Detection of Native Protein Ions in Aqueous Solution under Ambient

J. Presented at the 55th SMS Conference on Mass Spectrometry, Indianapolis, IN, June 3−7, 2007; Poster TPA 18. ...... Bioanalysis 2015 7 (15), 1...
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Anal. Chem. 2008, 80, 4845–4852

Detection of Native Protein Ions in Aqueous Solution under Ambient Conditions by Electrospray Laser Desorption/Ionization Mass Spectrometry Jentaie Shiea,*,†,‡ Cheng-Hui Yuan,† Min-Zong Huang,† Sy-Chyi Cheng,† Ya-Lin Ma,† Wei-Lung Tseng,†,‡ Hui-Chiu Chang,‡,§ and Wen-Chun Hung‡,| Department of Chemistry, National Sun Yat-Sen University, National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center, Graduate Institute of Medicine, Kaohsiung Medical University, and Institute of Biomedical Science, National Sun Yat-Sen University, Kaohsiung, Taiwan Liquid electrospray laser desorption/ionization (ELDI) mass spectrometry allows desorption and ionization of proteins directly from aqueous solutions and biological fluids under ambient conditions. Native protein ions such as those of myoglobin, cytochrome c, and hemoglobin were obtained. A droplet (ca. 5 µL) containing the protein molecules and micrometer-sized particles (e.g., carbon graphite powder) is irradiated with a pulsed UV laser. The laser energy adsorbed by the inert particles is transferred to the surrounding solvent and protein molecules, leading to their desorption; the desorbed gaseous molecules are then postionized within an electrospray (ESI) plume to generate the ESI-like protein ions. With the use of this technique, we detected only the protonated protein ions in various biological fluids (including human tears, cow milk, serum, and bacterial extracts) without interference from their corresponding sodiated or potassiated adduct ions. In addition, we rapidly quantified the levels of glycosylated hemoglobin present in drops of whole blood obtained from diabetic patients without the need of sample pretreatment.

ion signals using these mass spectrometric techniques usually requires tedious sample pretreatment processes. In ESI, to obtain a stable electrospray from a particular solution, certain chemical additives, such as an organic solvent or acid, must added to the sample prior to analysis.9–11 In general, these chemicals will induce protein denaturation and interfere with the protein-protein and protein-substrate interactions. In MALDI, the analyte ions are generated from a cocrystal of the analyte and organic matrix under vacuum (a highly artificial environment for protein analysis).12–14 The acidity of the MALDI matrix, desorption and ionization induced by laser irradiation, and the use of drying and crystallization processes usually result in protein denaturation, aggregation, or subunit dissociation. Crystallization also causes the unpredictable phenomena of “sweet spots” in MALDI analysis. Recently, two ionization techniquessdesorption electrospray ionization (DESI) and electrospray-assisted laser desorption/ ionization (ELDI)shave emerged to allow protein ions to be produced directly from the solid phase under ambient conditions.15–18 Unlike the situation for MALDI analysis, no organic matrix is required for either of these techniques, but drying of the sample solution prior to subjecting it to desorption/ionization is still

Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry combined with chromatographic and electrophoretic systems have become essential techniques for protein and peptide identification and for the study of protein structure and protein-protein interactions in proteomics.1–8 Nevertheless, obtaining good protein and peptide

(5) Englander, J. J.; Mar, C. D.; Li, W.; Englander, S. W.; Kim, J. S.; Stranz, D. D.; Hamuro, Y.; Woods, V. L., Jr Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 7057–7062. (6) Shevchenko, A.; Schaft, D.; Roguev, A.; Pijnappel, W. W. M. P.; Stewart, A. F.; Shevchenko, A. Mol. Cell. Proteomics 2002, 1, 204–212. (7) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64–71. (8) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37–70. (9) Loo, J. A.; Loo, R. R. O.; Udseth, H. R.; Edmonds, C. G.; Smith, R. D. Rapid Commun. Mass Spectrom. 1991, 5, 101–105. (10) Feng, R.; Konishi, Y. J. Am. Soc. Mass Spectrom. 1993, 4, 638–645. (11) Ramalingam, K.; Aimoto, S.; Bello, J. Biopolymers 1992, 32, 981–992. (12) Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299–2301. (13) Tanaka, K.; Waki, H.; Ido, Y.; Akita, S.; Yoshida, Y.; Yoshida, T.; Matsuo, T. Rapid Commun. Mass Spectrom. 1988, 2, 151–153. (14) Strupat, K.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1991, 111, 89–102. (15) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471–473. (16) Cooks, R. G.; Ouyang, Z.; Takats, Z.; Wiseman, J. M. Science 2006, 311, 1566–1670. (17) Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. Rapid Commun. Mass Spectrom. 2005, 19, 3701–3704. (18) Huang, M. Z.; Hsu, H. J.; Lee, J. Y.; Jeng, J.; Shiea, J. J. Proteome Res. 2006, 5, 1107–1116.

