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Technical Note

Development of Miniaturized Sorbent Membrane Funnel-based Spray Platform for Biological Analysis HoiSze Yeung, Xiangfeng Chen, Wan Li, Ze Wang, Y. L. Elaine Wong, and T.-W. Dominic Chan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5045324 • Publication Date (Web): 13 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Analytical Chemistry

TECHNICAL NOTE

Development of Miniaturized Sorbent Membrane Funnel-based Spray Platform for Biological Analysis Hoi Sze Yeung,† Xiangfeng Chen,†‡,* Wan Li,† Ze Wang,† Y. L. Elaine Wong,† T.-W. Dominic Chan†,* †

Department of Chemistry, The Chinese University of Hong Kong, Hong Kong SAR ‡

Shangdong Academy of Sciences, Jinan, Shandong, P. R. China

* Address reprint requests to Professor T.-W. D. Chan, Department of Chemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR. E-mail: [email protected], Dr. X. F. Chen, Shandong Academy of Sciences, Jinan, China. E-mail: [email protected].

ACS Paragon Plus Environment


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ABSTRACT In this work, a miniaturized SPE platform, called sorbent membrane funnel, which permits in situ clean-up prior to membrane funnel-based spray analysis was developed. The fabrication of funnel and the mounting of SPE sorbent were simple and straightforward by homemade punching system. Using different sorbents, the SPE sorbent funnel has been successfully applied in spray analysis of drug molecules spiked in human plasma, trypsin digested solution of bovine serum albumin in the presence of high concentration of chaotropic reagents and phosphopeptides in the tryptic digested solution of casein. The results demonstrated that SPE sorbent attached membrane funnels can be a useful tool in common metabolomic and proteomic applications.

Keywords: solid phase extraction, funnel-based spray, human plasma, trypsin digest, clean-up


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Mass spectrometry (MS) has become a powerful and widely applied analytical method in various disciplines because of its high sensitivity, selectivity and speed. In order to generate high quality MS data, proper sample preparation is critical to alleviate ion suppression effect by the matrix interferences during the sample ionization.1-3 Examples of sample pretreatment prior to mass spectrometry include dialysis, liquid-liquid extraction, solid-phase extraction, liquid chromatography and capillary electrophoresis, etc. Among them, solid phase extraction (SPE) remains one of the most important and widely used sample preparation approaches.4 SPE allows simultaneous sample clean-up and trace enrichment. Automated SPE provides high throughput and the processing time is largely reduced. SPE is suitable for different experimental goals as there are various types of sorbent material available. The development of ambient ionization techniques highlights the possibility of analyzing samples directly from their native substrates, i.e. requiring no or minimal sample preparation.5-7 Many analytes could be MS-analyzed in situ from their native substrates under suitable experimental conditions.8-10 These results had led to the development of a whole new field of MS research. Despite this exciting development, it had been reported that some sorts of sample pretreatment might be advantageous in analyzing relatively complex samples. Chipuk et al. reported that chemically modified mesh could selectively capture sulfhydryl analytes from plasma and urine.11 With the use of in-house developed SPE set up, desorption electrospray ionization (DESI) sensitivity could be enhanced by 6 orders of magnitude with sufficient amount of sample.12 Solid-phase microextraction was also applied in conjunction with DESI for the analysis of drugs from urine,13 and warfare agents from indoor office media.14 It has also been demonstrated that the commercially available TriVersa NanoMate®



