Derivatized Cellulose Combined with MALDI-TOF MS: A New Tool for

Nov 18, 2005 - To further compress and denoise the data discrete wavelet transformation (DWT) was utilized. Finally, the experiment was repeated 200 t...
0 downloads 0 Views 214KB Size
Download PDF
Derivatized Cellulose Combined with MALDI-TOF MS: A New Tool for Serum Protein Profiling Isabel Feuerstein,† Matthias Rainer,† Katussevani Bernardo,‡ Gu1 nther Stecher,† Christian W. Huck,† Kurt Kofler,§ Alexandre Pelzer,§ Wolfgang Horninger,§ Helmut Klocker,§ Georg Bartsch,§ and Gu1 nther K. Bonn*,† Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University, Innrain 52a, A-6020 Innsbruck, Austria, Biocrates Life Sciences GmbH, Innrain 66, A-6020 Innsbruck, Austria, and Department of Urology, Medical University of Innsbruck, Anichstraβe 35, A-6020 Innsbruck, Austria Received July 21, 2005

Providing a rapid and sensitive protein profiling method for biomarker discovery from a variety of biological samples is crucial for the introduction of new markers that improve cancer patient diagnosis at early tumor stages, thus increasing the chances of curative treatment. We report here the development and application of derivatized cellulose particles for selective serum protein profiling. For immobilized metal ion affinity chromatography (IMAC), cellulose was derivatized with glycidyl methacrylate (GMA) and subsequently with iminodiacetic acid (IDA). To investigate the application of this material for generating protein profiles of human serum samples, the serum samples were agitated with the derivatized cellulose particles to a suspension and incubated for 2 h at 30 °C. After washing, 1 µL of the IDA-Cu2+-cellulose suspension was applied directly onto a MALDI-target, mixed with sinapinic acid (SA) and analyzed with MALDI-TOF MS. Consistent serum specific data were obtained from aliquoted samples analyzed several times, indicating the reliability of the method. However, the serum fingerprints obtained proved to be specific for any given serum. The technique presented allows a high enrichment of sample on the developed target leading to a high sensitivity and reproducibility without depletion of albumin and immunoglobulin, and sample elution prior to MS-analysis. The study demonstrates for the first time that derivatized cellulose particles combined with MALDI-TOF MS represent a simple, economical, and rapid approach to generate serum protein profiles for biomarker identification. Keywords: spherical cellulose • protein profiling • direct MALDI-TOF • prostate cancer

Introduction In many cases, cancer can be cured when detected at an early, organ-confined stage, and there are considerable efforts do develop new biomarkers that improve current diagnosis and prognosis methods for cancer diseases. The identification and analysis of proteins associated with disease is a major challenge. Although several biomarkers for tumor diseases have been identified and introduced successfully into clinical practice, like the prostate-specific antigen (PSA), the carcinoembryonic antigen (CEA), or the alpha-fetoprotein (AFP), their sensitivity and specificity is limited. A good example is prostate cancer, the most frequently diagnosed cancer and the second leading cause of cancer death in men in Western countries.1 The prostate marker PSA is quite sensitive, however, it does not correctly differentiate benign from malignant prostate disease, and can miss some significant prostate cancers.2-4 It is war* To whom correspondence should be addressed. Tel.: ++43/(0)512/5075171. Fax: ++43/(0)512/507-2943. E-mail: [email protected]. † Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University. ‡ Biocrates Life Sciences GmbH. § Department of Urology, Medical University of Innsbruck.

2320

Journal of Proteome Research 2005, 4, 2320-2326

Published on Web 11/18/2005

ranted to search for additional biomarkers in order to improve cancer specificity. Most likely, multiple biomarkers will be required to improve early detection, diagnosis, and prognosis. The classical technique for discovering disease-associated proteins is two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) followed by the detection and identification of multiple protein species by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF MS).5-9 This technique is unchallenged in its ability to resolve thousands of proteins, but it is laborious, requiring large quantities of protein, lacking critical reproducibility standards, and lacking the ability easily convert the results into a routinely used diagnostic test. Therefore, more timesaving and robust techniques are needed. One technique is the ProteinChip approach produced by Ciphergen Biosystems Inc. (Fremont, CA). This method uses surface enhanced laser desorption/ ionization (SELDI) TOF-MS to detect proteins affinity-bound to a protein chip array.10,11 There have been many examples of the use of SELDI for the determination of disease biomarkers, with the primary focus being diagnostics for all forms of cancer.12-14 Compared to conventional MS-applications, the SELDI-technology is much easier and timesaving regarding 10.1021/pr050227z CCC: $30.25