* To whom correspondence should be addressed. E-mail: jetea@ mail.nsysu.edu.tw. † Department of Chemistry, National Sun Yat-Sen University. ‡ National Sun Yat-Sen University-Kaohsiung Medical University Joint Research Center. § Graduate Institute of Medicine, Kaohsiung Medical University. | Institute of Biomedical Science, National Sun Yat-Sen University. (1) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466–469. (2) Ho, Y.; Gruhler, A.; Heilbut, A.; Bader, G. D.; Moore, L.; Adams, S. L.; Millar, A.; Taylor, P.; Bennett, K.; Boutilier, K. Nature 2002, 415, 180– 183. (3) Coon, J. J.; Ueberheide, B.; Syka, J. E. P.; Dryhurst, D. D.; Ausio, J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9463– 9468. (4) Maleknia, S. D.; Downard, K. M. Mass Spectrom. Rev. 2001, 20, 388–401. 10.1021/ac702108t CCC: $40.75  2008 American Chemical Society Published on Web 05/30/2008

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necessary; thus, some changes in the conformations of protein molecules could occur. An ideal approach toward avoiding the protein conformational changes caused by the presence of chemical additives during sample pretreatment and drying processes would be to desorb and ionize the protein molecules directly from liquid solutions without drying the sample. Herein, we report a novel ionization techniquesliquid electrospray laser desorption/ionization (liquid ELDI) mass spectrometrysthat allows native protein ions to be generated directly from aqueous solutions and biological fluids.19,20 In essence, liquid ELDI is a combination of two ionization methods: ELDI and surface-assisted laser desorption/ionization (SALDI).17,18,21–25 The possible mechanisms of ELDI and SALDI have been discussed previously.17,18,21,25 The ELDI technique combines ESI with laser desorption (LD): the particles, molecules, or clusters generated on the solid upon laser ablation at atmospheric pressure are entrained in the ESI plume to react with the charged solvent species to produce ESI-like mass spectra. Recently, the principle of ELDI has been adapted in the techniques, including matrix-assisted laser desorption electrospray ionization (MALDESI) and laser ablation electrospray ionization (LAESI).26–28 In SALDI, a sample solution is mixed with an “inorganic liquid matrix” comprising a suspension of micrometer-sized carbon powder in a mixture of glycerol, sucrose, and methanol.21,22 Because SALDI is performed under vacuum, the sample solution must be mixed with a viscous liquid matrix, such as the glycerol/ sucrose solution, to prevent the sample solution from drying in the source. It has been proposed that in SALDI, the carbon powder is the energy transfer medium that couples the laser UV energy into the molecules in the viscous liquid solution under vacuum.21,22 Although the mechanisms of desorption and ionization in liquid ELDI are not well-understood yet, on the basis of the obtained experimental results, the processes can be described as follows: a sample droplet containing inert particles is irradiated with a pulsed UV laser; the laser energy is adsorbed by the inert particles; subsequently, the energy is transferred to the surrounding solvent and analyte molecules for desorption; the desorbed molecules enter an ESI plume and are postionized through their reactions with the charged species to generate ESI-like ions (e.g., multiply charged protein ions). Figure 1a illustrates the possible desorption/ionization processes occurring during liquid ELDI. Figure 1b displays a photograph of the laser irradiation of a sample droplet. Unlike SALDI, the liquid ELDI technique is performed under ambient conditions; no viscous liquid matrix is necessary: the (19) Yuan, C. H.; Hung, W. C.; Chang, H. C.; Lin, H. Y.; Liu, J. J.; Shiea, J. Presented at the 5th HUPO Annual World Conference, Long Beach, CA, Oct 28-Nov 1, 2006; Poster W 203. (20) Yuan, C. H.; Ma, Y. L.; Shiea, J. Presented at the 55th SMS Conference on Mass Spectrometry, Indianapolis, IN, June 3-7, 2007; Poster TPA 18. (21) Sunner, J.; Dratz, E.; Chen, Y. Anal. Chem. 1995, 67, 4335–4342. (22) Kraft, P.; Alimpiev, S.; Dratz, E.; Sunner, J. J. Am. Soc. Mass Spectrom. 1998, 9, 912–924. (23) Chen, Y.; Shiea, J.; Sunner, J. J. Chromatogr., A 1998, 826, 77–86. (24) Han, M.; Sunner, J. J. Am. Soc. Mass Spectrom. 2000, 11, 644–649. (25) Dale, M. J.; Knochenmuss, R.; Zenobi, R. Anal. Chem. 1996, 68, 3321– 3329. (26) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. J. Am. Soc. Mass Spectrom. 2006, 17, 1712–1716. (27) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8098–8106. (28) Sampson, J. S.; Hawkridge, A. M.; Muddiman, D. C. Rapid Commun. Mass Spectrom. 2007, 21, 1150–1154.

