Profiling of Endogenous Serum Phosphorylated Peptides by Titanium

Anal. Chem. 2009, 81, 94–104

Profiling of Endogenous Serum Phosphorylated Peptides by Titanium (IV) Immobilized Mesoporous Silica Particles Enrichment and MALDI-TOFMS Detection Lianghai Hu,† Houjiang Zhou,† Yinghua Li,‡ Shutao Sun,† Lihai Guo,⊥ Mingliang Ye,† Xiaofeng Tian,‡ Jianren Gu,§ Shengli Yang,†,| and Hanfa Zou*,† Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, 457 Zhongshan Road, Dalian 116023, China, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai 200032, China, Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, Shanghai 200032, China, Asia Pacific Application Support Center, Applied Biosystems, Shanghai 200040, China Phosphorylation is one of the most important posttranslational modifications of proteins, which modulates a wide range of biological functions and activities of proteins. The phosphorylation of proteins is also associated with the pathway of cancer cells. We have previously enriched the low molecular weight proteome from human plasma based on the combination of size exclusion and adsorption mechanism by using highly ordered mesoporous silica particles. Herein, highly ordered mesoporous silica particles were modified with titanium phosphonate to selectively capture the phosphopeptides from complex peptide and protein mixtures. The limit of detection for phosphopeptides from β-casein and standard phosphopeptide spiked in human serum was as low as 1.25 fmol based on MALDI-TOFMS detection. The modified mesoporous silica particles were further used to enrich phosphopeptides from serum of hepatocellular carcinoma patients and healthy individuals and then analyzed with MALDI-TOFMS. The combination of isobaric tagging for relative and absolute quantitation labeling with MALDITOFMS/MS was further applied to validate the serum phosphopeptide profiling result of MALDI-TOFMS. The profiling of the serum phosphopeptides between the cancer patients and healthy persons was distinguishingly different, which indicated the potential ability of this technique for cancer diagnosis and biomarker discovery. The approach developed here would be applicable to other biological samples and a wide variety of diseases. Protein phosphorylation plays important roles in the regulation of cellular functions, such as growth, metabolism and differentia* To whom correspondence should be adressed. Phone: +86-411-84379610. Fax: +86-411-84379620. E-mail: [email protected]. † Dalian Institute of Chemical Physics. ‡ The Second Affiliated Hospital of Dalian Medical University. ⊥ Applied Biosystems. § Shanghai Jiao Tong University. | Shanghai Research Center of Biotechnology.

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tion, and the phosphorylation of proteins is associated with the pathways of cancer cell.1 Studies in protein phosphorylation may hold particular promise for elucidation of signaling pathways, molecular classification of diseases, and profiling of novel kinase inhibitors.2 The peptidome, which is the name given to a low molecular weight proteome,3 has attracted increasing attention in recent years for the discovery of biomarkers due to the simplicity of sample preparation (without digestion).4 Compared with cells and tissues, due to its easy accessibility and recording of the current physiological states of the body, serum has become one of the best resources for biomarker discovery, with thousands of different types of peptides either degraded from larger proteins or secreted from cells and tissues.4 The degraded fragments of proteins are generated by the proteolytic enzymes in the body, which can be considered as the metabolic products of proteins.5,6 Circulating protein fragments generated in the body fluid or tissues may reflect biological events and provide a rich bank for diagnostic biomarkers.7 Cancer diagnosis has been successfully achieved by profiling of the peptides in serum. Petricoin et al.8 first reported the diagnosis of ovarian cancer by using SELDITOFMS to profile the serum peptidome in 2002. Recently, Villanueva et al.9 successfully used MALDI-TOFMS-based peptides profiling to distinguish prostate, bladder, and breast cancer patients from healthy persons. However, the endogenous phosphopeptides, which exist in the native state, are less reported up (1) (2) (3) (4) (5) (6) (7) (8)

(9)

Hunter, T. Cell 2000, 100, 113–127. Pawson, T.; Scott, J. D. Trends Biochem. Sci. 2005, 30, 286–290. Schrader, M.; Schulz-Knappe, P. Trends Biotechnol. 2001, 19, S55–S60. Petricoin, E. F.; Belluco, C.; Araujo, R. P.; Liotta, L. A. Nat. Rev. Cancer 2006, 6, 961–967. Hu, L. H.; Li, X.; Jiang, X. N.; Jiang, X. G.; Zhou, H. J.; Kong, L.; Ye, M. L.; Zou, H. F. J. Proteome Res. 2007, 6, 801–808. Soloviev, M.; Finch, P. Proteomics 2006, 6, 744–747. Diamandis, E. P. J. Proteome Res. 2006, 5, 2079–2082. Petricoin, E. F.; Ardekani, A. M.; Hitt, B. A.; Levine, P. J.; Fusaro, V. A.; Steinberg, S. M.; Mills, G. B.; Simone, C.; Fishman, D. A.; Kohn, E. C.; Liotta, L. A. Lancet 2002, 359, 572–577. Villanueva, J.; Shaffer, D. R.; Philip, J.; Chaparro, C. A.; Erdjument-Bromage, H.; Olshen, A. B.; Fleisher, M.; Lilja, H.; Brogi, E.; Boyd, J.; SanchezCarbayo, M.; Holland, E. C.; Cordon-Cardo, C.; Scher, H. I.; Tempst, P. J. Clin. Invest. 2006, 116, 271–284. 10.1021/ac801974f CCC: $40.75  2009 American Chemical Society Published on Web 12/02/2008

