Enrichment of Phosphopeptides by Fe3+-Immobilized Magnetic

Enrichment of Phosphopeptides by Fe3+-Immobilized Magnetic Nanoparticles for Phosphoproteome Analysis of the Plasma Membrane of Mouse Liver Feng Tan, Yangjun Zhang, Wei Mi, Jinglan Wang, Junying Wei, Yun Cai, and Xiaohong Qian* State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 100850, China Received October 10, 2007

Immobilized metal ion affinity chromatography (IMAC) is a commonly used technique for phosphoprotein analysis due to its specific affinity for phosphopeptides. In this study, Fe3+immobilized magnetic nanoparticles (Fe3+-IMAN) with an average diameter of 15 nm were synthesized and applied to enrich phosphopeptides. Compared with commercial microscale IMAC beads, Fe3+-IMAN has a larger surface area and better dispersibility in buffer solutions which improved the specific interaction with phosphopeptides. Using tryptic digests of the phosphoprotein R-casein as a model sample, the number and signal-to-noise ratios of the phosphopeptides identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) following Fe3+-IMAN enrichment greatly increased relative to results obtained with direct MALDITOFMS analysis. The lowest detectable concentration is 5 × 10-11 M for 100 µL of pure standard phosphopeptide (FLTEpYVATR) following Fe3+-IMAN enrichment. We presented a phosphopeptide enrichment scheme using simple Fe3+-IMAN and also a combined approach of strong cation exchange chromatography and Fe3+-IMAN for phosphoproteome analysis of the plasma membrane of mouse liver. In total, 217 unique phosphorylation sites corresponding to 158 phosphoproteins were identified by nano-LC-MS/MS. This efficient approach will be very useful in large-scale phosphoproteome analysis. Keywords: enrichment • Fe3+-immobilized magnetic nanoparticles • mass spectrometry • phosphorylation • plasma membrane

Introduction Protein phosphorylation is one of the most important posttranslational modifications in mammalian cells. It regulates numerous biological processes, including cell proliferation, differentiation, metabolism, communication, and signal transduction.1–3 Global analysis of protein phosphorylation is very significant for exploring these critical processes. Proteomics techniques based on mass spectrometry, in which the peptide products of proteolytic digestion of proteins are identified by MS or tandem MS/MS, have been widely applied for analysis of protein phosphorylation. However, large-scale analysis of phosphoproteins by MS is limited due to the low abundance of phosphoproteins in tissues or cells and because of the ion suppressive effect of the nonphosphopeptides in digests. The detection of phosphopeptides has been improved by many enrichment approaches that reduce sample complexity and increase the relative concentration of phosphopeptides including the introduction of affinity tags following β-elimination, immobilized metal affinity chromatography (IMAC), TiO2, ZrO2, Fe3O4/TiO2 nanoparticles, zirconium phosphonate enrich* Corresponding author. Beijing Proteome Research Center, 33 Life Park Road, Zhongguancun, Beijing 102206, China. Tel.: +86-10-80725055. Fax: +86-10-80705155. E-mail: [email protected].

1078 Journal of Proteome Research 2008, 7, 1078–1087 Published on Web 02/12/2008

ment, and strong cation/anion exchange chromatography (SCX/SAX).4–22 Among these approaches, IMAC is the most convenient and has been used for phosphoproteome analysis of tissues, whole cells, and cellular organelles, in which hundreds or even thousands of phosphorylation sites have been identified.18–22 Despite these great achievements, phosphoproteome analysis on a large scale requires further improvements in enrichment selectivity and efficiency. Solutions may follow two courses: (1) to optimize the enrichment procedures, including loading, washing, and elution, for enhancing the selectivity to phosphopeptides23–25 and (2) to optimize the materials for IMAC, including selection of different metal ions, affinity functional groups, and supporting matrixes.16,26–29 In this paper, we synthesized Fe3+-immobilized magnetic nanoparticles (Fe3+-IMAN) which have an average diameter of 15 nm. We first evaluated the performance of Fe3+-IMAN for enriching phosphopeptides with tryptic digests of standard proteins as model samples. Then, Fe3+-IMAN was applied for phosphoproteome analysis of plasma membranes from mouse liver, with the successful identification of a large number of phosphorylation sites. 10.1021/pr700655d CCC: $40.75