 2005 American Chemical Society

Derivatized Cellulose Combined with MALDI-TOF-MS

research articles

Figure 1. Derivatization of cellulose particles and sample preparation. Derivatization procedure for preparation of modified cellulose particles (A). Schematic illustration of the sample preparation (B). Sample loading (1), incubation (2), removal of unbound proteins (3), on-target sample preparation (4) and MALDI-TOF analysis (5).

sample preparation and analysis.15 Other well-established profiling techniques are based on different functionalized magnetic-particles and MALDI-TOF-MS16 and on the direct MALDI-TOF analysis of tissue sections.17 In addition to those MS-based proteomics approaches, we report here the development and optimization of material enhanced laser desorption/ionization (MELDI) by introducing derivatized cellulose beads for the selective serum-protein profiling with a high-resolution MALDI-TOF MS instrument. For derivatization, glycidyl methacrylate (GMA) was grafted onto 8 µm cellulose beads to bind iminodiacetic acid (IDA) through the epoxide of GMA in a second step. The functionalized beads were loaded with copper ions and mixed with serum samples. After binding of specific proteins (e.g., histidine, tryptophan, or cysteine containing proteins), unbound proteins were removed and 1 µL of this “protein-cellulose-suspension” was directly applied onto a MALDI-target, mixed with sinapinic acid (SA) and directly analyzed with MALDI-TOF MS.

Experimental Section Materials and Reagents. Iminodiacetic acid (IDA), acetonitrile (HPLC grade), sinapinic acid (SA), and glycidyl methacrylate (GMA) were purchased from Sigma Aldrich (St. Louis, MO). Spherical cellulose (8 µm particles), so-called Celluflow C-25 was from Collaborative Laboratories (New York, USA). Trifluoroacetic acid (TFA, for protein sequence analysis) was obtained from Fluka (Buchs, Switzerland). Serum samples of histological confined prostate cancer and age matched unaffected healthy men (50 years old, PSA e 0.5 ng/mL) were provided by the Department of Urology, at the Medical University of Innsbruck, Austria. All these serum samples had been procured from consenting patients according to standard

protocols, what means each sample has been taken off in the same work flow and directly stored at -80 °C. None of the samples has been thawed more than twice. Preparation of IDA-Cu2+-Cellulose. Cu2+ was loaded on derivatized cellulose particles. To derivatize cellulose, 10 g of Celluflow was dispersed with strong agitation in 230 mL of water for 2 min. A 328-mM portion of monomer glycidyl methacrylate was added and the reaction mixture took place for 2 h at 70 °C with 6.5 mM of ammonium persulfate and 8.6 mM of sodium thiosulfate as redox catalysts. The polymerization and grafting reaction was finalized by raising the temperature to 80 °C for a further 2 h. The suspension was then filtered and washed with a large volume of deionized water.18 The residual cellulose matrix was dispersed in 113 mM iminodiacetic acid (IDA) solution, prepared by dissolving IDA in 200 mL of a 2 M sodium carbonate solution followed by the addition of 86 mM of sodium chloride for 5 h at 75 °C with vigorous stirring. The final product was vacuum filtered, washed with deionized water and saturated with the designated Cu2+ ions by incubation of the derivatized cellulose with 50 mM metal solution for at least 2 h at room temperature. A 3-mg portion of the respective IDA-Cu2+-cellulose was filled in a 0.5 mL micro centrifuge tube, and activated with 50 mM of sodium acetate buffer for 5 min at room temperature and centrifuged at 13 000 rpm for 1 min. After centrifugation, the supernatant was discarded and the residue was equilibrated two times with 200 µL of PBS. Serum Sample Preparation. A 40-µL portion of serum was treated with 30 µL of 8 M urea containing 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in phosphate buffered saline (PBS) for a few minutes by shaking. Afterward, 100 µL of 1 M urea containing 0.125% CHAPS were Journal of Proteome Research • Vol. 4, No. 6, 2005 2321

research articles

Feuerstein et al.