Figure 1. (a) Scheme for preparation of the Ti(IV)-immobilized mesoporous silica particles and (b) flowchart for the enrichment of the phosphopeptides followed by MALDI-TOFMS detection.

to now.10,11 One of the major reasons is the huge complexity and extremely high dynamic range of human serum, which makes the analysis of human serum peptidome a very challenging task. Immobilized metal ion (such as Fe3+, Ga3+, Ti4+, and Zr4+) affinity chromatography (IMAC),12-18 is a commonly used method for the enrichment of phosphorylated peptides from a tryptic digest of a protein sample for phosphoproteome analysis. Recently, metal oxide particles of titanium,19-22 zirconium,23-25 and aluminum26-28 have also been used to selectively enrich phosphopeptides and phosphoproteins from complex mixtures. Among these techniques, Ti4+-IMAC has been demonstrated to have the highest performance for isolation of phosphopeptides.18 In our previous report, highly ordered mesoporous silica particles were successfully applied for enrichment of the peptidome from human plasma with their highly ordered pores and nearly 95% in-pore surface area to ensure the highly (10) Li, Y.; Leng, T. H.; Lin, H. Q.; Deng, C. H.; Xu, X. Q.; Yao, N.; Yang, P. Y.; Zhang, X. M. J. Proteome Res. 2007, 6, 4498–4510. (11) Li, Y.; Qi, D. W.; Deng, C. H.; Yang, P. Y.; Zhang, X. M. J. Proteome Res. 2008, 7, 1767–1777. (12) Feng, S.; Pan, C. S.; Jiang, X. G.; Xu, S. Y.; Zhou, H. J.; Ye, M. L.; Zou, H. F. Proteomics 2007, 7, 351–360. (13) Pan, C. S.; Ye, M. L.; Liu, Y. G.; Feng, S.; Jiang, X. G.; Han, G. H.; Zhu, J. J.; Zou, H. F. J. Proteome Res. 2006, 5, 3114–3124. (14) Posewitz, M. C.; Tempst, P. Anal. Chem. 1999, 71, 2883–2892. (15) Zhou, H. J.; Xu, S. Y.; Ye, M. L.; Feng, S.; Pan, C.; Jiang, X. G.; Li, X.; Han, G. H.; Fu, Y.; Zou, H. F. J. Proteome Res. 2006, 5, 2431–2437. (16) Feng, S.; Ye, M. L.; Zhou, H. J.; Jiang, X. G.; Jiang, X. N.; Zou, H. F.; Gong, B. L. Mol. Cell. Proteomics 2007, 6, 1656–1665. (17) Blacken, G. R.; Volny, M.; Vaisar, T.; Sadilek, M.; Turecek, F. Anal. Chem. 2007, 79, 5449–5456. (18) Zhou, H. J.; Ye, M. L.; Dong, J.; Wu, R. A.; Zou, H. F. J. Proteome Res. 2008, 7, 3957–3967. (19) Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Mol. Cell. Proteomics 2005, 4, 873–886. (20) Chen, C. T.; Chen, Y. C. Anal. Chem. 2005, 77, 5912–5919. (21) Cantin, G. T.; Shock, T. R.; Park, S. K.; Madhani, H. D.; Yates, J. R. Anal. Chem. 2007, 79, 4666–4673. (22) Lin, H. Y.; Chen, C. T.; Chen, Y. C. Anal. Chem. 2006, 78, 6873–6878. (23) Kweon, H. K.; Hakansson, K. Anal. Chem. 2006, 78, 1743–1749. (24) Zhou, H. J.; Tian, R. J.; Ye, M. L.; Xu, S. Y.; Feng, S.; Pan, C. S.; Jiang, X. G.; Li, X.; Zou, H. F. Electrophoresis 2007, 28, 2201–2215. (25) Lo, C. Y.; Chen, W. Y.; Chen, C. T.; Chen, Y. C. J. Proteome Res. 2007, 6, 887–893. (26) Wolschin, F.; Wienkoop, S.; Weckwerth, W. Proteomics 2005, 5, 4389– 4397. (27) Chen, C. T.; Chen, Y. C. J. Mass Spectrom. 2008, 43, 538–541. (28) Chen, C. T.; Chen, W. Y.; Tsai, P. J.; Chien, K. Y.; Yu, J. S.; Chen, Y. C. J. Proteome Res. 2007, 6, 316–325.