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water molecules in the aqueous solution are used as the medium for transferring the adsorbed laser energy from the inert particles to the analyte molecules. This feature greatly simplifies the sample pretreatment process. Because organic solvent, acid, and reactive and acidic organic matrixes are not used in liquid ELDI, the conformation of the protein can be maintained. In this paper, we report the use of liquid ELDI MS for direct protein analyses from aqueous solutions and describe some of the parameters that affect the protein ion signals and protein structure. Instead of an indepth analysis of one system, herein, we present examples where liquid ELDI can be applied to the analysis of proteins in aqueous solutions and various biological fluids. EXPERIMENTAL SECTION The chemicals and protein standards, including insulin, lysozyme, cytochrome c, and myoglobin, and organic solvents (HPLC grade) were purchased from Sigma or Aldrich (Milwaukee, WI) and used without further purification. Carbon graphite powder was purchased from Merck (Darmstadt, Germany). The solution containing gold nanoparticles (Au NPs, 56 nm) was synthesized in our laboratory following procedures reported elsewhere.29,30 The biological fluids were obtained from healthy donors. The proteins in Escherichia coli were extracted ultrasonically in 70% acetonitrile solution containing 0.25% trifluoroacetic acid. The sample preparation process for liquid ELDI is extremely simple: merely suspend a small amount of the fine carbon powder in the sample solution and then subject it to liquid ELDI analysis without any further sample pretreatment. Commonly, a small amount of sample solution (5-10 µL) deposited on an acrylic sample plate [5 (L) × 2 (W) cm2] using a micropipette was sufficient for complete liquid ELDI analysis. The sample plate was positioned on an acrylic plate placed on an XYZstage, which was set in front of the sampling capillary of an ion trap mass spectrometer. The sample droplet was then exposed to a pulsed nitrogen laser operating at a wavelength of 337 nm, a frequency of 10 Hz (controlled by a sweep function generator), a pulsed energy of ca. 150 µJ, and length of 4 ns. The laser beam (spot size: ca. 100 µm × 150 µm) was focused through an objective lens. The strongest ion signal (using myoglobin as the standard for evaluation) was obtained at an incident laser angle of ca. 45° and a focal length of ca. 25 cm. The laser-ablated molecules were postionized in the electrospray plume. The configuration of the ESI source for liquid ELDI is similar to those used in ELDI, pyrolysis/ESI, and fused-droplet electrospray ionization (FD-ESI) sources.31–36 The capillary electrosprayer was aligned parallel to the acrylic sample plate and placed ca. 3.5 mm above the sample plate. The ESI plume was directed toward the ion sampling orifice (i.e., parallel to the sample plate) (Figure 1a). The electrospray needle and the sample plate potentials were both held at 0 V (grounded); the sampling cone (29) Neiman, B.; Grushka, E.; Lev, O. Anal. Chem. 2001, 73, 5220–5227. (30) Huang, Y. F.; Chang, H. T. Anal. Chem. 2006, 78, 1485–1493. (31) Lee, C. C.; Chang, D. Y.; Jeng, J.; Shiea, J. J. Mass Spectrom. 2002, 37, 115–117. (32) Shiea, J.; Chang, D. Y.; Lin, C. H.; Jiang, S. J. Anal. Chem. 2001, 73, 4983– 4987. (33) Shiea, J.; Wang, C. H. J. Mass Spectrom. 1997, 32, 247–250. (34) Lee, C. Y.; Shiea, J. Anal. Chem. 1998, 70, 2757–2761. (35) Shiea, J.; Hong, C. M.; Tsai, F. C. Anal. Chem. 2000, 72, 1175–1178. (36) Hsu, H. J.; Kuo, T. L.; Wu, S. H.; Oung, J. N.; Shiea, J. Anal. Chem. 2005, 77, 7744–7749.

