Biomolecular Analysis and Biological Tissue Diagnostics by

Dec 17, 2013 - School of Chemical and Life Sciences, Shenyang Normal University, Shenyang 110034, China ... Christian W. Klampfl , Markus Himmelsbach...
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Biomolecular Analysis and Biological Tissue Diagnostics by Electrospray Ionization with a Metal Wire Inserted Gel-Loading Tip Mridul Kanti Mandal,*,† Kentaro Yoshimura,‡ Subhrakanti Saha,† Zhan Yu,†,§ Sen Takeda,‡ and Kenzo Hiraoka*,† †

Clean Energy Research Center, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan Department of Anatomy and Cell Biology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898, Japan § School of Chemical and Life Sciences, Shenyang Normal University, Shenyang 110034, China ‡

S Supporting Information *

ABSTRACT: A metal wire-inserted disposable gel-loading tip was examined as an electrospray emitter. Its performance was similar to that of conventional electrospray ionization (ESI) with a relatively low flow rate (∼100 nL/min) and without the need for solvent pumps. It was also used as an emitter for solid probe-assisted ESI (SPA-ESI) (e.g., biofluid was sampled from the biological tissue by a needle and was inserted into the solventpreloaded gel-loading tip). Selective detection of lipids and proteins, such α and β chains of hemoglobin could be accomplished by choosing appropriate solvents. A suitable protocol for cancer diagnosis was established by this method. A good figure of merit of this method is its applicability to biological tissue diagnostics with high cost efficiency and on a disposable basis.

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A simple spot-sampling method was developed by Van Berkel et al. using a liquid microjunction surface sampling probe/ESIMS (LMJ-SSP/ESI-MS) system,20 and later a fully automated surface sampling device coupled with chip-based infusion nanoESI system has been reported by the same group.21 Recently, Raoch et al. reported on a method called nanospray desorption ESI (Nano-DESI) for liquid extraction surface sampling in mass spectrometry.22 In 2007, a modified version of ESI using a solid needle was reported from our laboratory, namely, probe ESI-mass spectrometry (PESI-MS).23 Prior to this development ESI was also performed from a solid metal probe and a pointed carbon by Hong et al. and Liu et al., respectively.24,25 The new feature of PESI was the adoption of discontinuous sampling, making it possible to electrospray sample components in the order of their surface active values.26 Because of this unique feature, high-quality PESI mass spectra could be obtained for proteins and peptides, with reasonably less interference from high-concentration salts and detergents.27,28 Because of its high reproducibility and ease in handling, PESI has been used for direct biomolecular analysis and cancer diagnosis.29,30 For realtime and direct chemical constituents analysis from dry samples, we have developed a sheath-flow (SF) PESI-MS.31,32

lectrospray ionization (ESI) has been used as a routine biomolecules analysis since the development by Fenn and colleagues.1,2 Further, direct sampling and ionization techniques based on ESI and atmospheric pressure chemical ionization (APCI) have been developed [e.g., desorption electrospray ionization mass spectrometry (DESI-MS),3,4 direct analysis in real time (DART),5 fused droplet ESI (FD-ESI),6 and extractive ESI (EESI)].7 The use of biological tissues as a direct electrospray emitter has also been developed; for example, tissue spray,8,9 leaf spray,10 and needle biopsy and direct electrospray from the biopsied tissue.11 Mizuno et al. analyzed a live, single mammalian cell using a nano-ESI tip under a video microscope.12 Pagnotti et al. reported an inlet ionization method requiring no voltage or laser, which can produce similar mass spectra to those obtained with ESI for small molecules, peptides, and proteins, with sensitivity that surpasses ESI.13 Prior to this development, an atmosphericpressure solids analysis probe (ASAP) was reported by the same group.14 A paper spray ionization technique was developed for mass spectrometric analysis of a wide variety of compounds, including small organic compounds, peptides, and proteins.15 Hu et al. reported a simple and economical ESI technique using disposable wooden tips that can be used simultaneously for loading and ionizing the samples.16 In addition, ESI has been used as a subsidiary ionization source [e.g., electrospray-assisted laser desorption/ionization (ELDI),17 laser ablation with ESI (LAESI),18 and laser-induced acoustic desorption (LIAD) with ESI-MS (LIAD/ESI-MS)].19 © 2013 American Chemical Society