selective enrichment based on a combination of adsorption and a size-exclusion mechanism.29 Therefore, we speculated that the mesoporous particles with immobilized metal ion should be able to specifically enrich endogenous phosphorylated peptides from highly complex protein samples because of the integration of affinity chromatography and a size exclusion mechanism. In this work, the ordered mesoporous silica MCM-41 was synthesized and functionalized with titanium phosphonate,18 which made the modified particles possessed of both a size-exclusion ability and a high adsorption specificity toward phosphopeptides. The titanium ion-immobilized mesoporous silica particles were further used with MALDI-TOFMS detection to enrich the endogenous phosphopeptides from human serum that were associated with hepatocellular carcinoma patients and healthy individuals. The phosphorylated fragments degraded from fibrinogen protein were observed with different expression profiles between the two groups. The investigation of the expression level of phosphorylated peptides may help us to understand the pathogenesis process of tumor growth. We believe that this technique could be further extended to a variety of diseases for biomarker discovery. EXPERIMENTAL SECTION Reagents and Materials. Proteins of β-casein, alkaline phosphatase, and (TPCK)-treated trypsin were purchased from Sigma (St. Louis, MO). Model singly tyrosine phosphorylated peptide (RRLIEDAEpYAARG, MW 1599 Da) was obtained from Upstate Co. (New York). Chemical reagents of 2,5-dihydroxybenzoic acid (2,5-DHB), formic acid (FA), trifluoroacetic acid (TFA) and (3aminopropyl) trimethoxysilane were obtained from Sigma (St. Louis, MO). Acetonitrile (ACN) was chromatographic grade from Merck (Darmstadt, Germany). Ti(SO4)2 was obtained from Sinopharm (Shanghai, China). Deionized water used for all experiments was purified with a Milli-Q water system (Millipore, Milford, MA). All other chemicals, including hydrochloric acid (HCl), ethanol, pyridine, toluene, and phosphorus oxychloride (POCl3), were of analytical grade. Preparation of Titanium (IV)-Immobilized Mesoporous Silica Particles. The procedures for preparation of titanium (IV)immobilized mesoporous silica particles are shown in Figure 1a. MCM-41 silica particles were prepared as reported previously.29,30 A 1.0 g portion of MCM-41 was added to 20 mL of 6 M HCl Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Figure 2. MALDI-TOFMS spectra for the (a) β-casein before enrichment, (b) β-casein with enrichment by Ti(IV)-IMAC, (c) human serum with enrichment by MCM-41 without modification, and (d) human serum with enrichment by Ti(IV)-modified MCM-41 (insert is the zoomed sight between 1300 and 1700 Da).

solution, and the mixture was stirred gently at room temperature for 5 h. The material was then washed with water until pH 7. Then the material was washed with ethanol and subsequently dried at 96


120 °C in vacuum for chemical modification. Aminopropylfunctionalized MCM-41 was prepared as follows: 0.5 g of activated MCM-41 was added to 25 mL of dry toluene and 1.5 mL of (3-

Table 1. Phosphopeptides Detected from β-Casein and Phosphorylated Fibrinogen Fragments from Human Serum with Their Location in Protein Fibrinogen r/r-E Chain Precursor (IPI00021885.1)a no.

mol wt phosphorylation (Da) sites

β1 β2 β3

2061.83 2556.09 3122.27

1 1 4

F1 F2 F3 F4

1389.31 1460.39 1545.50 1616.57

1 1 1 1

amino acid sequence β-Casein FQ[pS]EEQQQTEDELQK FQ[pS]EEQQQTEDELQDKIHPF RELEELNVPGEIVE[PS]L[pS][PS][pS] EESITR

Fibrinogen D[pS]GEGDFLAEGGGV AD[pS]GEGDFLAEGGGV D[pS]GEGDFLAEGGGVR AD[pS]GEGDFLAEGGGVR

a MFSMRIVCLVLSVVGTAWTAD[pS]GEGDFLAEGGGVRGPRVVERHQSACKDSDWPFCSDEDWNYKCPSGCRMKG LIDEVNQDFTNRINKLKNSLFEYQKNNKDSHSLTTNIMEI LRGDFSSA... (from website of www.phosphosite.org).