 2008 American Chemical Society

Enrichment of Phosphopeptides by Nanoparticles

Experimental Section Materials and Reagents. Bovine R-casein and serum albumin (BSA), iminodiacetic acid (IDA), 2-[4-(2-hydroxyethyl)-1piperazinyl]ethanesulfonic acid (HEPES), 2,5-dihydroxybenzoic acid (DHB), and sucrose were purchased from Sigma-Aldrich (MO, USA). Sequencing grade trypsin was purchased from Promega (WI, USA). 1,4-Dithio-DL-threitol (DTT) and iodoacetamide were obtained from Pierce (IL, USA). Iron(III) chloride hexahydrate (FeCl3 · 6H2O), iron(II) chloride tetrahydrate (FeCl2 · 4H2O), ethylenediaminetetraacetic acid (EDTA), sodium orthovanadate (Na3VO4), sodium pyrophosphate (Na4P2O7 · 10H2O), 25∼28% NH3 · H2O, and sodium fluoride (NaF) were obtained from Beijing Chemical Factory (Beijing, China). 99 % standard phosphopeptide (FLpTEYVATR, m/z ) 1179.55) was obtained from SBS Genetech (Beijing, China). HPLC grade acetonitrile (ACN) was purchased from Fisher (MA, USA). Tetraethyl orthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane (GLYMO) were purchased from Acros (NJ, USA). POROS IMAC beads were obtained from Applied Biosystems (MA, USA). All reagents are of analytical grade except special illustration. Preparation of Fe3+-IMAN. Magnetic Fe3O4 nanoparticles were synthesized based on a literature procedure30 with minor modifications. FeCl2 · 4H2O (1.08 g, 5.44 mmol) and FeCl3 · 6H2O (2.95 g, 10.92 mmol) were dissolved in 50 mL of deionized water with vigorous stirring under an N2 atmosphere. The solution was heated to 60 °C, followed by addition of 6 mL of NH3 · H2O. After agitation for 30 min, the resulting Fe3O4 nanoparticles were harvested by applying a magnet adjacent to the reaction vial. The isolated particles (∼1 g) were rinsed with deionized water four times to remove unreacted chemicals. Fe3O4 nanoparticles (∼20 mg) were resuspended in 60 mL of isopropanol and 6 mL of deionized water, followed by ultrasonication for 30 min. Then, NH3 · H2O (7 mL) and TEOS (1 mL) were consecutively added under continuous stirring for reaction for 4 h at room temperature (RT). This reaction process coated the Fe3O4 nanoparticles with a thin layer of SiO2. The resulting SiO2/Fe3O4 nanoparticles were rinsed with deionized water four times prior to further usage. An amount of 100 mg of the SiO2/Fe3O4 nanoparticles was prepared by this procedure. IDA (4.25 g, 18.71 mmol) was dissolved in 50 mL of deionized water and adjusted to pH 11.0 by 10 M NaOH, then transferred into a flask immersed into an ice-bath. GLYMO (1.4 mL) was slowly added, with stirring, and the mixture was heated to 65 °C and then allowed to react for 6 h. The solution was chilled to 0 °C in an ice-bath; an additional 1.6 mL of GLYMO was added; and the temperature was elevated to 65 °C for another 6 h of reaction. The resulting GLYMO-IDA silane solution was adjusted to pH 6.0 by 6 M HCl. Subsequently, 40 mg of SiO2/ Fe3O4 nanoparticles was added into 50 mL of the silane solution under an N2atmosphere for reaction for 4 h at 75 °C with stirring. The resulting IMAN were isolated by applying a magnet. They were rinsed with deionized water three times prior to storage in deionized water. Finally, about 5 mg of IMAN was rinsed with 0.5 M EDTA-1 M NaCl and deionized water in turn, followed by adding 5 mL of 0.1 M FeCl3/5 mM HCl under vortex for 0.5 h at RT and rinsing with deionized water and enrichment loading buffer (60% ACN/1% HAc). The resulting Fe3+-IMAN was used for enrichment of phosphopeptides. Digestion of r-Casein and BSA. BSA was prepared in 50 mM NH4HCO3/8 M urea at pH 8.0. An amount of 1 µL of 0.5 M DTT was added for 4 h at 37 °C, followed by adding 2 µL of 1