Received: October 9, 2013 Accepted: December 17, 2013 Published: December 17, 2013 987

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Supporting Information). Then, a 0.1 mm metal wire (SUS-304 stainless steel or titanium or a 0.12 mm acupuncture needle with ∼700 nm tip diameter) was inserted into the capillary from the back side to enable the application of the high voltage. In an alternative but similar manner to SPA-nanoESI,33 a 0.1 mm o.d. metal wire with a sharp tip (tangentially cut or etched) or an acupuncture needle was inserted about 1−2 mm into the biological tissue. The needle carrying the extracted sample on the tip was inserted into a gel-loading tip, containing 2 μL preloaded solvent. Alternatively, the solvent can also be loaded after inserting the needle into the capillary. Next, the plastic capillary was positioned 3 mm away from a mass spectrometer (MS) inlet, and electrospray was generated by applying a high voltage (∼3.0 kV) to the wire. The human kidney tissues stored at −80 °C were thawed at room temperature prior to the experiments. Conventional ESI Source. A commercially available pneumatically assisted ESI emitter (JEOL, Akishima, Japan) was used for the conventional ESI experiments. The emitter was perpendicular to the axis of the ion sampling orifice of the MS at a 4 mm distance. A syringe pump (PHD 4400, Harvard Apparatus, Holliston, MA) was used to deliver the sample solution with variable flow rates in the range of 100−2000 nL/ min. The high voltage applied to the needle was 2.5 kV. MS Measurements. MS analyses were performed using a time-of-flight (TOF) MS (AccuTOF, JEOL, Tokyo, Japan). The exact mass analysis was performed with Orbitrap Exactive (Thermo Scientific, Bremen, Germany) for identification of observed peaks with our home-developed lipid identification software. Sample Preparation. All reagents and solvents used in this work were of analytical grade or higher and were used without further purification. Water was purified and deionized by a Milli-Q system (Millipore, Bedford, MA). High-performance liquid chromatography (HPLC) grade organic solvents were purchased from Kanto Chemicals (Tokyo, Japan). All proteins, peptides, and lipids were obtained from Sigma-Aldrich. Stock solutions of proteins and peptides were prepared in water and

To perform remote, direct microextraction and sampling for MS, the solid probe-assisted (SPA) nanoESI method was also developed in our laboratory.33 In this method, the biological tissue sample is extracted either by sticking the tip of a needle into the sample or by direct biopsy, after which the needle is inserted into the solvent-preloaded nanoESI capillary from the backside. SPA-nanoESI follows the principle of ultralow-level extraction and nano electrospray directly from the untreated biological tissue and biomaterial.33 In this work, a fine plastic gel-loading tip was used instead of a nanoESI glass capillary, termed solid probe-assisted ESI (SPA-ESI). This technique was successfully applied to peptides, proteins, and biological tissue analysis, as well as selective detection/ionization of biomolecules directly from the biological tissues.

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EXPERIMENTAL SECTION SPA-ESI Method. A schematic of the present method and the image of a gel-loading pipet tip are shown in Figure 1. A 2

Figure 1. Schematic showing solid probe assisted-ESI (SPA-ESI) with the use of a gel loading tip and a metal wire.

μL liquid sample was sucked in the gel-loading pipet tip (GELoader, epT.I.P.S., 20 μL, Eppendorf, Germany) with internal diameter (i.d.) of about 0.135 mm and outer diameter (o.d.) of 0.2 mm. After sampling, the backside of the capillary was cut to a total length of about 20 mm (Figure 1 of the

Figure 2. Positive mode SPA-ESI mass spectra of 10 μM (a) cytochrome c, (b) hemoglobin, (c) ubiquitin, and (d) insulin using MeOH/H2O (1/2) as solvent. 988

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then diluted with organic solvent/H2O (1/2) or pure organic solvent. Lipids were prepared by following the company’s protocols and diluted with organic solvent/H2O (1/2) or pure organic solvent. The kidney tissues were about 10 × 4 × 2 mm3 in size. Cancer tissues from patients diagnosed as clear cell renal cell carcinoma (ccRCC) and their uninvolved kidney tissues were obtained from the Department of Surgery, Faculty of Medicine, University of Yamanashi. All the procedures relating to the handling of human specimens were approved by the Ethical Committee of the Faculty of Medicine, University of Yamanashi. Appropriate precautions should be taken when handling cancerous biomaterials.