aminopropyl)trimethoxysilane under argon gas protection. The mixture was stirred at room temperature for 5 h and then heated to 110 °C by refluxing for 16 h. After that, the material was recovered, washed with toluene and ethanol, and dried at 110 °C in vacuum. Then 0.2 g of aminopropyl-functionalized MCM-41 was dispersed in 15 mL dry toluene with addition of 0.5 mL dry pyridine. After that, 0.5 mL POCl3 was added, and the mixture was incubated for 18 h at ambient temperature to prepare phosphonate-modified particles. After rinsing with toluene and water, the particles were dried at 60 °C in vacuum. Finally, 10 mg of phosphonate-modified particles was added to 10 mL of 100 mM Ti(SO4)2 solution at room temperature overnight under gentle stirring. The obtained Ti (IV) immobilized mesoporous silica particles were centrifuged at 20000g for 2 min. After the supernatant was removed, distilled water was used to wash the Ti(IV) immobilized mesoporous silica particles several times to remove the residue of titanium ion. The obtained Ti(IV)immobilized mesoporous silica particles were dispersed in 30% ACN containing 0.1% TFA solution before usage. Enrichment of the Phosphopeptides Using Ti(IV) Immobilized Mesoporous Silica Particles. β-Casein (1 mg) was dissolved in 1 mL of ammonium bicarbonate (50 mM, pH 8.2) and digested for 16 h at 37 °C with trypsin at an enzyme-to-protein ratio of 1:40 (w/w). The obtained peptide solution was lyophilized by a vacuum concentrator and redissolved in 0.1% TFA at a concentration of 1 pmol/µL. The procedures for the isolation and detection of phosphopeptides are shown in Figure 1b. For enrichment of phosphopeptides from the β-casein digest, 1 µL of sample solution was mixed with 5 µL of Ti(IV)-immobilized mesoporous silica particles (10 mg/mL) in 80% ACN and 6% TFA with vibration for 30 min. The supernatant was removed after centrifugation at 25000g for 5 min. The particles with the captured phosphopeptides were then washed with 30 µL of 50% ACN/6% TFA containing 200 mM NaCl, and 30 µL of 30% ACN/0.1% TFA in turn. Finally, the bound peptides were eluted with 10 µL of 10% NH3 · H2O under sonication for 10 min. After centrifugation (29) Tian, R. J.; Zhang, H.; Ye, M. L.; Jiang, X. G.; Hu, L. H.; Li, X.; Bao, X. H.; Zou, H. F. Angew. Chem., Int. Ed. 2007, 46, 962–965. (30) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S. Nature 1992, 359, 710–712.

at 25000g for 5 min, the supernatant was collected and lyophilized to dryness. We lyophilized the sample because the sample is eluted with NH3 · H2O, whereas the samples need to be analyzed with other techniques, such as LC-MS and isotope labeling under different conditions. If only MALDI analysis is performed, we can directly analyze the sample on-bead and do not need to elute the sample from the particle. Five microliters of DHB solution (25 mg/mL in 70% ACN) containing 1% H3PO4 (v/v) was added to redissolve the captured phosphopeptides because it was reported that the addition of H3PO4 into the matrix solution will improve the detection of phosphopeptides,31 and 0.5 µL of the mixture was directly deposited on the target for MALDI-TOFMS analysis. Human serum samples were collected from 12 healthy adults and 12 patients with hepatocellular carcinoma (HCC). The HCC samples were from mid stage (tumor size between 5 and 10 cm) to late stage (tumor size larger than 10 cm). All the samples (patient and healthy individuals) were collected in The Second Affiliated Hospital of Dalian Medical University according to their standard clinical procedures. Briefly, blood samples from volunteer subjects with no known malignancies and from HCC patients were collected in PET vacuum blood collection tubes (ST750CG, Beijing Sekisui Trank Medical Technology Co., China) and centrifuged for 5 min at 2000g. After collection, the serum samples were stored at -80 °C until further use. The flowchart for the extraction of the phosphopeptides from the serum sample is shown in Figure 1b. Ten microliters of the serum sample was first diluted 10 times with 50% ACN, 0.1% TFA before enrichment, then the diluted serum sample was first incubated with 10 µL of Ti(IV)-immobilized mesoporous silica particles (10 mg/mL) in 30% ACN/0.1% TFA with vibration for 30 min. The supernatant was removed after centrifugation at 25000g for 5 min. The particles with captured phosphopeptides were then washed with 50 µL of 50% ACN/0.1% TFA containing 200 mM NaCl and 50 µL of 30% ACN/0.1% TFA in turn. The bound phosphopeptides were then eluted with 30 µL of 10% NH3 · H2O under sonication for 10 min. After centrifugation at 25000g for 5 min, the supernatant was collected and lyophilized to dryness. To test the detection limit of this method, a stock sample solution was prepared by spiking 1 pmol of pY into 1 µL of serum sample. The mixture was then diluted to 100 µL with 30% ACN/0.1% TFA. Appropriate volumes of the stock solution containing different amounts (50, 20, 10, 5 fmol) of pY were used for analysis. Two microliters of DHB solution (25 mg/mL in 70% ACN) containing 1% H3PO4 (v/v) was added to redissolve the captured phosphopeptides, and 0.5 µL of the mixture was directly deposited on the target for MALDI-TOFMS analysis. The same procedures were adopted for β-casein digest. For isobaric tagging for relative and absolute quantitation (iTRAQ) labeling of the serum sample, 2 µL of the pooled serum samples from a mixture of five HCC patients and five healthy individuals, respectively, were added to 18 µL of dissolution buffer (iTRAQ Reagent Kit, Applied Biosystems). iTRAQ reagents of 114 and 117 dissolved in 40 µL ethanol were then added to the serum samples of HCC patients and healthy individuals, respectively. After 1 h incubation at room temperature, 10 µL of 5% TFA was added to quench potentially unreacted iTRAQ reagents. Finally, (31) Kjellstrom, S.; Jensen, O. N. Anal. Chem. 2004, 76, 5109–5117.