research articles M iodoacetamide for 30 min away from light at RT. The solution was diluted ten times by 50 mM NH4HCO3, and trypsin was added with a substrate/trypsin ratio of 50:1 (w:w) at 37 °C for 16 h. R-Casein was digested using the same procedures. Protein Solubilization and Digestion. Plasma membranes (PM) were isolated from mouse liver with the method in the literature.31 PM protein solubilization was based on the method in the literature32 with minor modifications. A sample of the isolated PM preparation (∼5 mg) was resuspended in 100 mM NH4HCO3 (40 mL) at pH 8.0 with occasional vortexing. The suspension was kept for 20 min in a 90 °C water-bath for thermal denaturation of proteins, cooled by an ice-bath, and diluted by addition of methanol to achieve a sample solution containing 60% methanol. Protein concentration was measured by the Bio-Rad method. Digestion was performed with a substrate/trypsin ratio of 50:1 (w:w) at 37 °C for 6 h. The supernatant was collected following centrifugation at 10 000g for 5 min at 4 °C. The pellet was resuspended in 200 µL of 40 mM NH4HCO3/60% methanol and subjected to another 6 h digestion at the same conditions. The two supernatants were pooled and dried under vacuum, then resuspended in 1% formic acid, followed by desalting with a C18 SepPak cartridge. Eluted peptides were lyophilized and stored at -80 °C. Strong Cation Exchange Chromatography (SCX) Separation. SCX separation was carried out using a Yilite HPLC apparatus with 5 µm dimension of BioBasic SCX column 4.6 × 250 mm (ThermoElectron, MA, USA) with a flow rate of 0.5 mL/min and UV monitoring at 214 nm. We collected one fraction per 3 min. The elution conditions were: a linear gradient from 0% to 15% buffer B over 20 min, then 1 min to attain 100% B, and then 100% B for 20 min. The system was changed to 100% A in 1 min and held for 18 min. In our HPLC system, there is an approximately 12 min gradient delay, and thus, those fractions from 1 to 12 min are eluted by 100% buffer A. The first 10 fractions that eluted were collected, lyophilized, and redissolved in 1% formic acid, followed by desalting with C18 SepPak cartridges. Eluted peptides were lyophilized and stored at -80 °C. Enrichment of Phosphopeptides by Fe3+-IMAN. Digests of standard proteins were resuspended in an Eppendorf vial containing 100 µL of loading buffer, followed by addition of 1 mg of Fe3+-IMAN. After incubation for 0.5 h under vortexing, Fe3+-IMAN was separated from the buffer by application of a magnet to the Eppendorf vial and then washed with 200 µL of 0.1 M NaCl/60% ACN/1% HAc once and 200 µL of 60% ACN/ 1% HAc twice. The phosphopeptides at the Fe3+-IMAN were eluted with 3 µL of 0.2 M NH3 · H2O with vortexing for 3 min, and 0.8 µL of eluate was used for MALDI-TOFMS analysis. An amount of 1 mg of IMAC beads was place in a 0.6 mL Eppendorf vial and washed subsequently with 50 mM EDTA/1 M NaCl, H2O, and 0.1 M FeCl3 in 10 mM HCl for 10 min, two times with H2O, and 60% ACN/1% HAc. Enrichment of phosphopeptides with the activated IMAC beads was carried out in the Eppendorf vial by centrifugation using the same washing buffer and elution buffers as Fe3+-IMAN enrichment above. The resulting elutes from the IMAC beads were analyzed by MALDI-TOFMS. Each of the ten lyophilized fractions from SCX and 40 µg digests of PM proteins were, respectively, enriched by Fe3+IMAN using the same procedures as those described above except for the use of 800 µL of loading buffer and washing buffer and 5 mg of Fe3+-IMAN. The phosphopeptides were eluted with 20 µL of 0.2 M NH3 · H2O three times. The three Journal of Proteome Research • Vol. 7, No. 3, 2008 1079