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RESULTS AND DISCUSSION Aksyonov and Williams reported that peptide analysis (e.g., bradykinin) can be performed with metal-inserted pipet tips (e.g., plastic capillary, i.d.: ∼0.3 mm) based ESI.34 To evaluate the present method using a finer gel-loading tip than the plastic capillary used by Aksyonov and Williams, several peptides with a concentration of 10 μM (10 pmol/μL) in MeOH/H2O (1/2) were examined (see Figure 2 of the Supporting Information). Four peptides were detected with reasonably high intensities. The mass spectra of four proteins, cytochrome c, hemoglobin, ubiquitin, and insulin, using MeOH/H2O (1/2) as a solvent are shown in Figure 2 (panels a−d). Strong signals of peptides and proteins were observed. Schmidt et al. reported that in nanoESI, the flow rates have a strong influence on ion signals.35 To measure the flow rate of this system, the duration of the electrospray was measured for 1.5 μL of 10 μM (10 pmol/μL) ubiquitin in MeOH/H2O (1/2), containing 1% acetic acid (AcOH) as a solvent. The electrospray lasted about 15 min (see Figure 3 of the Supporting Information), and the flow rate was determined to be ∼100 nL/min. In addition, we performed a series of experiments with conventional ESI with the use of analytes in the same solvents with different flow rates. The sensitivity of the current technique was about the same as that of conventional ESI (see Figure 4 of the Supporting Information). We have also checked the limits of detection (LODs) by SPA-ESI for standard peptides and proteins using bradykinin and ubiquitin. The LODs for bradykinin and ubiquitin were 100 fmol/μL (10−7 M) and 500 fmol/μL (5 × 10−7 M), respectively, with the signal-tonoise (S/N) ratio better than 8 (see Figures 5 and 6 of the Supporting Information). Selective Ionization of a Mixture of Lipids and Proteins. To investigate the effect of solvents using a gelloading tip, two solvent systems, MeOH/H2O (1/2) and 90% organic solvent were examined for the solution of 10 μM L-αphosphatidylcholine and hemoglobin. As shown in Figure 3, when an organic solvent [90% MeOH or ACN/MeOH/ isopropanol (1/1/1)] was used, only L-α-phosphatidylcholine was detected but no hemoglobin. That is, hemoglobin was almost totally suppressed in this solvent system. On the other hand, only hemoglobin but little L-α-phosphatidylcholine could be detected when MeOH/H2O (1/2) or ACN/MeOH/ isopropanol/H2O (1/1/1/6) was used, indicating that a H2Orich solvent system preferentially gives proteins, but a pure organic solvent system gives only lipids. To examine whether this finding could be generalized to real-world samples, normal and cancerous kidney tissues were examined using MeOH/ H2O (1/2) and a pure organic solvent system, as described in the following section.

Figure 3. Positive mode SPA-ESI mass spectra obtained for mixtures of hemoglobin and L-α-phosphatidylcholine using (a) ACN/MeOH/ isopropanol (1/1/1) solvent and (b) MeOH/H2O (1/2), respectively.