97

Figure 3. MALDI-TOFMS spectra for the phosphopeptides enriched with (a) β-casein digest and (b) pY spiked into serum at amounts of 50, 20, 10, and 5 fmol.

the samples were mixed and enriched with Ti(IV)-IMAC as the procedures described above for MALDI-TOF/TOFMS analysis. Mass Spectrometric Analysis. MALDI-TOF mass spectrometry analysis was performed on a Bruker Autoflex time-of-flight mass spectrometer (Bruker, Bremen, Germany). The instrument was equipped with a delayed ion-extraction device and a pulsed nitrogen laser operated at 337 nm; its available accelerating potential is in the range of ± 20 kV. The MALDI uses a ground98


steel sample target with 384 spots. The range of laser energy was adjusted to slightly above the threshold to obtain good resolution and signal-to-noise ratio (S/N). All mass spectra reported were obtained in the positive ion linear mode with delayed extraction for 90 ns. External mass calibration was obtained by using two points that bracketed the mass range of interest, and each mass spectrum was typically summed with 30 laser shots. MALDI-TOF/ TOFMS analysis for iTRAQ labeling samples was carried out on 4800 Plus MALDI TOF/TOF analyzer (Applied Biosystems, Foster

Figure 4. MALDI-TOFMS spectra of the enriched serum phosphopeptides (a) before and (b) after being treated with alkaline phosphatase.

City, CA) at Applied Biosystems Inc. (Asia Pacific Application Support Center, Shanghai, China). Partial least-squares discriminant analysis (PLS-DA) was used as the classification method for modeling the discrimination between the hepatocellular carcinoma patients and healthy controls on the basis of serum phosphopeptides. Multivariate analysis was performed using the SIMCA-P software (demo version 11, Umetrics AB, Umeå, Sweden).32 PLS-DA is a multivariate classification method based on PLS, the regression extension of PCA. Whereas PCA works to explain the maximum variation between samples, PLS-DA explains the maximum separation between defined class samples in the data (X) using a Y matrix that represents an orthogonal unit vector for each class. PLS-DA is done by a PLS regression against a “dummy matrix” (Y), which describes variation according to class. Variation is interpreted in terms of X scores (T) and X weights (W, C). Once a PLS-DA model is calculated and validated, it can be used for prediction of class membership for unknown samples. RESULTS AND DISCUSSION EDX (energy dispersive X-Ray) and ICP-AES (inductively coupled plasma-atomic emission spectrometry) experiments were (32) Eriksson, L.; Johansson, E.; Kettaneh-Wold, H.; Wold, S. Introduction to Multi-and Megavariate Data Analysis Using Projection Methods (PCA & PLS); Umetrics AB: Box 7960, SE 90719 Umeå, Sweden, 1999.

carried out to characterize the loading amount of Ti(IV) on mesoporous silica particles, which reveal that the Ti(IV) was 35 mg/g and 78.5 mg/g (corresponding to 0.73 and 1.64 mmol/g), respectively. The difference is probably caused by EDX being a semiquantitative analysis method and can only characterize part of the surface element compositions, as shown in Figure S1 of the Supporting Information. β-Casein, a standard protein with known phosphorylated sites, was used for enrichment experiments with the Ti(IV)-immobilized mesoporous silica particles to characterize the specificity of the materials. The MALDI-TOFMS spectra of the β-casein digest before and after enrichment by Ti(IV)-immobilized mesoporous silica particles made from ordered mesoporous silica MCM-41 are shown in Figure 2a and b. After enrichment by Ti(IV)-immobilized mesoporous silica particles, it can be clearly seen that the three dominant peaks of the phosphopeptides (β1, β2, β3 with m/z of 2061.577, 2555.286, and 3120.850 Da, respectively) were successfully observed with a clean background, which indicated the abundant non-phosphopeptides were not adsorbed by this material. The sequence information of the detected phosphopeptides is listed in Table 1. To have a comparison, we also directly analyzed the β-casein digest without enrichment. As shown in Figure 2a, the three phosphopeptides in β-casein digest can also be detected by MALDI-TOFMS; however, there were many highly abundant nonphosphopeptide peaks present on the spectra. To further examine Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

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Figure 5. (a) 3D view of the MALDI-TOFMS profiling of phosphopeptides enriched from serum of HCC cancer and healthy persons. (b) PLSDA score plot showing the separation between the HCC cancer and healthy groups.

the specificity of the Ti(IV)-MCM-41, we analyzed the serum sample enriched with the native MCM-41 and Ti(IV)-modified MCM-41 particles, respectively. As shown in Figure 2d, only four peaks at m/z of 1389.609, 1460.605, 1545.698, and 1616.889 Da (proved to be phosphorylated peptides) can be observed after enrichment with Ti(IV)-MCM-41. One important reason that Ti(IV)-MCM-41 could specifically enrich phosphopeptides from serum with a huge amount of HMW proteins is that these proteins are not accessible to the in-pore surface of the materials. The MCM-41 without modification was also used to enrich the peptides from human serum, as in our previous report.29 Due to the high complexity of human serum, there were a number of peaks for enrichment with MCM-41 without modification, as shown in Figure 2c. However, none of the four phosphopeptides can be found when enrichment with native MCM-41 for it has no specific affinity to phosphopeptides. The sensitivity of phosphopeptides enriched using the Ti(IV)-MCM-41 was investigated for both the tryptic digest of 100