research articles eluates were pooled and acidified with 10 µL of 20% formic acid, lyophilized, and redissolved in 90 µL of 2% ACN/0.1% formic acid for nano-LC-MS/MS analysis. Mass Spectrometry Analysis. MALDI-TOFMS analysis was performed on a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems, MA, USA) equipped with an Nd:YAG laser, which has an excited wavelength of 355 nm. All mass spectra (1600 laser shots for every spectrum) were obtained in positive ion reflection mode and analyzed by data Explorer Version 4.5. 2,5Dihydroxybenzoic acid prepared in 10% methanol/1% phosphoric acid was used as the matrix. Nano-LC-MS/MS analysis was carried out on an Agilent 1100 series system coupled with a hybrid linear ion trap-7 T Fourier transform ion cyclotron resonance mass spectrometer (LTQFTMS). The sample (40 µL) was loaded by a loading pump onto a fused silica capillary column prepared in-house with C18 packing materials (SP-ODS-AP, 5-µm, 120-Å pore size, Jinouya, Beijing). A 45 min linear gradient was applied ranging from 0% B to 45% B with a flow rate of 200 nL/min. The mobile phase consisted of: A, 0.1% formic acid/2% ACN, and B, 0.1% formic acid/80% ACN. For each cycle, one full MS scan [400–2000 m/z, 105 resolution in ICR cell and automatic gain control (AGC) of 5 × 105] was followed by 10 data-dependent MS/MS spectra in the linear ion trap from the 10 most abundant ions, and with dynamic exclusion for 30 s. Singly charged ions were rejected. Two replicate nano-LC-MS/MS analyses were completed for every sample at the same conditions. Database Searching and Evaluation of MS/MS Spectra. All MS/MS spectra were searched with the Sequest algorithm at Bioworks 3.2 against a composite database containing the mouse IPI database (v.3.28, download from ftp.ebi.ac.uk/pub/ databases/IPI/current, containing 53 847 proteins) and its reversed database. Searching parameters include: full tryptic specificity, two missed cleavage sites, dynamic modifications of 79.96633 Da on serine, threonine, tyrosine and 15.99491 Da (oxidation) on methionine, precursor mass tolerance of 10 ppm, and fragment ions mass tolerance of ( 0.6 Da. The results were filtered by setting Xcorr g 2.5 and 3.5 corresponding to 2+ and 3+ charge states, respectively, DeltaCn g 0.08, and Rsp e 4. This strategy obtained a rate of less than 1% for false-positive values for all identified peptides. The filtered phosphopeptides were validated by manually interpreting MS/MS spectra, including at least four continuous y or b ions above baseline noise, and exact location of phosphorylation sites in sequences.

Results and Discussion Figure 1a shows a model of the synthesized Fe3+-IMAN, which is a three-layer composite including the inner Fe3O4 core, middle SiO2 layer, and outer affinity functional groups. From the TEM image of Fe3+-IMAN (Figure 1b, 100 k magnification), an average diameter of 15 nm was estimated. The enrichment of IMAC beads to phosphopeptides is based on the specific interaction between the Fe3+-immobilized at the beads and phosphate groups of phosphopeptides; increasing the surface area of IMAC beads will result in more Fe3+ immobilized, achieving a large capture capacity. Fe3+-IMAN will present a high capacity to phosphopeptides since nanoparticles have a very large surface area. Because Fe3+-IMAN are strongly hydrophilic and have very small particle size, they dispersed easily in buffer solutions; this characteristic will improve the interaction between Fe3+ immobilized at IMAN and the phosphate groups of phosphopeptides, resulting in enhanced 1080

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Figure 1. Model (a) and TEM image (b) of the synthesized Fe3+IMAN. In Figure 1a, the inner core is Fe3O4 nanoparticles; the middle is the SiO2 layer; and the outer is affinity functional groups consisting of iminodiacetic acid and Fe3+, which are linked with the SiO2 layer through GLYMO. The TEM result in Figure 1b clearly shows the appearance of the prepared Fe3+IMAN nanoparticles with an average diameter of 15 nm.

efficiency. We used a pure standard phosphopeptide (FLTEpYVATR) for determination of the capture capacity of Fe3+-IMAN (analyzing the supernatant and eluate following Fe3+-IMAN enrichment with HPLC. The resulting capture capacity of Fe3+IMAN to the phosphopeptide is 64.2 pmol/mg, which is about 1.4 times of that (44.2) of microscale IMAC beads (see method S-1, Supporting Information). Another advantage is that Fe3+IMAN enriched phosphopeptides can be easily isolated from buffer solutions by applying a magnet adjacent to Eppendorf vials. The isolation of microscale IMAC beads in Eppendorf vials by centrifugation during the washing processes is hampered by the light density of the beads relative to the washing buffers, especially when the washing buffers do not contain ACN, resulting in the loss of IMAC beads carrying phosphopeptides during aspiration of the supernatants. Enrichment of Phosphopeptides in Digests of r-Casein. Tryptic digests of the phosphoprotein R-casein were used to evaluate the enrichment efficiency of Fe3+-IMAN toward phosphopeptides. Casein tryptic digests contain many phosphopeptides and have been used as typical samples to evaluate novel enrichment approaches or affinity materials. In direct analysis of the digest, six phosphopeptides (labeled as R1, R2, R3, R4, R5, and R6) are observed in the MALDI-TOF mass spectrum of the digests of 2 pmol of R-casein (Figure 2a). The presence of a number of nonphosphopeptides in the spectrum seriously suppressed the detection of the phosphopeptides, leading to low signal-to-noise ratios (S/N). However, after the digests were enriched by Fe3+-IMAN using the described procedures, the MALDI-TOF mass spectrum indicates that the nonphosphopeptides in the digests are effectively eliminated, and 17 phosphopeptides are detected (Figure 2b). After enrichment, the S/N of the phosphopeptides at m/z 1466.66, 1660.85, 1927.77, and 1952.02 obviously increased, and they constituted the most abundant peaks in the spectrum. In addition, most