Figure 2. Reproducibility of serum protein fingerprinting on IDA-Cu2+-cellulose combined with MALDI-TOF analysis. Human serum samples from healthy patients were prepared as described in Material and Methods, aliquoted and stored frozen at -20 °C. Samples were analyzed directly after preparation (A), one week later (B) and three weeks later (C). The triangles indicate the most prominent peaks found in all measurements of the same sample.

added and again briefly shaken. The mixture was diluted 1:5 in PBS and vortexed at 4 °C for 10 min. Sample Analysis Using IDA-Cu2+-Cellulose. Four-hundred microliters of the prepared serum sample was added to the equilibrated IDA-Cu2+-cellulose, and the whole suspensionmixture was incubated on a platform shaker at 1500 rpm for 2 h at 30 °C. To remove unbound material, IDA- Cu2+-cellulose was washed three times with 200 µL of equilibration buffer followed by a quick washing step with 200 µL of deionized water. One microliter of serum IDA-Cu2+-cellulose mixture was loaded onto a stainless steel target and 1 µL of saturated sinapinic acid in 50% acetonitrile and 0.1% trifluoroacetic acid was added. It is important to add the matrix solution before the sample is air-dried. MALDI-TOF MS Analysis. Proteins bound to the derivatized cellulose were analyzed by MALDI-TOF-MS (Ultraflex MALDITOF-TOF, Bruker Daltonics, Bremen, Germany). All serum samples were measured in linear mode and the detector energy was set to 1623 V. Data were collected by averaging 120 laser shots and analyzing mass region from 2000 to 10 000 Da. The validation of all data obtained, including baseline subtraction of the TOF data, external calibration using Protein Standard I 2322

Journal of Proteome Research • Vol. 4, No. 6, 2005

(Bruker Daltonics, Bremen, Germany) and all further data processing, was carried out by using Flex analysis 2.0 post analysis software and for data acquisition by Flex control 2.0.

Results Derivatization of Cellulose. In order to develop new selective and high capacity binding phases for serum profiling, we derivatized spherical cellulose particles, which can be normally used in chromatography.18,19 The characterization of derivatized cellulose beads as well as the derivatization procedure is given in Table 1 and Figure 1, panel A, respectively. To establish a reliable sample preparation method for routine serum profiling, different buffer systems20-22 and variable amounts of derivatized IDA-Cu2+-cellulose and R-cyano-4hydroxycinnamic acid (HCCA) or sinapinic acid (SA) were explored for material enhanced LDI MS (MELDI). Moreover, different modes for acquiring MALDI-TOF MS spectra (e.g., linear or reflector modes) were tested. The best serum sample preparation is summarized in the Experimental Section and illustrated in Figure 1, panel B. HCCA as a matrix was found to provide satisfying results in a mass range of 1-4 kDa in reflector mode (data not shown). The matrix sinapinic acid in

Derivatized Cellulose Combined with MALDI-TOF-MS

research articles

Figure 3. Biomarker profiling on IDA-Cu2+-cellulose combined with MALDI-TOF MS. Analysis of different serum samples for biomarker profiling was done as described in Materials and Methods. Control sample (A), cancer patient (B). The circles indicate potential biomarker peaks. Table 1. Characterization of Derivatized Cellulose Beads particle size (wet state) surface functionality metal capacity (Cu2+) UV/visa pore size a

8-10 µm IDA 360 µmol/g 0.34 µm

Average of three independent measurements.

linear acquisition mode was found to produce the best results in terms of signal-to-background ratios as well as reproducibility (Figure 2) and was used for further experiments. Reproducibility of Serum Protein Profiles Obtained with Derivatized Cellulose. To investigate the variability of the derivatized IDA-Cu2+-cellulose for monitoring serum biomarkers, the reproducibility of the discovered method was tested. The reproducibility was proved by repeated MALDI-TOF analysis of one given sera at different times. A serum sample was prepared and stored frozen in aliquots. Three representative spectra were then recorded in duplicates within a time frame of four weeks (Figure 2). MALDI-serum profiles of these

three representative spectra showed consistent peaks at distinct positions (of distinct masses) and clearly showed a stable spectrum (Figure 2, triangle marked peaks), when using this analytical technique. Similarly, stable spectra were obtained by repeating the experiment using sera provided from different control persons (data not shown). To demonstrate the capability of this method for detection and/or identification of new biomarkers, serum samples of a control person and a cancer patient were analyzed. As shown in Figure 3 the serum fingerprints of the respective sera differed significantly. The most prominent differences in the two spectra are labeled with circles in Figure 3. Although the differences of these two spectra are obvious, in most cases cancer and control samples are indistinguishable at first sight. Therefore, MALDI spectra were processed using different statistical tests to differentiate prostate cancer from non cancer, within a group of 137 cancer and 163 control serum samples. First, data were processed by a two-sample KolmogorovSmirnov goodness-of-fit test (briefly, KS-test) to reduce data in order to pick out useful features. In a second step, an Journal of Proteome Research • Vol. 4, No. 6, 2005 2323

research articles

Feuerstein et al.