β-casein and standard phosphopeptide pY spiked into human serum. Figure 3a shows the MALDI-TOFMS spectra of the enriched phosphopeptides from β-casein digest with amounts of 50, 20, 10, and 5 fmol. It can be seen that when the amount of β-casein digest is 5 fmol (1.25 fmol was deposited on each spot of the MALDI target), the ion signals from the phosphopeptides can still be detected, which indicates the high detection sensitivity of this approach. To further study whether the same detection limit for phosphopeptide can be obtained with the presence of highly abundant background proteins, a stock sample solution was prepared by spiking 1 pmol standard phosphopeptide pY (RRLIEDAE[pY]AARG) into 1 µL of serum sample and then diluting the sample to 100 µL with 30% ACN/0.1% TFA. Different amounts of the stock solution containing pY (50, 20, 10, and 5 fmol) were used for further enrichment and MALDI-TOFMS detection. As shown in Figure 3b, the pY can also be detected with the amount of 5 fmol, which means that the highly efficient and selective enrichment ability for phosphopeptides can be

Figure 6. (a) MALDI-TOFMS spectrum of the phosphopeptides enriched from pooled human serum after being labeled with iTRAQ reagents; MS/MS spectra for the precursor ions of (b) D[pS]GEGDFLAEGGGV, 1533.49 Da; (c) AD[pS]GEGDFLAEGGGV, 1604.52 Da; (d) D[pS]GEGDFLAEGGGVR, 1689.57 Da; and (e) AD[pS]GEGDFLAEGGGVR, 1760.60 Da. Low-mass region (insert) showed the signature ions of m/z 114 (HCC patients) and 117 (healthy individuals) used for quantitation. Analytical Chemistry, Vol. 81, No. 1, January 1, 2009

101

Table 2. Relative Abundance Ratio of the Identified Phosphopeptides from Human Serum (Healthy Persons/Cancer Patients) Based on MALDI Profling and iTRAQ Labeling Techniques peaks 1389.31 Da

1460.39 Da

1545.50 Da

1616.57 Da

methods

healthy (n ) 12)

HCC (n ) 12)

healthy (n ) 12)

HCC (n ) 12)

healthy (n ) 12)

HCC (n ) 12)

healthy (n ) 12)

HCC (n ) 12)

MALDI profiling (av/RSD) MALDI profiling (ratio) iTRAQ labelinga (ratio)

3.75/38% 0.06 0.17

60.44/39%

16.97/21% 0.55 1.29

30.95/61%

36.85/29% 0.38 0.69

97.62/8%

99.63/0.3% 4.97 6.62

20.06/71%

a

The samples for iTRAQ labeling were pooled serum samples from five healthy persons and five cancer patients, respectively.

achieved even from complex serum samples when using the Ti(IV)-immobilized mesoporous silica particles. We also titrated different amounts of β-casein digest into 1 µL of human serum, which showed that the detection limit of phosphopeptides digested from β-casein is about 10 fmol for β1 and 20 fmol for β2 and β3 even with the ion suppression of the high abundant phosphopeptides as shown in Figure S2 of the Supporting Information. Therefore, this material can effectively enrich phosphopeptides with high dynamic range of concentrations in complex samples. To validate whether the four peptides enriched from serum by Ti(IV)-immobilized mesoporous silica particles were phosphorylated, we treated the sample mixture with alkaline phosphatase, which can cleave the phosphonic group from phosphopeptides. The MALDI-TOFMS spectra of the phosphopeptides from serum before and after treatment with alkaline phosphatase are shown in Figure 4. We can see clearly that the peaks of the four peptides (m/z at 1388.836, 1459.811, 1544.714, and 1615.696 Da) in Figure 4a almost disappeared, and four new peaks (m/z at 1308.755, 1379.721, 1464.671, and 1535.738 Da) that lost a group of 80 Da (HPO3 ) 80 Da) were detected in Figure 4b, which indicated that all of the four peaks were singly phosphorylated peptides. To identify the sequence of the detected phosphopeptides enriched from human serum, we submitted the sample mixture to LC-MS for further analysis. The acquired MS2 and MS3 spectra were searched by the Sequest program, and the four phosphopeptides were identified from the same protein of fibrinogen, whose sequence information and molecular weight are listed in Table 1. The MS2 and MS3 spectra can be seen in Figures S3-S6 of the Supporting Information. We further searched the PhosphoSite Web site (www.phosphosite.org) to validate the reliability of phosphopeptide identification. The location of the phosphorylation sites identified according to the PhosphoSite is shown in Table 1. Hepatocellular carcinoma is a malignancy that accounts for over a million deaths per year in both underdeveloped and developing countries.33 Human serum is the most commonly used sample for clinical diagnosis due to its easy collection and stability. There have been numbers of reports on the use of proteomic profiling for the clinical diagnosis and searching of biomarkers, which rely on the use of SELDI or MALDI techniques.4,8,9 However, there are few reports to study the expression difference by directly enriching the phosphopeptides (33) Sun, W.; Xing, B. C.; Sun, Y.; Du, X. J.; Lu, M.; Hao, C. Y.; Lu, Z. A.; Mi, W.; Wu, S. F.; Wei, H. D.; Gao, X.; Zhu, Y. P.; Jiang, Y.; Qian, X. H.; He, F. C. Mol. Cell. Proteomics 2007, 6, 1798–1808.