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chip-based infusion

nanoelectrospray system

could read-out a SPE

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card

automatically.15 Our group has recently presented a variant of nanoelectrospray, named funnel-based spray.16 Funnel-based spray is a sensitive technique that is applicable to a wide range of compounds. The sample volume requirement for a funnel-based spray analysis could be as low as 50 nL. Direct coupling SPE and funnel-based spray might lead to a powerful tool for complex sample analysis. In this work, a miniaturized SPE platform called sorbent funnel was reported. Sorbent funnel is a low cost and conveniently prepared platform that allows in situ clean-up prior to funnel-based spray analysis. The performance of this sorbent funnel was demonstrated by directly analysis of pharmaceutical molecules (vildagliptin) spiked in human plasma. More challenging situations like peptide profiling of bovine serum albumin (BSA) tryptic digest using C18 and MIL-53 (Al),17 and selective capture of phosphopeptides using TiO2 sorbent were also evaluated. EXPERIMENTAL SECTION Reagents and Materials. All materials were obtained commercially and were used without further purification. Vildagliptin, angiotensin II, beta-casein, trypsin, dithiothreitol and iodoacetamide were purchased from Sigma Aldrich (St. Louis, MO, USA). BSA was obtained from Fluka BioChemika (Buchs, Switzerland). Acetic acid and ammonium hydrogen carbonate were purchased from Riedel-de Haën (Seelze, Germany). Tris buffer, sodium hydroxide, calcium chloride, ammonium hydroxide, ammonium bicarbonate and urea were bought from USB Corporation (Cleveland, OH, USA), Farco Chemical Supplies (Beijing, P. R. China), Acros (New Jersey, USA), BDH AnalaR (Poole, England), Junsei Chemical Co. Ltd. (Tokyo, Japan) and Panreac (Barcelona, Spain) respectively. The 0.9% sodium chloride solution was obtained


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from B. Braun Medical Industries (Penang, Malaysia). HPLC grade methanol and acetonitrile were supplied from Merck KGaA (Darmstadt, Germany) and Lab-Scan (Bangkok, Thailand). Bottled distilled water (Wastons, Hong Kong SAR) was used for experiments. Teflon (CS Hyde Company, Lake Villa, IL, USA) and self-adhesive book covering folie (Bantex, item no.18145-08) were used to fabricate an SPE sorbent funnel. Needles of 130 µm and 750 µm diameter were obtained from Hwato (Jiangsu, China) and a local store, respectively. C18 powder (50 µm) was obtained by disassembling a 3-mL GracePureTM SPE C18-Low cartridge (500 mg, Grace, Deerfield, IL). Five µm Titansphere® TiO2 bulk material was obtained from GL Sciences (Tokyo, Japan). MIL-53 (Al) powder was provided by Key Laboratory for Applied Technology of Sophisticated Analytical Instruments, Shandong Academy of Sciences (Shandong, P. R. China). Sample Preparation. Stock solutions of angiotensin II and vildagliptin were prepared at 2 mM in methanol-distilled water (1:1, v/v) and diluted to appropriate concentrations before use. Because of the presence of disulfide linkage in BSA, reduction and alkylation steps were applied prior to tryptic digestion. The digestion protocol of BSA was adopted from reported by Klammer and MacCoss.18 In brief, BSA was solubilized and denatured in 100 mM Tris, pH 8.5 and 8 M urea. Reduction was performed in 10 mM dithiothreitol for 30 minutes at 60 °C to break the disulfide bonds. Cysteines were then alkylated in dark in 25 mM iodoacetamide for 30 minutes at room temperature. The sample was diluted by adding an equal volume of 100 mM Tris buffer. Trypsin (in 50 mM acetic acid) was added in a ratio of 1:30 enzyme/protein along with 2 µL of 2 mM CaCl2. The reaction mixture was incubated overnight at 37 °C. The final digested sample concentration was 90 µM and was kept frozen prior to analysis. Digestion of beta-casein was performed by dissolving the