Figure 1. (a) Illustration of the possible desorption/ionization processes that occur during liquid ELDI. (b) Photograph of the surface of a sample droplet irradiated using a UV laser pulse in the liquid ELDI source. The photograph was taken by a Nikon D70 camera (exposure time: 3 s, with no additional illumination).

voltage in the ion trap mass spectrometer was maintained at -4.5 kV. The electrospray solutions comprised methanol and water mixtures with and without acetic acid. A typical composition of the ESI solution used in liquid ELDI analysis was H2O/MeOH (80%, v/v). The ESI solution was delivered through a capillary by a syringe pump at a flow rate of 120 µL/h. A nebulizing gas, commonly used in conventional ESI sources, was not used during liquid ELDI. The ions generated in this manner were further analyzed using a quadrupole/time-of-flight mass analyzer (Bruker Dalton Bio-TOF-q, Billerica, MA). The mass spectra were recorded at a scan rate of ca. 2 s/scan (i.e., 20 laser shots/scan), and five mass spectra were averaged for the presenting results. RESULTS AND DISCUSSION To study the effect of the inert particles on the desorption and ionization of protein ions in aqueous solution in liquid ELDI, we prepared a series of the aqueous protein solutions suspended with various amounts of carbon powder and then analyzed them using a water/methanol (80/20, v/v) solution as the ESI solution. Figure 2 displays the ELDI mass spectra obtained from small amount (ca. 10 µL) of the myoglobin solutions (10-4 M). Direct analysis of the sample solution without the addition of carbon powder indicated that the solution was transparent to the UV laser light: no myoglobin ion signal was detected (Figure 2a). Only after 0.2 mg/mL or more of the carbon powder was added into the sample solution did we observe the signals of the myoglobin ion (Figure

2b-e). The intensity of the myoglobin ion signals increased upon increasing the amount of carbon powder added in the sample solution, reaching a plateau when the concentration of carbon powder reached 0.6 mg/mL. The detection of the myoglobin ion signal only in the presence of carbon powder suggested that the laser energy adsorbed by the carbon powders led to the desorption of the protein molecules. The observation of the calculated molecular weight (m/z 17 567) and charge distribution (+9 to +15) of the detected myoglobin ions in the ELDI spectra suggested that native myoglobin ions were detected; only a trace of a signal for the apomyoglobin ion (m/z 16 951) was detected (Figure 2). The respective deconvoluted ELDI spectra in Figure 2b-e further confirm our hypothesis. Because of the inertness of the carbon powder, although no experiment is performed, it is rational to suspect that the presence of the carbon powder in the aqueous sample solution shall not affect the protein’s structure. This situation is unlike those occurring in the acidic organic matrixes used for MALDI. In addition, because the protein molecules were desorbed directly from the aqueous solution under ambient conditions, no protein denaturation occurred as a result of drying. It is possible that carbon powder may provide a surface that the protein may interact with yielding an influence on the protein conformation; the results shown in Figure 2 suggested that denaturation of myoglobin had not occurred. The combination of Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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Figure 2. Positive ELDI mass spectra of an aqueous myoglobin droplet (10-4 M, 10 µL) containing carbon powder at concentrations of (a) 0, (b) 0.2, (c) 0.4, (d) 0.6, and (e) 0.8 mg/mL (b, myoglobin; O, apomyoglobin). The insets display the respective deconvoluted mass spectra of the myoglobin ions.