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Figure 2. MALDI-TOF mass spectra of the tryptic digests of 2 pmol of R-casein without (a) and with (b) Fe3+-IMAN enrichment.

of the newly detected phosphopeptides contain multiple phosphorylation sites and have high mass-to-charge ratios. These phosphopeptides are difficult to find in ordinary MALDITOFMS analysis without enrichment treatment due to low ionization efficiency and the interference from nonphosphopeptides. This result demonstrates that single phosphopeptides and also multiple phosphopeptides can be effectively identified by MALDI-TOFMS following Fe3+-IMAN enrichment. The detailed information about the identified phosphopeptides is listed in Table S-1, Supporting Information. The selective enrichment of phosphopeptides by Fe3+-IMAN was further inspected using a complex mixture consisting of digests of R-casein (2 pmol) and BSA (20 pmol). Figure 3a shows the MALDI-TOF mass spectrum for direct analysis of the mixture. Only two phosphopeptides at m/z 1927.78 and 1952.01 with very low S/N were detected, and their signals were overwhelmed by a great number of nonphosphopeptides from R-casein and BSA. Figure 3b displays the MALDI-TOF mass spectrum of the mixture following Fe3+-IMAN enrichment. Most of the nonphosphopeptides have been effectively eliminated, and the 14 detected phosphopeptides dominate the

mass spectrum. When the mixture was subjected to enrichment with IMAC beads, the MALDI-TOF mass spectrum (Figure 3c) showed that seven phosphopeptides are detected, but the most abundant peak is the nonphosphopeptide with m/z 1749.75. Thus, Fe3+-IMAN can efficiently enrich phosphopeptides from complex digests of proteins. To reveal the ability of Fe3+-IMAN to trap phosphopeptides present in very low concentration, several samples with fixed volume but different concentration were used. Figures 4a-c show the MALDI-TOF mass spectra of 100 µL digests of R-casein with a concentration of 10-8, 10-9, and 2 × 10-10 M, in which thirteen, seven, and two phosphopeptides are detected, respectively. As far as we know, this is the lowest sample concentration for which the phosphopeptides from the digests of R-casein can be enriched. For comparison, we obtained the MALDI-TOF mass spectra (Figures 4d,e) of 100 µL of the digests at concentrations of 10-8 and 10-9 M using an equivalent amount of commercial microscale IMAC beads for the enrichment procedures. Only five phosphopeptides were detected at 10-8 M, and no phosphopeptides were found at 10-9 M. We also used a pure standard phosphopeptide (FLTEpYVATR) for Journal of Proteome Research • Vol. 7, No. 3, 2008 1081

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Figure 3. MALDI-TOF mass spectra of a tryptic digest mixture of 2 pmol of R-casein and 20 pmol of BSA. (a) direct MALDI-TOFMS analysis; (b) following Fe3+-IMAN enrichment; (c) following IMAC beads enrichment.