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protein in 100 mM ammonium bicarbonate solution. Trypsin was dissolved in 50 mM acetic acid. The two solutions were mixed such that the trypsin:protein ratio was 1:50. The mixtures were incubated overnight at 37 °C. The final digested sample concentration was 250 µM and was kept frozen prior to analysis. Membrane Funnel-based Spray Source. The details of the funnel-based spray source were described in earlier work.16. The Schematic diagrams of funnel-based spray and the fabrication device for membrane funnel-based spray were shown in the Figure S-1 and S-2. In brief, the funnel-based spray source consisted of a platform, a sample holder cart and a solvent sprayer cart. The platform table allowed the funnel-based spray source to be attached to the mass spectrometry inlet. The sample holder was held on a 1-D manual linear translational stage and a two-axis motorized stage (Sigma Koki, Tokyo, Japan) for the adjustment of the separation of the membrane funnel from the mass spectrometer inlet, and from the sampling position, respectively. The solvent spray cart consisted of a fused silica of i.d. 50 µm and o.d. 150 µm (Polymicro Technologies, Phoenix, AZ, USA), which was attached on a rotational and 3-D linear stage for position adjustment. A syringe pump (KD Scientific, Holliston, MA, USA) was connected to the fused silica capillary to deliver the spray solvent, which was grounded at the connection to the syringe pump. A CCD (Videosonic, Saitama-shi, Japan) camera and a digital microscope were used to monitor the operation of the ionization source. FTICR System. All the experiments were performed by using FTICR-MS system (4.7 Tesla APEX III and 9.4 Tesla Solarix, Bruker Instrument Inc., Billerica, MA). Data acquisition was performed by summing up 10 to 20 scans. The spectra were recorded using 128 K-byte and 1 M memory for 4.7 Tesla and 9.4 Tesla FTICR-MS, respectively. The spray voltage was set to -3.5 kV according to our previous


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optimized result. Heated stream of nitrogen gas (at ~ 250 °C) was used to warm up the dielectric capillary and to assist the desolvation process of the analyte ions from the sprayed droplets. All mass spectra were acquired in broadband mode. Zero-filling was performed once prior to Fourier transformation for BSA tryptic digest data. RESULTS AND DISCUSSION SPE Sorbent Funnel Fabrication. Fabrication of sorbent funnel is demonstrated in Figure 1a. It is a five steps process which mainly involves sticking SPE powder onto an adhesive surface and punching the funnel orifice. Step 1: A Teflon sheet and an adhesive membrane were mounted on separate plastic holders. Step 2: A mask was prepared by punching the Teflon sheet with a Ф 0.75-mm needle to give a 7×7 array of holes (spot to spot distance was 2.0 mm). The mask was stuck onto the adhesive membrane. Step 3: The sorbent material was added onto the surface of the mask to cover up the punched orifices. The sorbent powder was pressed against the adhesive membrane to ensure firm attachment. Step 4: Excess sorbent powder was removed and the Teflon mask was peeled off. Step 5: The SPE sorbent attached spots were then punched by a Ф 130 µm needle to give orifices with diameter of around 100 µm. The amount of sorbent for each funnel spot was estimated to be around 20 µg. Figure 1b shows the schematic diagram of the sample pretreatment procedure using the sorbent funnel. First, 50 nL of the sample was loaded onto the sorbent funnel. After the sample was dried under ambient condition, washing was performed to remove the undesired interferences. Washing solution was added on the sorbent funnel surface and then was removed by capillary action using a tissue paper from the bottom of the funnel. Washing step could be repeated based on the sample complexity. It is important to note that the necessary materials for the fabrication of SPE



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sorbent funnel were mostly available in a laboratory. Since the funnel was functionalized by adhering sorbent powder onto the membrane surface, the SPE sorbent material could be conveniently switched according to applications. The sample volume required for the SPE sorbent funnel clean-up was only 50 nL. After removal of matrix interferences, the purified sample could be eluted and analyzed in situ using the membrane funnel-based spray technique. Analysis of Spiking Drug Compound in Human Plasma. The in situ clean up function of miniaturized SPE platform was illustrated by using C18 sorbent funnel to clean up a drug in human plasma sample. Plasma is one of the most complex biological fluid samples. It contains many interferences like proteins, glucose, electrolytes, hormones and metabolites. These matrix components have higher concentration than the drug analyte and will cause severe ion suppression effect during the spray process. Figure 2 showed the funnel-based spray mass spectrum of 2.5 pmol of vildagliptin in human plasma. Without any pretreatment, funnel spray could not be generated. After the simple clean-up process by C18 sorbent funnel, the drug peak could be recorded. As shown in Figure S-3, using the newly installed 9.4 Tesla FTICR-MS system, 25 fmol on-spot sample loading was sufficient to provide a detectable signal (S/N>3). The two-fold enhancement in the sensitivity using the new FTMS system was tentatively attributed to the use of better ion transfer optics (e.g. dual ion funnel and multipole transfer electrodes) and higher magnetic field strength. Analysis of Trypsin Digested BSA using Different Sorbent Materials. In proteomics, proteins are usually analyzed by the bottom-up approach to obtain the sequence information.22-25 Prior to mass spectrometric analysis, a protein molecule is usually digested into small fragments. Normally, the protein has to be denatured and