carbon powder-assisted laser desorption and ESI postionization in the liquid ELDI technique is a soft process, as evidenced by the mass spectra of the native myoglobin ions in this aqueous solution. Because liquid ELDI is performed under ambient conditions, evaporation of the sample solution is slow and denaturation of protein ion usually will not occur as a result of drying of the sample solution during analysis. We did observe, however, that when the sample droplet approached dryness ca. 6 min after it had been deposited on the sample plate, both native and denatured myoglobin ion signals appeared in the spectra. Figure 3 displays the changes that occurred to the myoglobin ion signals upon decreasing the sample droplet volume through evaporation. The insets in Figure 3 present the deconvoluted mass spectra of the myoglobin ions. As the volume of the sample droplet decreased, more protein molecules condensed on the surface of the carbon powder. Direct laser energy input onto the carbon powder featuring adsorbed protein molecules would then induce protein denaturation and fragmentation during desorption. Previously, we reported that the protein ion signals arising from FD-ESI are strongly influenced by the composition of the ESI solutionseven when the time during which the desorbed protein molecules may interact with the charged species generated in the ESI plume is short.31 In this study, we observed similar phenomena upon changing the composition of the ESI solution. Figure 4 displays the change in the structure of the myoglobin ion with respect to the composition of the ESI solution. Denatured myoglobin ion signals were obtained when pure methanol was used as the ESI solution (Figure 4b). When using a water/ methanol (80/20) solution containing 0.1% acetic acid as the ESI solution, we detected signals for both the native and denatured

Figure 3. Positive ELDI mass spectra displaying the changes in the signals of myoglobin ions upon decreasing the sample droplet volume through the effects of evaporation and laser ablation (b, myoglobin; O, apomyoglobin). The mass spectra were recorded at (a) 0-0.2, (b) 6.4-6.6, and (c) 7.0-7.2 min after the sample droplet (10-4 M) had been deposited on the sample plate. The sample droplet was completely depleted after 7.5 min. The insets display the respective deconvoluted mass spectra of the myoglobin ions. 4848

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Figure 4. Positive ELDI mass spectra of myoglobin solutions (10-4 M) obtained using (a) water/methanol (80/20, v/v), (b) methanol, and (c) water/methanol (80/20, v/v) containing 0.1% acetic acid as electrospray solutions for postionization (b, myoglobin; O, apomyoglobin).

myoglobin ions (Figure 4c). However, when using a water/ methanol solution (80/20 by volume) as the ESI solution for liquid ELDI (Figure 4a), the major ions are from native myoglobin. These results reveal that although the reaction time for ionization between the myoglobin molecules and the charged species may be short, the structure of the protein is still affected by the presence of acid and organic solvent in the ESI solution. We examined other inert materials, such as gold nanoparticles (Au NPs), to see if they could also be used as matrixes for protein analysis in liquid ELDI. The sample was prepared by dissolving the myoglobin protein standard in a solution containing Au NPs (56 nm; 50 pM). Figure 5a displays the corresponding ELDI spectrum; the structure of the detected myoglobin ions was nearly entirely in the denatured form. Because citric acid was used to synthesize the Au NPs, we suspect that the conformation of the myoglobin ions was altered by its presence in the Au NP solution. We also examined whether the UV-absorbing organic matrixes [including sinnapinic acid (SA), R-cyano-4-hydroxycinnamic acid (R-CHC), and 2,5-dihydroxybenzoic acid (2,5-DHB)] used for MALDI analysis could be employed as matrixes in liquid ELDI. We only detected weak myoglobin ion signals from the sample solution containing SA and R-CHC (4 mg/mL; Figure 5, parts c and d). We did, however, detect strong ion signals of the denatured myoglobin ions when the sample solution reached neardryness and the SA molecules seemed to be precipitated out (inset to Figure 5c). We observed similar results when using R-CHC as the matrix (Figure 5d and its inset). These results indicate that although the SA molecules absorbed the UV laser energy, there was insufficient energy to desorb the protein molecules in the solution. Because the solubility of 2,5-DHB in water is much higher than that of SA and R-CHC, we detected signals for myoglobin ions only when the solution containing 2,5-DHB was completely dry (Figure 5b and its inset). Because all of these MALDI matrixes are innately acidic, the myoglobin ions obtained were denatured. Because sample pretreatment for liquid ELDI analysis is extremely simple, one application of liquid ELDI is the rapid determination of the predominant proteins present in biological

Figure 5. Positive ELDI mass spectra of the myoglobin solutions (10-4 M) containing (a) Au NPs (56 nm), (b) 2,5-DHB, (c) SA, and (d) R-CHC. No signals for myoglobin ions were detected in the solutions containing SA, R-CHC, or 2,5-DHB; they were observed only when the sample solution was nearly or completely dry (see the insets to panels b-d).