this experiment. The results showed, following Fe3+-IMAN enrichment, that the lowest sample concentration in which the phosphopeptide could be isolated and identified by MALDITOFMS is about 5 × 10-11 M (Figure 5). The results above indicate that the capture ability of Fe3+IMAN toward phosphopeptides is superior to that of the microscale IMAC beads due to large surface area and good dispersibility in buffer solutions. Application of Fe3+-IMAN to Phosphoproteome Analysis of the Plasma Membrane of Mouse Liver. Plasma membranes (PM) play important roles in maintaining intracellular environments, substance transport, signal transduction, cell signaling, communication, and recognition. Analysis of PM proteins has presented a great challenge due to the poor solubility in aqueous buffers. Surfactant- (SDS) or organic solvent- (methanol) assisted protein solubilization and digestion methods are usually used to circumvent this problem.30–34 Considering the compatibility with subsequent nano-LC-MS/MS analysis, the methanol-assisted method was used to treat PM proteins of mouse liver. The resulting digests of 240 µg pf PM proteins were divided into two parts. One sample (40 µg) was directly enriched by Fe3+-IMAN (see Experimental Section). The enriched peptides were analyzed by nano-LC coupled with a LTQFTMS. Previous studies indicated that only a small fraction of identified phosphorylation sites were identified from MS3 scans,20,35 so MS analysis in this study was carried out by ten data-dependent MS/MS scans in LTQ following one full MS scan in FTICR. In two replicate analyses, 38 unique phosphopeptides were identified. If a phosphopeptide was simultaneously identified in two replicate MS analyses, the identification result with a high Xcorr value was selected and counted. 1082


The coverage of two replicate results is 70%. The corresponding phosphorylated sites in these phosphopeptides could be exactly located by manual inspection of the MS/MS spectra. Because the digests of PM proteins are very complex, we further applied a combined strategy of strong cation exchange chromatography (SCX) and Fe3+-IMAN for the phosphoproteome analysis. SCX chromatography is mainly based on the net solution charge of peptides, with those phosphopeptides with +1 net charge eluting early relative to tryptic peptides with + g+2 net charge.20 SCX separation prior to Fe3 -IMAN enrichment will greatly reduce sample complexity. Here, using the remainder of the above PM protein digest, 200 µg of PM digests were separated by SCX at pH 2.7 (see Figure S-1, Supporting Information). The 10 eluted fractions were collected and individually enriched by Fe3+-IMAN. The enriched peptides were then analyzed by nano-LC-LTQ-FTMS. In total, 192 unique phosphopeptides were identified in two replicate analyses (the identification result with the higher Xcorr value was selected and counted), and the coverage of two replicate results is 73%. Figure 6 lists the number of detected unique phosphopeptides from every SCX fraction. The majority (80%) of the phosphopeptides is isolated from fractions 6 (15 to 18 min) and 7 (18 to 21 min), and they exclusively contain a solution charge of +1 except for two phosphopeptides with a solution charge of 0 in fraction 7 and of +2 in fraction 6. The largest percentage of identified phosphopeptides in whole peptides from SCX fractions 6–7 is 81.3%. The identified phosphopeptides in fractions 8–10 mainly represent g+2 net charges, which coeluted with many nonphosphopeptides with g+2 net charge. This result indicates that the SCX separation based on net solution charges is efficient in this system. The


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Figure 4. MALDI-TOF mass spectra of 100 µL tryptic digests of R-casein with different sample concentration following Fe3+-IMAN (a-c) and commercial 5 µm IMAC bead (d,e) enrichment. Sample concentration: (a) 10-8 M, (b) 10-9 M, (c) 2 × 10-10, (d) 10-8 M, (e) 10-9 M.

combination of SCX and Fe3+-IMAN resulted in a number of the phosphopeptides 5-fold greater more than that obtained using single Fe3+-IMAN enrichment, indicating that a combined enrichment strategy is necessary for such a complex system. In total, 207 unique phosphopeptides corresponding to 158 phosphoproteins were identified by the direct Fe3+IMAN enrichment and the combination of SCX and Fe3+-IMAN. Twenty-three phosphopeptides were simultaneously identified by the two methods. Of the identified phosphoproteins, 28 were identified from more than one phosphopeptide. Table S-1 lists the detailed information about the 207 phosphopeptides. The 158 identified phosphoproteins were analyzed by GO fact (http://www.hupo.org.cn). 97 have component annotation, while 29 (30%) are regarded as PM proteins. Contaminants from the nucleus, mitochondrion, and endoplasmic reticulum which

are usually present in PM preparations were found. 96 phosphoproteins with function annotation are categorized into ten groups, including transporter, translation regulator, transcription regulator, structural molecule, signal transducer, enzyme regulator, catalytic and motor activity, molecular function unknown, and binding proteins (see Figure S-2). Extracellular hydrophilic signal molecules such as neurotransmitters, proteohormones, and growth factors are transmitted by specific receptors at the PM to trigger intracellular cascade events. In this study, six receptors were identified, including the tumor necrosis factor receptor, adrenomedullin receptor, lipoprotein receptor precursor, F11r receptor, progesterone receptor, and receptor expression-enhancing protein. MS/MS spectra of phosphopeptides usually show strong neutral loss peaks, which are usually used as a criterion for Journal of Proteome Research • Vol. 7, No. 3, 2008 1083

