, T.; Oda, Y. Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis. Anal. Chem. 2005, 77 (16), 5144-54. (8) Kange, R.; Selditz, U.; Granberg, M.; Lindberg, U.; Ekstrand, G.; Ek, B.; Gustafsson, M. Comparison of different IMAC techniques used for enrichment of phosphorylated peptides. J. Biomol. Tech. 2005, 16 (2), 91-103. (9) Feuerstein, I.; Morandell, S.; Stecher, G.; Huck, C. W.; Stasyk, T.; Huang, H. L.; Huber, L. A.; Bonn, G. K. Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder. Proteomics 2005, 5 (1), 46-54. (10) Stensballe, A.; Jensen, O. N. Phosphoric acid enhances the performance of Fe(III) affinity chromatography and matrixassisted laser desorption/ionization tandem mass spectrometry for recovery, detection and sequencing of phosphopeptides. Rapid Commun. Mass Spectrom. 2004, 18 (15), 1721-30. (11) Smith, S. D.; She, Y. M.; Roberts, E. A.; Sarkar, B. Using immobilized metal affinity chromatography, two-dimensional electrophoresis and mass spectrometry to identify hepatocellular proteins with copper-binding ability. J. Proteome Res. 2004, 3 (4), 834-40. (12) Liu, H.; Stupak, J.; Zheng, J.; Keller, B. O.; Brix, B. J.; Fliegel, L.; Li, L. Open tubular immobilized metal ion affinity chromatography combined with MALDI MS and MS/MS for identification of protein phosphorylation sites. Anal. Chem. 2004, 76 (14), 422332. (13) Thompson, A. J.; Hart, S. R.; Franz, C.; Barnouin, K.; Ridley, A.; Cramer, R. Characterization of protein phosphorylation by mass spectrometry using immobilized metal ion affinity chromatography with on-resin beta-elimination and Michael addition. Anal. Chem. 2003, 75 (13), 3232-43. (14) Raska, C. S.; Parker, C. E.; Dominski, Z.; Marzluff, W. F.; Glish, G. L.; Pope, R. M.; Borchers, C. H. Direct MALDI-MS/MS of phosphopeptides affinity-bound to immobilized metal ion affinity chromatography beads. Anal. Chem. 2002, 74 (14), 3429-33. (15) Hart, S. R.; Waterfield, M. D.; Burlingame, A. L.; Cramer, R. Factors governing the solubilization of phosphopeptides retained on ferric NTA IMAC beads and their analysis by MALDI TOFMS. J. Am. Soc. Mass Spectrom. 2002, 13 (9), 1042-51. (16) Posewitz, M. C.; Tempst, P. Immobilized gallium(III) affinity chromatography of phosphopeptides. Anal. Chem. 1999, 71 (14), 2883-92. (17) Zheng, H.; Hu, P.; Quinn, D. F.; Wang, Y. K. Phosphotyrosine proteomic study of interferon alpha signaling pathway using a combination of immunoprecipitation and immobilized metal affinity chromatography. Mol. Cell. Proteomics 2005, 4 (6), 72130. (18) Stupak, J.; Liu, H.; Wang, Z.; Brix, B. J.; Fliegel, L.; Li, L. Nanoliter sample handling combined with microspot MALDI-MS for detection of gel-separated phosphoproteins. J. Proteome Res. 2005, 4 (2), 515-22. (19) Kim, J. E.; Tannenbaum, S. R.; White, F. M. Global phosphoproteome of HT-29 human colon adenocarcinoma cells. J. Proteome Res. 2005, 4 (4), 1339-46. (20) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20 (3), 3015. (21) Brill, L. M.; Salomon, A. R.; Ficarro, S. B.; Mukherji, M.; StettlerGill, M.; Peters, E. C. Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry. Anal. Chem. 2004, 76 (10), 2763-72. (22) Sano, A.; Nakamura, H. Chemo-affinity of titania for the columnswitching HPLC analysis of phosphopeptides. Anal. Sci. 2004, 20 (3), 565-6.