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the disulfide linkages present inter-/intra-molecularly should be broken. A large amount of chaotrope (e.g. urea) is usually added to disrupt the intra-molecular interactions. In addition, buffer solutions used for digestion are very often not compatible with mass spectrometric analysis. Therefore, some sort of clean-up process is usually needed for these digested samples. Apart from the most commonly used C18, we have also prepared sorbent funnels using MIL-53 (Al).17 This class of material has been reported to be applied to sample enrichment.26 Figure 3 shows the SPE sorbent funnel-based spray mass spectra of BSA tryptic digest sample using ordinary membrane funnel, C18 funnel and MIL-53 (Al). The data obtained from the sorbent funnel-based spray were submitted to Mascot search engine using SwissProt database. Search parameters and results were summarized in Table S-1. The MOWSE scores obtained were 55, 228 and 230 for ordinary membrane funnel, C18 funnel and MIL-53 (Al) funnel, respectively. The sequence of bovine serum albumin identified using MASCOT search were shown in Table S-2. The sequence coverages from ordinary membrane funnel, C18 funnel and MIL-53 (Al) were 12 %, 68 % and 68 %, respectively. Although the sequence coverages observed from C18 and MIL-53(Al) sorbent were similar, these sorbent were found to have certain degree of difference in sorption selectivity. Some peptides components could only be observed in C18 funnel or MIL-53(Al) funnels. Combining the results from both sorbent funnels, the sequence coverage was 82 %. Phosphopeptide Analysis. Phosphopeptide is one of the most common types of posttranslationally modified peptides. Phosphopeptides are typically masked by nonphosphorylated peptides present in greater abundance in a complex sample.27 To deal with this problem, a sorbent funnel was prepared using titanium dioxide powder to isolate and preconcentrate the phosphopeptides.28-30 The phosphate group(s) of the



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phosphopeptide interacts strongly with Ti4+ via metal-ligand chelation.31 In this experiment, bovine beta-casein was used as the probing analyte. In its tryptic digest, there

are

two

segments

containing

the

phosphate

functionalities,

FQpSEEQQQTEDELQDK and RELEELNVPGEIVEpSLpSpSpSEESITR. Without any sample pretreatment, only the fragment with one phosphate group could be detected under high non-phosphopeptide background (Figure 4a). The signal of phosphorylated peptide with four phosphate groups was completely suppressed. Upon clean-up by the TiO2 sorbent funnel, intense mono- and tetra-phosphopeptides signals could be detected with much less interference in peaks arising from other non-phosporylated peptides (Figure 4b). We have further demonstrated the application of the device by imaging analysis of an ink pattern in an automatic and high throughput format. In the experiment, a 14 × 10 funnel-array fabricated on an area (3.5 ݉݉ × 2.5 ݉݉) marked with a letter “M” was screened automatically spot by spot. The spatial resolution (i.e. inter-funnel distance) was 250 µm. After the acquisition of a mass spectrum at each funnel, a TTL pulse was sent from the console of FT-ICR-MS that would trigger one movement of the sample plate to the next funnel. One hundred and forty sampling spots with analyzed automatically over around 20 min. The chemical image was visualized by coupling the funnel coordinates and the relative signal intensities from 0% to 100% (representing absolute signal intensity 4.79E7) by default color. Figure S-4 showed the mass spectrometry imaging analysis of an ink pattern. This result demonstrates that the device has potential application on high throughput analysis. CONCLUSIONS A miniaturized sample processing platform which consists of an array of solid