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Figure 5. MALDI-TOF mass spectra of 100 µL pure standard phosphopeptide (FLTEpYVATR) with different sample concentration for direct analysis (a) and following enrichment with Fe3+-IMAN. Sample concentration: (a) 10-7 M, 2 × 10-8 M, 2 × 10-9 M, 2 × 10-10 M; (b) 2 × 10-8 M, 2 × 10-9 M, 2 × 10-10 M, 5 × 10-11 M.

information. Figure 7b shows the MS/MS spectrum of the phosphopeptide ALGVISNFQSS#PK containing two consecutive serine residues, in which y8+1 and y9+1 ions dominate the spectrum. Analysis of the Identified Phosphorylation Sites. Most of the identified phosphopeptides are singly phosphorylated except for 2 triply phosphorylated and 13 doubly phosphorylated peptides. 207 phosphopeptides contained 217 unique phosphorylation sites, with 175 (81.8%) located at serine residues, 29 (13.1%) at threonine residues, and 11 (5.1%) at tyrosine residues.

Figure 6. Number of the identified unique phosphopeptides from every SCX fraction. Phosphopeptides were identified in fractions 2 and 5–10, and peptides were not detected in fractions 1 and 3–4.

identification of phosphopeptides.7,26 On one hand, not all phosphopeptides are subject to fragmentation that produces a strong neutral loss in collision-induced dissociation (CID). The amide bonds adjacent to prolines in sequences are more susceptible to fragmenting than O-P bonds of phosphorylated serine, threonine, and tyrosine residues in CID, leading to either weak or no neutral loss, named the “proline effect”.36 In addition, when two or more serine, threonine, and tyrosine residues are closely spaced, β-elimination cleavage yielding neutral loss is difficult to detect. On the other hand, fragmentation of some nonphosphopeptides possibly undergoes loss of H2O, yielding fragment ions with the same m/z as peptides or fragment ions with loss of H3PO4.37 Of our data set, 67 phosphopeptides produced no obvious neutral losses. For example, the amide bond of the leucine at the fourth position adjacent to the proline at the fifth position in the phosphopeptide DEILPTT@PISEQK broke prior to that of the O-P bond of the seventh phosphothreonine, leading to the most abundant y9+ ion rather than any neutral loss peak from the precursor or b+ or y+ ions, as shown in Figure 7a. The phosphorylation position can be exactly located to the seventh threonine by observing continuous b+ and y+ ions containing backbone 1084


We classified the identified phosphorylated sites into three major general kinase motifs: pro-directed (P at +1), basophilic (R/K at -3 or g2 R/K at -1 to -6), and acidophilic (Sp/Tp-XX-E/D/Sp). Pro-directed and basophilic sites account for 35.5% and 37.3% of all the identified sites, respectively; however, the proportion of acidophilic sites is less relative to that in previous data sets.18,20 The phosphorylation sites were analyzed by the SCANSITE program (http://scansite.mit.edu).38 36 (16.6%) and 91 (41.9%) sites could be predicted at high (0.2%) and middle (1%) stringency, respectively. Table 1 shows the number of matches between the sites and several potential kinase motifs. The sites matching basophilic S/T kinase groups are most commonly predicted, followed by Pro-directed S/T kinase groups, which is similar with that of the manual classification above; however, many identified sites could not be predicted by SCANSITE even at middle stringency. At high stringency, several phosphorylation sites are matched by AKT, PKA, PKC, Erk1, and 14–3–3 mode 1 kinase motifs, respectively. These kinases play important roles in mediating extracellular and intracellular signaling events, including cell apoptosis, cell cycle, cell secretion, proliferation, differentiation, and in muscle contraction, transducing signal cascades. For example, in response to messengers induced by extracellular signals, AKT and PKC shift to the PM internal wall, where they are activated by a phosphoinositol-dependent kinase and diacylglycerol, respectively. The activated kinases phosphorylate serine and threonine residues of proteins, resulting in a series of cell processes.39,40 Erk1 is a key transducer of extracellular signals that promotes cell growth and movement and is critical for the