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from tissues or body fluids.10,11 Here, we enriched the phosphopeptides from human serum associated with HCC patients and healthy individuals. Twenty-four serum samples (12 from HCC patients and 12 from healthy adults) were used for further analysis. Both the patients’ and the healthy adults’ serum was sampled at the Second Affiliated Hospital of Dalian Medical University using the standard clinical protocols. The serum was then extracted and analyzed following the procedures as shown in Figure 1b. The 3D profiling of the phosphopeptides from cancer and control serums is shown in Figure 5a. We can clearly see that the two groups could be distinguished using the phosphopeptides profiling. When using PLS-DA to investigate the difference between the HCC patients and healthy individuals, the two groups were clearly separated as shown in Figure 5b. One phenomenon to be mentioned is that the healthy individuals clustered together, but the HCC ones spread around on the PLS-DA score plot. The reason may be that the healthy individuals are normal, with little difference, but the cancer patients differentiated from one with another for their different states of illness and treatments. Although MALDI/SELDI profiling of proteins/peptides can be used to identify the expression difference of the clinical samples with high throughput, the ion patterns may be affected by ionization efficiency, ion suppression effects, sweet spots, and even the sample preparation process. MALDI/SELDI profiling can be thought of as a semiquantitative method. For quantitative analysis with good accuracy, isotope labeling is most widely used. iTRAQ is an approach based on aminereactive isobaric tagging and MS/MS for quantitation, which has been used for both protein- and peptide-level quantitative analysis. The iTRAQ reagent consists of a reporter group, a balance group, and an amino-reactive group. Peptides of the same sequence labeled with iTRAQ reagents are identical in mass in single MS mode but produce strong, diagnostic, lowmass MS/MS signature ions. Thus, a multiplex set of identical but differentially labeled peptides will appear as a single ion signal in MS. Following MS/MS analysis of the precursor ion, the reporter groups appear as distinct ions, and quantification is based on the relative intensity ratio of reporter ions in the MS/MS spectrum. Here, we used the iTRAQ technique (reagents with reporter groups of 114 and 117 Da) to validate the reliability of the MALDI-TOFMS profiling results. The pooled serum samples mixed from five HCC patients and five healthy individuals were labeled with iTRAQ reagents with reporter groups of 114 and 117 Da, respectively. The two samples were then combined and enriched using the titanium

(IV)-immobilized mesoporous silica particles and submitted to MALDI-TOF/TOF for quantitative analysis. The MS and MS/ MS of the enriched phosphopeptides are shown in Figure 6. We can see from Figure 6a that the peptides were successfully labeled with iTRAQ reagents (the MW of peptides added with 144 Da of the labeling reagents). For quantitative analysis of the labeled peptides, we can see from Figure 6 that for HCC patients, the peptide D[pS]GEGDFLAEGGGV was evidently upregulated, and the peptide of AD[pS]GEGDFLAEGGGVR was down-regulated greatly. The other two peptides of AD[pS]GEGDFLAEGGGV and D[pS]GEGDFLAEGGGVR changed only a little among the two groups. From the CID fragments of the peptides, it can be seen clearly that each of the dominant peaks in MS/MS lose a H3PO4 group of 98 Da for the precursor ions, which indicated that the peptides were phosphorylated. From the MS/MS spectra, we can also see the b- and y-ion series that verified the identification of the phosphopeptides. We further compared the quantitative analysis results of the iTRAQ and MALDI profiling. The relative abundance ratio of phosphopeptides by iTRAQ and MALDI profiling is shown in Table 2. We can see that although there was some difference between two techniques, the trends of most phosphopeptides were consistent with each other. Triplicate analysis of serum sample and also pooled samples for iTRAQ experiments was also performed. Triplicate analysis of the same sample by parallel extraction and MALDI-TOFMS analysis has a RSD of less than 5% (0.15%, 2.49%,2.26%, and 4.54% for the four peaks). The iTRAQ data for triplicate labeling and MALDI-TOFMS/ MS measurements had a little higher RSD values due to the chemical labeling reaction but also less than 20% (19.946%, 17.89%, 10.75%, and 14.14% for the four peaks). Therefore, MALDI profiling is a fast, high-throughput screening approach with semiquantitative ability. However, the results need to be further validated with other accurate quantitative methods or classical biological approaches. Human fibrinogen is a phosphoprotein with a subunit composition of R, β, and γ chains, among which only the R-chain is phosphorylated in vivo to about 25% at two serine residues.34 It has been suggested that fibrinogen is synthesized in a highly phosphorylated form and that the phosphate groups are subsequently removed by a phosphatase.35 The degree of phosphorylation is increased up to 70% in conjunction with an