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phase extraction funnels for funnel-based spray analysis was reported. Fabrication of the sorbent funnel was simple and straightforward requiring no complex experimental facilities as in chip-based microfabrication. Small volume of sample (50 nL) solution was sufficient for in situ desalting (data not shown), clean-up of a complex tryptic digest and selective enrichment of pharmaceutical molecules and phosphopeptides. This miniaturized SPE set-up has potentials for other applications and deserves further exploration. For instance, implementation of automatic sample platform might allow high throughput analysis of complex samples. An optimization platform for SPE conditions using a minimal amount of sample could also be established. An array of funnels with rows of different sorbent materials could serve as a convenient platform to perform comprehensive characterization of chemicals present in complex mixtures.

ACKNOWLEDGEMENTS Financial supports from the National Natural Science Foundation of China (21205071), Research Grant Council of the Hong Kong Special Administrative Region (Research Grant Direct Allocation, Ref. 2060351), Natural Science Foundation of Shandong Province (ZR2012BQ009) and Shandong Academy of Science are gratefully acknowledged. The authors thank the staff in the mechanical workshop of the Chinese University of Hong Kong for preparing the funnel-based spray set up. REFERENCES [1] Annesley, T. M. Clin. Chem. 2003, 49, 1041-1044. [2] Jessome, L. L.; Volmer, D. A. LCGC North Am. 2006, 24, 83-89. [3] King, R.; Bonfiglio, R.; Fernandez-Metzler, C.; Miller-Stein, C.; Olah, T. J. Am. Soc. Mass Spectrom. 2000, 11, 942-950. [4] Majors, R. E. LCGC North Am. 2002, 20, 1098-1113. [5] Chen, H.W.; Gamez, G.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2009, 20,



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1947-1963. [6] Ifa, D. R.; Wu, C.P.; Ouyang, Z.; Cooks, R. G. Analyst 2010 135, 669-681. [7] Weston, D. J. Analyst 2010, 135, 661-668. [8] Dill, A. L.; Ifa, D. R.; Manicke, N. E.; Costa, A. B.; Ramos-Vara, J. A.; Knapp, D. W.; Cooks, R. G. Anal. Chem. 2009, 81, 8758-8764. [9] Peng, Y.; Zhang, S.; Wen, F.; Ma, X.; Yang, C.; Zhang, X. Anal. Chem. 2012, 84, 3058-3062. [10] Chen, H. W.; Talaty, N. N.; Takats, Z.; Cooks R. G. Anal. Chem. 2005, 77, 6915-6927. [11] Chipuk, J. E.; Gelb, M.H.; Brodbelt, J. S. Anal. Chem. 2010, 82, 4130-4139. [12] Denes, J.; Katona, M.; Hosszu, A.; Czuczy, N.; Takats, Z. Anal. Chem. 2009, 81, 1669-1675. [13] Kennedy, J. H.; Aurand, C.; Shirey, R.; Laughlin, B. C.; Wiseman, J. M. Anal. Chem. 2010, 82, 7502-7508. [14] D'Agostino, P. A.; Chenier, C. L.; Hancock, J. R.; Lepage, C. R. J. Rapid Commun. Mass Spectrom. 2007, 21, 543-549. [15] Walworth, M. J.; ElNaggar, M. S.; Stankovich, J. J.; Witkowski, C.; Norris, J. L.; Berkel, G.J. V. Rapid Commun. Mass Spectrom. 2011, 25, 2389-2396. [16] Yeung, H.S.; Li, W.; Wang, Z.; Wong, Y.L.; Chen, X.; Chan, T.-W. D. Rapid Commun. Mass Spectrom. 2015, 29, 336-342. [17] Loiseau, T.; Serre, C.; Huguenard, C.; Fink, G.; Taulelle, F.; Henry, M.; Bataille, T.; Ferey, G. A. Chem. Eur. J. 2004, 10, 1373-1382. [18] Klammer, A. A.; MacCoss, M. J. J. Proteome Res. 2006, 5, 695-700. [19] Warren, M. E.; Brockman, A. H.; Orlando, R. Anal. Chem. 1998, 70, 3757-3761. [20] Dahlin, A. P.; Bergstrom, S. K.; Andren, P. E.; Markides, K. E.; Bergquist, J. Anal. Chem. 2005, 77, 5356-5363. [21] Tan, A. M.; Benetton, S.; Henion, J. D. Anal. Chem. 2003, 75, 5504-5511. [22] Butt, A.; Davison, M. D.; Smith, G. J.; Young, J. A.; Gaskell, S. J.; Oliver, S. G.; Beynon, R. J. Proteomics 2001, 1, 42-53. [23] Dwivedi, R. C.; Spicer, V.; Harder, M.; Antonovici, M.; Ens, W.; Standing, K. G.; Wilkins, J. A.; Krokhin, O. V. Anal. Chem. 2008, 80, 7036-7042. [24] VerBerkmoes, N. C.; Bundy, J. L.; Hauser, L.; Asano, K. G.; Razumovskaya, J.;