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Figure 7. MS/MS spectra of the phosphopeptides DEILPTT@PISEQK and ALGVISNFQSS#PK without obvious neutral loss peaks. The strongest peaks are the y9+ ion for the phosphopeptide DEILPTT@PISEQK and y8+1 and y9+1 for the phosphopeptide ALGVISNFQSS#PK.

initiation and progression of vascular lesions. Many transcription factors are potential targets of activated ERK1.41 Because there are no published phosphoproteome data sets of the PM of mouse liver, we compared our data set with the

public database of UniProtKB/Swiss-Prot and found that 63.5% of them are newly identified phosphorylation sites. Some phosphorylated proteins at the PM with novel phosphorylation sites play an important role in mediating cellular functions. Journal of Proteome Research • Vol. 7, No. 3, 2008 1085

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Table 1. Scansite Predictions at High (0.2%) and Middle (1%) Stringency for Kinase Phosphorylation and Binding Motifs from the Identified Phosphorylation Sites kinases

hits (1%)

hits (0.2%)

basophilic S/T kinase groups AKT PKC PKA Cam2 Clk2 proline-dependent S/T kinase groups Erk1 p38 Cdc2 Cdk5 pS/T binding groups 14–3–3 Mode 1 acidophilic S/T kinase groups GSK3 Casn2

54 18 18 7 6 5 45 13 12 12 8 11 11 10 7 3

19 10 3 3 2 1 10 5 1 2 2 5 5 4 3 1

For example, the organic cation/carnitine transporter 2 (OCTN2) which is expressed in kidney, liver, and other tissues is a Na+independent organic cation transporter as well as a Na+dependent carnitine transporter. Mouse OCTN2 protein has 83% sequence homology with human OCTN2. A systemic carnitine deficiency in mice showed critical symptoms of fatty liver, hyperammonemia, and hypoglycemia. These symptoms are also the phenotype of systemic carnitine deficiency in humans.42 In addition, some organic cations which OCTN2 recognized as substrates are pharmacologically active and are currently used as therapeutic agents, and thus this transporter may have significant functions of pharmacological and therapeutic relevance in the body.43 In this study, OCTN2 has been first identified as a phosphoprotein (phosphorylation of S548 in sequences). Further study is necessary to reveal whether these significant functions of OCTN2 are associated with the phosphorylation. Archvillin, located in the PM, is among the first costameric proteins to assemble during myogenesis. It forms a high-affinity link between the Actin cytoskeleton and the membrane and contributes to myogenic membrane structure and differentiation.44 Except for the previously identified sites at S218, S227, S632, S634, and S1011, T81, and T1131, a novel phosphorylation site at S960 is identified in this current study. These newly identified phosphorylation sites will be useful in the research of signal transduction processes involving the plasma membrane.

Conclusions

of mouse liver. With nano-LC-MS/MS, we successfully identified 217 phosphorylation sites including many novel sites with only 240 µg of PM proteins. This efficient approach will be very useful in large-scale phosphoproteome analyses.

Acknowledgment. This work was supported by the National Natural Science Foundation (20505019, 20505018, 20635010, and 20735005), National Key Program for Basic Research (2006CB910803, 2006CB910801) and Hi-Tech Research and Development Program of China (2006AA02A312). Supporting Information Available: Supporting Information (SI) included in the text. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) (2) (3) (4) (5) (6) (7)

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3+

Fe -immobilized magnetic nanoparticles with an average diameter of 15 nm for enriching phosphopeptides were synthesized. Fe3+-IMAN captured phosphopeptides could be easily separated from loading buffer by using a magnet adjacent to Eppendorf vials and then eluted by NH3 · H2O for MS analysis. Compared with commercial microscale IMAC materials, Fe3+IMAN has a larger surface area and better dispersibility in buffer solutions, improving the specific interaction between the Fe3+immobilized nanoparticles and the phosphopeptide molecules. The resulting efficiency is obviously superior to that of microscale IMAC beads. We have presented a phosphopeptide enrichment scheme for using simple Fe3+-IMAN and a combination of strong cation exchange chromatography and Fe3+IMAN for phosphoproteome analysis of the plasma membrane 1086


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PR700655D

Journal of Proteome Research • Vol. 7, No. 3, 2008 1087

Enrichment of Phosphopeptides by Fe3+-Immobilized Magnetic

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