acute phase reaction. It is of interest that also in the healthy fetus or newborn, the degree of phosphorylation is considerably increased, which is the same case with the alphafetoprotein, a famous tumor marker for HCC. There have been reports on the change of the fibrinogen and its fragments in human serum associated with cancer. Fibrinogen peptide A (FPA) is found to be up-regulated in gastric cance36 and other diseases.37,38 Phosphorylated FPA was also found to be up-regulated in the low molecular weight of serum from ovarian cancer patients.39,40 In other reports by Tempst et al.,9,41 by using reversed magnetic particle extraction and MALDI-TOFMS detection, they found decreased levels of FPA and its fragments in prostate, bladder, and breast cancer patients, which is different from other reports. However, they did not observe the phosphorylated FPA in either cancer patient or control samples, which may be due to the nonspecific extraction of reversed-phase retention of magnetic particles toward phosphopeptides. Although many reports have shown the existence of different polypeptides in body fluids, to date, no one has shown the mechanism underlying the presence of the diagnostic peptides in blood. In our experiments, the phosphorylated peptides in human serum were selectively enriched and identified by our developed approach. We found that the phosphopeptides from human serum expressed differently between the cancer and healthy groups. The phosphorylated FPA was found to be down-regulated, which was similar to Tempst’s reports.9,41 We also detected another three isoforms of phosphorylated FPA, which may be the degraded fragments from phosphorylated FPA by proteases, and this has not been reported previously. It has been well-known that FPA is the proteolytic product of fibrinogen by thrombin.38 Other proteases, such as neutrophil elastase, tryptase, matrix metalloproteinases, and cathepsin D/G may also degrade the fibrinogen into fragments.36 The expressed levels of fibrinogen and its fragments in cancer serum may therefore reflect the expression and activation of enzymes including kinase, phosphatase, and protease, which have been implicated in tumor biology by previous reports.42 We thought the expression of the phosphorylated fibrinogen fragments is dynamic with a balance of phosphatase, kinase, and disease-specific proteases. Investigation of the expression level of phosphorylated fibrinogen isoforms may help in the understanding of the pathogenesis process of tumors.

(34) Maurer, M. C.; Peng, J. L.; An, S. S.; Trosset, J. Y.; Henschen-Edman, A.; Scheraga, H. A. Biochemistry 1998, 37, 5888–5902. (35) Henschen-Edman, A. H. In Fibrinogen; New York Acad Sciences: New York, 2001; Vol. 936, pp 580-593. (36) Ebert, M. P. A.; Niemeyer, D.; Deininger, S. O.; Wex, T.; Knippig, C.; Hoffmann, J.; Sauer, J.; Albrecht, W.; Malfertheiner, P.; Rocken, C. J. Proteome Res. 2006, 5, 2152–2158. (37) Theodorescu, D.; Wittke, S.; Ross, M. M.; Walden, M.; Conaway, M.; Just, I.; Mischak, H.; Frierson, H. F. Lancet Oncol. 2006, 7, 230–240. (38) Seydewitz, H. H.; Matthias, F. R.; Schondorf, T. H.; Witt, I. Thromb. Res. 1987, 46, 437–445. (39) Bergen, H. R.; Vasmatzis, G.; Cliby, W. A.; Johnson, K. L.; Oberg, A. L.; Muddiman, D. C. Dis. Markers 2003, 19, 239–249. (40) Ogata, Y.; Hepplmann, C. J.; Charlesworth, M. C.; Madden, B. J.; Miller, M. N.; Kalli, K. R.; Cilby, W. A.; Bergen, H. R.; Saggese, D. A.; Muddiman, D. C. J. Proteome Res. 2006, 5, 3318–3325. (41) Villanueva, J.; Martorella, A. J.; Lawlor, K.; Philip, J.; Fleisher, M.; Robbins, R. J.; Tempst, P. Mol. Cell. Proteomics 2006, 116, 1840–1852. (42) Matrisian, L. M.; Sledge, G. W.; Mohla, S. Cancer Res. 2003, 63, 6105– 6109.

A method for profiling of the endogenous phosphorylated peptides from human serum has been developed and applied to the analysis of serum samples from HCC patients and healthy persons. The phosphopeptide expressions were found to be distinguishingly different between cancer and healthy individuals. The four identified phosphorylated peptides were further validated to be degraded fragments from the same protein of fibrinogen. The peptide expression differences may reflect the activities of enzymes between the blood of HCC patients and healthy persons. However, the mechanism needs to be further validated in the future. We thought this approach would be effective for the profiling of phosphopeptides in complex mixtures of tissues and body fluids, and may provide a new approach for biomarker discovery and elucidation of the pathogenesis process of tumors.

CONCLUSIONS


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ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Nos. 20675081, 20735004), the China State Key Basic Research Program Grant (2007CB914104), the China High Technology Research Program Grant (Nos. 2006AA02A309 and 2008ZX10002-017), the Knowledge Innovation program of CAS (KJCX2.YW.HO9), and the Knowledge Innovation program of DICP (to H. Zou).

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SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review July 13, 2008. Accepted November 11, 2008. AC801974F

Profiling of Endogenous Serum Phosphorylated Peptides by Titanium

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