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Larimer, F.; Hettich, R. L.; Stephenson, J. L. J. Proteome Res. 2002, 1, 239-252. [25] Kitagawa, S.; Kitaura, R.; Noro, S. Angew. Chem., Int. Ed. 2004, 43, 2334-2375. [26] Gu, Z. Y.; Chen, Y. J.; Jiang, J. Q.; Yan, X. P. Chem. Commun. 2011, 47, 4787-4789. [27] Steen, H.; Jebanathirajah, J. A.; Rush, J.; Morrice, N.; Kirschner, M. W. Mol. Cell. Proteomics 2006, 5, 172-181. [28] Jensen, S. S.; Larsen, M. R. Rapid Commun. Mass Spectrom. 2007, 21, 3635-3645. [29] Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. D. Mol. Cell. Proteomics 2005, 4, 873-886. [30] Yu, Y. Q.; Fournier, J.; Gilar, M.; Gebler, J. C. J. Sep. Sci. 2009, 32, 1189-1199. [31] Han, G. H.; Ye, M. L.; Zou, H. F. Analyst 2008, 133, 1128-1138.



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Figure Captions Figure 1. (a) A flow chart showing the fabrication process of sorbent funnel (1-5), and a photograph of a C18 sorbent funnel (6). (b) Schematic diagram illustrating the sample pretreatment procedure using sorbent funnel.

Figure 2. Funnel-based spray mass spectrum of vildagliptin spiked in human plasma. (a): Normal funnel-based spray (2.5 pmol); (b): SPE funnel with C18-coated membrane funnel (2.5 pmol).

Figure 3. Funnel-based spray mass spectra of bovine serum albumin tryptic digest from (a) ordinary membrane funnel, (b) C18 funnel, and (c) MIL-53 (Al) funnel.

Figure 4. Funnel-based spray mass spectra of bovine beta-casein tryptic digest, (a) without any sample clean, and (b) purified using TiO2 sorbent funnel.


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Figure 1. (a) A flow chart showing the fabrication process of sorbent funnel (1-5), and a photograph of a C18 sorbent funnel (6). (b) Schematic diagram illustrating the sample pretreatment procedure using sorbent funnel.



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Figure 2. Funnel-based spray mass spectrum of vildagliptin spiked in human plasma. (a): Normal funnel-based spray (2.5 pmol); (b): SPE funnel with C18-coated membrane funnel (2.5 pmol).


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Figure 3. Funnel-based spray mass spectra of bovine serum albumin tryptic digest from (a) ordinary membrane funnel, (b) C18 funnel, and (c) MIL-53 (Al) funnel.

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Figure 4. Funnel-based spray mass spectra of bovine beta-casein tryptic digest, (a) without any sample clean, and (b) purified using TiO2 sorbent funnel.

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