Selective Detection of Lipids and Proteins from Biological Tissue. For direct analysis of biological tissue, a sharp wire tip or an acupuncture needle was used, which could easily be inserted into the biopsied tissue or organ of a living animal with less pain during biofluid sampling. In our previous study, the biofluid sampled by a needle was inserted into a nanoESI glass capillary.33 A similar procedure was adopted here by using a plastic gel-loading tip. The needle was inserted into the dissected human kidney tissues and then immersed into a gel-loading tip (SPA-ESI) that was preloaded with a MeOH/ H2O (1/2) solvent. Figure 4 (panels a and b) shows the SPAESI mass spectra measured for normal and cancerous kidney tissues obtained by using a MeOH/H2O (1/2) solvent. Intriguingly, only α and β chains of hemoglobin with heme were detected, with little appearance of lipids for both noncancerous and cancerous kidney tissues. It should be noted that a cancerous tissue has a relatively much smaller amount of hemoglobin. It is well-known that blood flow rates and water content decrease with increasing fatty content in cancer tissues.36,37 In our previous study,33 we found that a mixture of ACN/ MeOH/isopropanol (1/1/1) solvent gives the most abundant signal intensities from lipid molecules from among various solvents.33 Thus, we also used this solvent as an organic solvent for SPA-ESI (see Figure 4, panels c and d). In these mass spectra, only phosphatidylcholines (PCs) and triacylglycerols (TAGs) were detected, but no hemoglobin. It was evident that this organic solvent is the most useful for lipid detection. In Figure 4c (normal kidney tissue), ion signals due to potassiated cholesteryl ester such as CE[18:3]K+ and protonated and PCs, such as PC[32:2]H+, PC[34:2]Na+, PC[34:1]Na+, PC[36:2]Na+, PC[36:1]Na+, as major ions and TAGs, such as TAG[52:2]Na+ as minor ions were observed. On the other hand, in Figure 4d (kidney cancer tissue), ion signals due to sodiated and potassiated TAGs, such as TAG[50:2]K+, TAG[50:2]Na + , TAG[52:3]K + , TAG[52:2]K + , and TAG[54:3]K+ as major ions and PCs as minor ions were observed, and these results were in line with our previous study using nanoESI.33 989

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Figure 4. Positive mode SPA-ESI mass spectra of (a) normal and (b) cancerous kidney tissues using the mixture of MeOH/H2O (1/2) as the solvent. Positive mode SPA-ESI mass spectra of (c) normal and (d) cancerous kidney tissues with the use of a mixture of ACN/MeOH/isopropanol (1/1/1) as the solvent. (e) Principal component analysis (PCA) from cancerous regions and uninvolved tissues for 10 patients.

exhaustion of the liquid, 2 μL of ACN/MeOH/isopropanol (1/ 1/1) was newly loaded to the gel-loading tip. The liquid loading was performed by connecting the used cut gel-loading tip with a new gel loading tip (see Figure 8 of the Supporting Information). The tip of the cut gel-loading tip was wrapped in parafilm to protect solvent leaking. The mass spectrum obtained for the second electrospray is shown in Figure 7b of the Supporting Information. Only lipid, but no hemoglobin was detected, which is similar to Figure 4c. These experimental results indicate that the adsorption of analytes on the wall of the gel-loading tip and/or metal needle has a significant effect on the analysis of mixed samples, which is easily envisaged from the principles of solid phase microextraction or chromatographic separation taking place between the solid and liquid phases. For sophisticated and precise analysis it is advisable to use a glass capillary with 1 μm tip diameter (either surfacemodified or not) with very low flow rates. As in our previous study,33 for sampling procedures that may require long-term sample preservation, we have frozen the kidney sample loaded on the metal needles in glass vials at −30 °C for 1−7 days. We took out the needles from the freezer sequentially one by one, thawed at room temperature and performed the experiment. The obtained mass spectra before and after freezing were found to be almost identical for lipid analysis (Figure 9 of the Supporting Information and Figure 4). For protein profiling using aqueous solvents, we found that for normal kidney tissues (1st−7th day) all the frozen needles gave almost equivalent mass spectra of hemoglobin, as shown in Figure 10 of the Supporting Information. As described