fluids. Figures 6 and 7 display the ELDI spectra recorded from human tears, whole cow milk, human serum, and bacterial extracts; for comparison, conventional ESI and MALDI mass spectra of these biological fluids are also presented. One of the problems of direct analysis of biological fluids using conventional ESI MS or MALDI MS is the interference caused by the high concentrations of alkali salts present in most biological fluids. Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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Figure 6. Positive ELDI mass spectra of human tears and whole cow milk; for comparison, conventional ESI and MALDI mass spectra of these biological fluids are also presented. The insets to panels a, c, d, and f display deconvoluted protein ion signals of the ESI and ELDI mass spectra. We detected three predominant protein ions in the human tears analyzed using liquid ELDI MS (c). We obtained exact masses of the molecular ions of lysozyme (m/z 14 695), lipocalin-1 (m/z 17 442), and an unknown protein (m/z 16 872, not shown in panel c), calculated directly from the ion peaks in the ELDI mass spectra, and identified the former two proteins through databank searching (Swiss Prot). Interestingly, direct analysis of the whole cow milk sample using ESI and MALDI mass spectrometry provided only lipid-like small ions and protein ion signals, respectively; in contrast, both sets of ions appeared in the ELDI mass spectrum.

Figure 7. Positive ELDI spectra of human serum and bacterial extracts; for comparison, conventional ESI and MALDI mass spectra of these biological fluids are also presented. The insets to panels a, c, d, and f display deconvoluted protein ion signals of the ESI and ELDI mass spectra. The ELDI mass spectrum of human serum was predominated by the ions of the most abundant proteins: albumin (m/z 66 567) and apolipoprotein A1 (m/z 28 082).

Their mass spectra will feature the predominant protein-sodium and protein-potassium adducts ions if the samples are not subjected to desalting prior to ESI MS (cf., Figure 6, parts a and d, and Figure 7, parts a and d) and MALDI MS analyses (cf., Figure 6, parts b and e, and Figure 7, parts b and e). With the use of liquid ELDI, however, we observed that even though the salt contents were high in these biological fluids, all of the protein ions obtained were in their protonated forms; that is, we detected no sodiated or potassiated protein ion adducts (see Figure 6, parts c and f, and Figure 7, parts c, and f, and their insets). Accurate molecular weights of the protein molecules could be calculated 4850

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from the ions in the ELDI spectra. We have observed similar phenomena from previous studies using ESI to postionize myoglobin molecules generated by nebulizing the protein solution with 1 M NaCl.31,37–40 We suggest that the desalting effect of the protein ions might be due to the low solubility of sodium and

(37) (38) (39) (40)

Shieh, I. F.; Lee, C. Y.; Shiea, J. J. Proteome Res. 2005, 4, 606–612. Lee, C. C.; Chang, D. Y.; Shiea, J. Anal. Chem. 2002, 74, 2465–2469. Chen, H.; Venter, A.; Cooks, R. G. Chem. Commun. 2006, 2042–2044. Chen, H.; Wortmann, A.; Zhang, W.; Zenobi, R. Angew Chem., Int. Ed. 2007, 46, 580–583.

Figure 8. (a) Positive ELDI mass spectrum of human whole blood (g, HBA-R; f, HBA1c-R; 3, HBA-β; 1, HBA1c-β). The inset displays the deconvoluted protein ion signals (A, peak area) of the ELDI mass spectra. (b) Ratio of the peak area of glycosylated to nonglycosylated + glycosylated R-hemoglobin ion signal [Af/(Af + Ag)] using ELDI plotted with respect to the absolute amount of glycosylated hemoglobin determined through conventional spectroscopic measurement of single drops of whole blood obtained from 20 diabetic patients.

potassium ions in the charged droplets (comprising water and methanol) when the protein molecules were postionized in the ESI plume. We detected at least three predominant protein ions in the human tear sample analyzed using liquid ELDI MS (Figure 6c). Without the interference of alkali adduct ions, we calculated the exact molecular weights of two of these proteins from the ELDI spectra and identified them through data bank searching (Swiss Prot) using the top-down strategy (m/z 14 695 for lysozyme; m/z