Figure 4. Stability of serum protein fingerprints generated on IDA-Cu2+-cellulose combined with MALDI-TOF analysis. Stability and durability of human serum sample loaded on IDA-Cu2+-cellulose were determined by analyzing one serum sample at different conditions. Direct MALDI-TOF-MS analysis (A) and MALDI-TOF-MS analysis 4 h after sample processing (B).

empirical cumulative distribution of the coefficient of variation (CV), which suggests a threshold to reduce the effect of other diseases affecting some of the high-risk controls, was performed. To further compress and denoise the data discrete wavelet transformation (DWT) was utilized. Finally, the experiment was repeated 200 times independently, each time the classifier was trained on a 10-fold x-validation, which resulted in a sensitivity and specificity of more than 90%. Stability of Human Serum Sample. Reproducibility of serum profiling is a main problem starting from drawing the blood sample to the varying time periods until serum is centrifuged, stored, and frozen. This can result in autolysis and degradation fragments and has to be taken into consideration for further data processing. In this study, all blood samples were equally treated by a well-developed routine working process and stability tests were performed. The same cellulose-serum suspension was analyzed immediately and after about 4 h storage at room temperature by MALDI-TOF MS. Figure 4 impressively depicts the effects of time between sample preparation and analysis. Prepared serum samples should be immediately analyzed to guarantee the reproducibility. Additionally, sample preparation was performed at varying temperatures 4, 21, and 30 °C, respectively. As shown in Figure 5 temperature also is important for reliable sample preparation. Sample preparation performed at 4 °C and 21 °C, illustrated in Figure 5 panel A and B, yielded almost identical profiles but these were different form the profile of the sample prepared at 30 °C. In the latter case, the signal-to-noise ratio is enhanced, resulting in difficulty distinguishing noise from true peaks (Figure 5 panel C). 2324

Journal of Proteome Research • Vol. 4, No. 6, 2005

Discussion Analysis and identification of serum proteins is an important first step for the discovery of biomarkers that are useful in the development of more effective treatments and novel cancer therapies. New biomarkers are required for the improvement of diagnosis and prognosis of cancer patients as well as for decision making regarding the introduction of new candidate drugs in order to speed up this process and bring new medicines to the right patients faster than possible today. MALDI-TOF mass spectrometry in combination with IDA-Cu2+cellulose based protein adsorption material allows the resolution of a broad range of serum proteins/peptides, from 2 to 20 kDa based on the ability of chemically activated copper to selectively bind proteins through histidine, tryptophan or cysteine. Compared with the hundreds to thousands of proteins being separated by two-dimensional gel electrophoresis, the method described here has the advantage to effectively resolve peptides and proteins smaller than 20 kDA which are difficult to monitor by 2D-SDS-PAGE. The versatility and reliability of IDA-Cu2+-cellulose combined with MALDI-TOF MS was demonstrated by a number of different experiments. The procedure described is easy to perform, blood drawn from patients is directly applied onto our newly developed cellulose material and directly analyzed by MALDI-TOF MS. Preliminary studies indicated that serum from cancer patient produced MALDITOF MS fingerprints that could be readily distinguished from those of control persons using standard bioinformatics tools. The results of our study demonstrate that MALDI-TOF MS combined with derivatized cellulose is a simple, beneficial, and

research articles

Derivatized Cellulose Combined with MALDI-TOF-MS

Figure 5. Influence of temperature on protein serum profiles. A serum sample was aliquoted and sample preparation was done at 4 °C (A), at 21 °C (B), and at 30 °C (C), respectively. Protein profiles were then generated by MALDI-TOF-MS analysis in linear mode averaging 120 laser shots. Obtained spectra were baseline subtracted and calibrated using an external protein calibration standard.

rapid approach to generate serum protein profiles for biomarker identification at high level of sensitivity. It provides proteins/peptides enrichment with a minimum of sample handling. This leads also increased sensitivity and reproducibility without prior albumin and immunoglobulin depletion, elution, and desalting steps that are mandatory for other MSanalysis techniques. Our approach eliminates possible sample loss associated with eluting proteins/peptides from binding material and removal of salts. Moreover, the cellulose particles can be prepared easily and at very low costs. They can be used for automated routine analysis, e.g., with a lab robotic system, which should allow the analysis of more than 200 samples per day.