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Hsiao et al. (23) Sano, A.; Nakamura, H. Titania as a chemo-affinity support for the column-switching HPLC analysis of phosphopeptides: application to the characterization of phosphorylation sites in proteins by combination with protease digestion and electrospray ionization mass spectrometry. Anal. Sci. 2004, 20 (5), 861-4. (24) Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 2004, 76 (14), 3935-43. (25) Kuroda, I.; Shintani, Y.; Motokawa, M.; Abe, S.; Furuno, M. Phosphopeptide-selective column-switching RP-HPLC with a titania precolumn. Anal. Sci. 2004, 20 (9), 1313-9. (26) Chen, C. T.; Chen, Y. C. Fe3O4/TiO2 core/shell nanoparticles as affinity probes for the analysis of phosphopeptides using TiO2 surface-assisted laser desorption/ionization mass spectrometry. Anal. Chem. 2005, 77 (18), 5912-9. (27) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 2005, 4 (7), 873-86. (28) Zhang, X.; Leung, S. M.; Morris, C. R.; Shigenaga, M. K. Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma. J. Biomol. Tech. 2004, 15 (3), 16775. (29) Villanueva, J.; Philip, J.; Entenberg, D.; Chaparro, C. A.; Tanwar, M. K.; Holland, E. C.; Tempst, P. Serum peptide profiling by magnetic particle-assisted, automated sample processing and MALDI-TOF mass spectrometry. Anal. Chem. 2004, 76 (6), 156070. (30) Baumann, S.; Ceglarek, U.; Fiedler, G. M.; Lembcke, J.; Leichtle, A.; Thiery, J. Standardized approach to proteome profiling of human serum based on magnetic bead separation and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. Clin. Chem. 2005, 51 (6), 973-80. (31) Tummala, R.; Limbach, P. A. Serum protein profiling using surfactant-aided matrix-assisted laser desorption/ionization mass spectrometry. Anal. Chim. Acta 2005, 551 (1-2), 137-41. (32) Bruce, I. J.; Sen, T. Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations. Langmuir 2005, 21 (15), 7029-35. (33) Qhobosheane, M.; Santra, S.; Zhang, P.; Tan, W. Biochemically functionalized silica nanoparticles. Analyst 2001, 126 (8), 12748. (34) Lee, C. L.; Hsiao, H. H.; Lin, C. W.; Wu, S. P.; Huang, S. Y.; Wu, C. Y.; Wang, A. H.; Khoo, K. H. Strategic shotgun proteomics approach for efficient construction of an expression map of targeted protein families in hepatoma cell lines. Proteomics 2003, 3 (12), 2472-86. (35) Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 2003, 75 (3), 663-70. (36) Ishihama, Y.; Rappsilber, J.; Mann, M. Modular stop and go extraction tips with stacked disks for parallel and multidimensional peptide fractionation in proteomics. J. Proteome Res. 2006, 5 (4), 988-94. (37) Kim, J. S.; Kim, J. Y.; Kim, H. J. Suppression of matrix clusters and enhancement of peptide signals in MALDI-TOF mass spectrometry using nitrilotriacetic acid. Anal. Chem. 2005, 77 (22), 7483-8. (38) Smirnov, I. P.; Zhu, X.; Taylor, T.; Huang, Y.; Ross, P.; Papayanopoulos, I. A.; Martin, S. A.; Pappin, D. J. Suppression of alphacyano-4-hydroxycinnamic acid matrix clusters and reduction of chemical noise in MALDI-TOF mass spectrometry. Anal. Chem. 2004, 76 (10), 2958-65. (39) Imanishi, S. Y.; Kochin, V.; Eriksson, J. E. Optimization of phosphopeptide elution conditions in immobilized Fe(III) affinity chromatography. Proteomics 2007, 7 (2), 174-6. (40) Kishimoto, A.; Nishiyama, K.; Nakanishi, H.; Uratsuji, Y.; Nomura, H.; Takeyama, Y.; Nishizuka, Y. Studies on the phosphorylation of myelin basic protein by protein kinase C and adenosine 3′:5′monophosphate-dependent protein kinase. J. Biol. Chem. 1985, 260 (23), 12492-9.

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