17 442 for lipocalin-1). Interestingly, direct analysis of the whole cow milk sample using ESI MS and MALDI-TOF gave only lipidlike and protein ion signals, respectively (Figure 6, parts d and e); both sets of these ions were detectable in the ELDI spectrum (Figure 6f). The ELDI mass spectrum of human serum (Figure 7c) featured predominant ions from the most abundant proteins: albumin (m/z 66 567) and apolipoprotein A1 (m/z 28 082). Figure 7f displays the results of direct liquid ELDI analysis of the protein extracts from an E. coli culture. Strong and stable signals were obtained for the ions of four major proteins; Figure 7f displays their calculated accurate molecular masses. We successfully identified three of the bacterial proteins through Swiss Prot data bank searching based on these accurate molecular masses. Thus, even though we performed no prior sample cleanup, interference from salt and other chemicals in the cultural broth was avoided in the liquid ELDI MS analysis. The extremely high salt tolerance for the analysis of predominant proteins in biological fluids suggested that it would be possible to use liquid ELDI MS to rapidly detect glycosylated hemoglobin directly from samples of whole blood. The level of glycosylated hemoglobin in whole blood is currently the most important measure of long-term control of the glycermic state of diabetes. As a result of the interference of high concentrations of salts in whole blood, tedious sample cleanup procedures must be performed prior to subjecting the sample solutions to conventional LC/MS and MALDI analyses. In this study, we found that the direct analysis of one drop of whole blood using liquid ELDI resulted in the observation of only the signals for multiply protonated hemoglobin and glycosylated hemoglobin ions (Figure 8a); we detected no sodiated or potassiated hemoglobin and glycosylated hemoglobin ions. Quantification of the levels of glycosylated hemoglobin in the whole blood of a diabetes patient was possible by calculating the ratio of the peak intensities of the

Figure 9. (a) Positive ELDI mass spectrum obtained from a small drop (ca. 5 µL) of an aqueous solution containing a mixture of four protein standards ((, insulin; 9, cytochrome c; 2, lysozyme; b, myoglobin; O, apomyoglobin) with a mole ratio of 1:2:5:10 (insulin/cytochrome c/lysozyme/ myoglobin). The addition of a small amount of carbon powder (g4 mg/mL) was necessary to obtain the protein ion signals. (b) Positive ESI mass spectrum of the protein solution in (a); (c) positive MALDI mass spectrum of the protein solution in (a). The insets in (a) and (b) display the deconvoluted protein ion signals. Analytical Chemistry, Vol. 80, No. 13, July 1, 2008

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glycosylated and nonglycosylated hemoglobin ions. Figure 8b indicates that a linear relationship exists between the concentrations of glycosylated hemoglobin analyzed from blood samples collected from 20 diabetic patients using liquid ELDI with those determined through conventional spectroscopic measurement. Because the protein molecules were desorbed directly from the aqueous solution, we expected that the intensities of the detected protein ion signals might reflect their concentrations in the solution. Figure 9 displays ELDI mass spectra obtained from a small drop (ca. 5 µL) of an aqueous solution containing a mixture of four protein standards (insulin, cytochrome c, lysozyme, and myoglobin; mole ratio, 1:2:5:10; the actual concentration of insulin is 2.7 × 10-5 M). The signals for the protein ions in the deconvoluted mass spectrum (inset in Figure 9a) correspond well with the concentrations of each protein standard in the solution. This situation differs from those obtained using conventional ESI and MALDI analyses (Figure 9, parts b and c).

biological fluids under ambient conditions. Soft ionization was achieved by using a water dominant ESI solution without acid. Commonly, a small amount of the sample solution (5-10 µL) was sufficient for complete liquid ELDI MS analysis. The presence of fine particles of carbon powder or Au NPs in the sample solution is required for the desorption of the protein molecules upon irradiating their sample solutions with a nitrogen laser. The signals of protein ions in ELDI spectra also correspond well with their concentrations in protein standard solutions. Other analytical merits for the method include the following: sample pretreatment for liquid ELDI MS is extremely simple; analysis is performed under ambient conditions, allowing rapid characterization of the predominant proteins present in a biological fluid; sample switch is also rapid. The extremely high salt tolerance for protein analysis also makes it possible to use liquid ELDI MS to rapidly quantify the levels of glycosylated hemoglobin in whole blood samples.

CONCLUSION We have demonstrated that liquid ELDI can be used to directly desorb and ionize native proteins in aqueous solutions and in

Received for review October 13, 2007. Accepted April 29, 2008.

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