Acknowledgment. This work was supported by the Austrian Genome Program (GEN-AU), of the Austrian Ministry of Education, Science and Culture and the Special Research Program “Cell Proliferation and Cell Death in Tumors” (SFB021) of the Austrian Science Funds (FWF).

References (1) Jemal, A.; Tiwari, R. C.; Murray, T.; Ghafoor, A.; Samuels, A.; Ward, E.; Feuer, E. J.; Thun, M. J. CA-Cancer J. Clin. 2004, 54 (1), 8-29. (2) Oesterling, J E. J. Urology 1991, 145 (5), 907-923. (3) Pannek, J.; Partin, A. W. Semin. Urol. Oncol. 1998, 16 (3), 100105. (4) Crawford, D. E. Lancet 2005, 365, 1447-1448. (5) Hillenkamp, F.; Karas, M.; Beavis, R. C.; Chait, B. T. Anal. Chem. 1991, 63 (24), 1193A-1203A. (6) Sheffield, L. G.; Gavinski, J. J. J. Anim. Sci. 2003, 81 (3), 48-57. (7) Tamura, J.; Kunihiro, F. Reza Kenkyu 2003, 31 (1), 21-24. (8) Rappsilber, J.; Moniatte, M.; Nielsen, M. L.; Podtelejnikov, A. V.; Mann, M. Int. J. Mass Spectrom. 2003, 226 (1), 223-237. (9) Zhao, S.; Somayajula, K. V.; Sharkey, A. G.; Hercules, D. M.; Hillenkamp, F.; Karas, M.; Ingendoh, A. Anal. Chem. 1991, 63 (5), 450-453. (10) Issaq, H.; Veenstra, T. D.; Conrads, T. P.; Felschow, D. Biochem. Res. Commun. 2002, 292, 587-592. (11) Petricoin, E. F.; Liotta, L. A. Curr. Opin. Biotech. 2004, 15 (1), 2430. (12) Adam, B.-L.; Vlahou, A.; Semmes, O. J.; Wright, G. L., Jr. Proteomics 2001, 1 (10), 1264-1270. (13) Tang, N.; Tornatore, P.; Weinberger, S. R. Mass Spectrom. Rev. 2003, 23 (1), 34-44.

Journal of Proteome Research • Vol. 4, No. 6, 2005 2325

research articles (14) Shahid, M.; Ball, G.; Hornbuckle, J.; Holding, F.; Carmichael, J.; Ellis, I.; Ali, S.; Li, G.; McArdle, S.; Creaser, C.; Rees, R. Proteomics 2003, 3, 1725-1737. (15) Fung, E.; Diamond, D.; Simonsesn, A. H.; Weinberger, S. R. Methodol. Mol. Med. 2003, 86 (Renal Disease), 295-312. (16) Zhang, X.; Leung, S.-M.; Morris, C. R.; Shigenaga, M. K. J. Biomol. Technol.: JBT 2004, 15 (3), 167-175. (17) Chaurand, P.; Schwartz, S. A.; Caprioli, R. M. J. Proteome Res. 2004, 3 (2), 245-252. (18) Yang, L.; Jia, L.; Zou, H.; Kong, L.; Zhang, Y. Chin. J. Chrom. 1997, 15 (4), 292-295. (19) Bonn, G. K.; Kalghatgi, K.; Horne, W. C.; Horvath, C. Chromatographia 1990, 30, 484-488.

2326

Journal of Proteome Research • Vol. 4, No. 6, 2005

Feuerstein et al. (20) Qu, Y.; Adam, B. L.; Yasui, Y.; Ward, M. D.; Cazares, L. H.; Schellhammer, P. F.; Feng, Z.; Semmes, O. J.; Wright, G. L., Jr. Clin. Chem. (Washington, DC, United States) 2002, 48 (10), 18351843. (21) Adam, B.-L.; Davies, J. W.; Ward, M. D.; Clements, M. A.; Cazares, L. H.; Semmes, O. J.; Schellhammer, P. F.; Yasui, J.; Feng, Z.; Wright, G. L., Jr. Cancer Res. 2002, 62 (13), 3609-3614. (22) Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Nat. Biotechnol. 2002, 20 (3), 301-305.

PR050227Z