In a previous study,38 adjacent tissue sections were stained with hematoxylin and eosin (H&E) to distinguish between normal and cancerous parts. Although H&E staining allows diagnosis, no information concerning the molecular information of the tissue is obtained.39 We found that SPA-ESI can be used to distinguish normal and cancerous tissue from the mass spectra (see Figure 4, panels c and d). While ccRCC may be distinguished from the normal tissue on the basis of the individual mass spectrum (i.e., relative intensities of PC and TAG),29 the use of statistical methods increases the confidence of diagnosis.39 It has also been known that ccRCC accumulates TAG in the cytoplasm.40 To classify normal and cancerous kidney tissues for 20 samples from 10 patients, principal component analysis (PCA) was performed using an open source41 and a commercial software (XLSTAT 2013). The peaks of the positive mode mass spectra at m/z in the range of 700−950 were used for PCA. Two-dimensional plots of PC1 × PC2 for the data obtained from the cancerous regions and uninvolved tissue for 10 patients are presented in Figure 4e. As shown in the Figure 4e, spectra from the normal and cancerous kidney tissues were reasonably separated. The separate detection of lipids and proteins with different solvents is likely to have been caused by the possible adsorption of either analytes on the needle and on the plastic tip’s inner wall. To clarify this point, two consecutive ESI runs were conducted using the same biological sample. The SPA-ESI mass spectrum for normal kidney tissue obtained by using 2 μL of MeOH/H2O (1/2) as a solvent is shown in Figure 7a of the Supporting Information. Hemoglobin appears as a major ion but only little lipid ions are detected. After the complete 990

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previously, for the frozen needles with cancerous kidney tissues, the hemoglobin peak was not detected properly.36,37 We examined the applicability of negative mode SPA-ESI for biomolecular analysis. For the model experiment, we first analyzed 10 μM hemoglobin and 5 μM bradykinin. Both peptide and protein were detected with reasonably high sensitivity (see Figure 11 of the Supporting Information). Finally, we checked the applicability of this technique to direct analysis of biological tissue in the negative mode using cancerous and their uninvolved kidney tissues. Several kinds of lipids with their different species such as phosphatidylethanolamines, sulfatides, phosphatidylserine, and phosphatidylinositol have been detected for the cancerous and uninvolved tissues (see Figure 12 of the Supporting Information). One drawback of the system was that in the negative mode, no selective ionization of proteins and lipids could be observed, as both aqueous (50% alcohols) and organic solvents only gave the abundant ions of only lipid molecules.

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CONCLUSION As ESI sources remain at the forefront of ion production in biomedical MS, it is very important to develop a source for a cost-effective and easily disposable procedure. Herein, we report a cost-effective technique using a gel-loading tip that can be widely used, from liquid samples to biological tissue analysis, with potential applications in clinical diagnostics. By using a plastic gel-loading tip, proteins such as α and β chains of hemoglobin and lipids could be selectively detected by choosing an aqueous or nonaqueous solvent. The selective detection of proteins and lipids was attributed to the adsorption of analytes on the surface of the gel-loading tip and/or metal needle. Organ dissection would not be prerequisite prior to analysis in this method, as the needle (e.g., acupuncture) used in this technique may also be applicable to the less invasive needle biopsy for off-line MS.33 Therefore, SPA-ESI could be a powerful tool by which the identification and characterization of lipids, proteins, and other biomolecules can be achieved within a minute in a very simple manner and using disposable equipment.

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ASSOCIATED CONTENT

S Supporting Information *

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

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: +81-55-220-8709. *E-mail: [email protected]. Tel: +81-55-220-8572. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS M. K. Mandal gratefully acknowledges the financial support from the Japan Society for the Promotion of Science (JSPS). REFERENCES

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(37) Pedrosa, I.; Alsop, D. C.; Rofsky, N. M. Cancer 2009, 115, 2334−2345. (38) Yoshimura, K.; Chen, L. C.; Mandal, M. K.; Nakazawa, T.; Yu, Z.; Uchiyama, T.; Hori, H.; Tanabe, K.; Kubota, T.; Fujii, H.; Katoh, R.; Hiraoka, K.; Takeda, S. J. Am. Soc. Mass Spectrom. 2012, 23, 1741− 1749. (39) Dill, A. L.; Eberlin, L. S.; Zheng, C.; Costa, A. B.; Ifa, D. R.; Cheng, L.; Masterson, T. A.; Koch, M. O.; Vitek, O.; Cooks, R. G. Anal. Bioanal. Chem. 2010, 398, 2969−2978. (40) Hoffmann, K.; Blaudszun, J.; Brunken, C.; Höpker, W.-W.; Tauber, R.; Steinhart, H. Lipids 2005, 40, 1057−1062. (41) Kamakura’s Free Software Downloads. http://wak2.web.rice. edu/bio/WagnerKamakuraDownloads.htm (accessed Nov 20, 2013).

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