Detection of biomolecules from solutions with high concentration of salts using probe electrospray and nano-electrospray ionization mass spectrometry

Abstract

Probe electrospray ionization (PESI) is a recently developed ionization technique which uses a solid needle or wire as sampling probe and ESI emitter instead of capillary. PESI is free from the clogging problem and it has high tolerance to salts and urea. We present herein a comparative study of the probe electrospray ionization (PESI) and nano-electrospray ionization (nano-ESI) for the measurement of biomolecules in the sample solutions with high concentrations of salts and urea. Our results show that PESI could provide equivalent ionization performance with nano-ESI, and in certain cases it could be superior to nano-ESI for the samples with high concentration (>100 mM) of salts and urea. Therefore, PESI can be useful for the direct analysis of cell/tissue extracts and protein digestion without or reduced procedures in sample purification.

---

Introduction

Over the decades electrospray ionization mass spectrometry (ESI-MS) has been a powerful tool for quantitative and qualitative analyses of biomolecules in many biological research fields.^1,2

Biological samples usually contain high concentration of salts and the removal of salts and the related sample purification processes play a critical role in successful proteomics analysis with mass spectrometry. Multilayer gel electrophoresis and polyacrylamide gel electrophoresis (PAGE) are usually used for this purpose.^3,4 Other de-salting methods include gel cartridge based inline chromatography,⁵ nanoparticle based microextraction, and microdialysis.^6,7

Urea is widely used as denaturant for protein digestion,⁸ but its elimination is crucial before mass spectrometry analysis for successful sequence coverage of proteins or peptides. HPLC (high performance liquid chromatography),⁹ and PAGE based separation are commonly used for urea removal from samples. Though online/inline desalting and urea purification approaches are proved to be successful, they may produce memory effect and plug the membrane frequently.

Several ionization methods have been developed which allow the direct analysis of native sample without purification step. For example in 2002, Shiea's group reported a method called fused-droplet electrospray ionization (FD-ESI), and by mixing charged acidic methanol droplets with sample aerosol, they successfully detected the analytes from the concentrated NaCl and sodium phosphate solution.¹⁰ Another similar method, extractive electrospray ionization, developed by Chen and co-workers also allows the mass spectrometric analysis to be performed without the tedious clean-up steps for salts or other contaminants.^11,12

Micro- and nano-electrospray ion sources using very fine capillaries have been applied to the analysis of small amounts of samples.^13–15 Due to the much reduced flow rate, nano-electrospray produces much finer droplets compared to the standard electrospray. Owing to the high surface charge density of the initial charged droplets, nano-electrospray had also been theoretically and experimentally proven to have high tolerance towards high concentration of salts present in the sample solution.¹⁶

Besides using capillary, electrospray ionization of biomaterials can also be realized by simply picking up the sample (liquid or bio-fluid) with a solid needle probe and electrospray it directly. Non-capillary based ESI had previously been used by Shiea and co-workers by depositing the sample solution to various types of solid ESI emitters.^20–23 Instead of continuous operation, the ion source, which we refer as probe electrospray ionization (PESI) is performed in a repetitive pick-and-spray process. When the fine needle is used, electrospraying condition and the ionization efficiency are equivalent to those of nano-electrospray that has a high tolerance to inorganic salts.^17–19 Compared to the capillary, solid needle is completely free from clogging, much cheaper to fabricate and commercially available in bulk for disposable use.

The objective of this paper is to make the comparative study of nano-electrospray and probe electrospray on the effect of salts and urea for the detection of proteins and peptides. Tolerance to high salt concentrations and resultant high throughput analysis of analytes for “salty” solutions are advantageous, because (i) sodium chloride is usually present in the native sample, (ii) phosphate buffer is used for cell culture washing and re-suspension of cells, and (iii) urea is commonly used for protein denaturation prior to digestion analysis.

Experimental

The PESI experimental procedures were similar to those described in our previous papers.^17–19 Briefly, the needle was moved up and down along a vertical axis using a custom made linear actuator system. When the needle was at the bottom position, the tip of the needle was adjusted to touch the surface of the liquid sample. When the needle or wire was moved up to the highest position, a high voltage of about 2–3 kV was applied to it. The distance of the needle stroke was 10 mm. As electrospray emitters, disposable acupuncture needles (Seirin, Shizuoka, Japan) with sub-micromere tip diameter, titanium wire (Nilaco Corporation, Japan) with 100 µm diameter, and etched tungsten wire (Nilaco Corporation, Japan) with 30 nanometre tip diameter were used throughout the PESI-MS experiments. The tungsten needle was fabricated in the laboratory with anodic etching using 30% (w/v) potassium hydroxide (KOH). The PESI mass spectra were obtained with an acquisition time of 1 s with 3 Hz of the probe motion.

The nano-ESI emitters (EconoTip™, New Objectives, USA) with inner diameter of 1 µm were used for nano-ESI experiments. The sample was loaded to the emitter by gel loading pipette (Eppendorf, Germany). The emitter was positioned axially towards the ion sampling orifice of the mass spectrometer with the distance of 3 mm. No syringe pump was used. The liquid sample was electrosprayed spontaneously when high voltage (0.5–2.5 kV) was applied to the tip. The tips of the nano-ESI capillaries were found to be easily plugged when sample solutions with high concentrations of salts/urea were used, and when that happened, the diameter of the tip was slightly enlarged by touching the tip with the counter electrode or glass plate to break away the plugged part of the tip. The ions generated from the electrospray (either by PESI or nano-ESI) were sampled through the ion-sampling orifice with a diameter of 0.4 mm into the vacuum chamber and mass analyzed by an orthogonal time-of-flight mass spectrometer (AccuTOF; JEOL, Akishima, Japan).

Sample preparation

Horse heart myoglobin, gramicidin S, leu-enkephalin (Tyr-D-Ala-Gly-Phe-Leu), urea, potassium dihydrogen phosphate (KH₂PO₄), dipotassium hydrogen phosphate (K₂HPO₄), sodium chloride (NaCl), acetic acid (HAc) and methanol (MeOH) were purchased from Sigma. Solvents and buffer solutions were of HPLC grade, and were used without further purification. Pure water was prepared using a Milli-Q system (Millipore, USA). Phosphate buffer was prepared by using equal amount of KH₂PO₄ and K₂HPO₄ in pure water. Stock solutions of proteins and peptides were initially prepared in pure water. The proteins and peptides were then added to the 25% (v/v) methanolic aqueous solution with different concentrations of urea, NaCl and phosphate buffer. The sample solutions with phosphate buffer or NaCl contained 1% (v/v) HAc. The sample in the urea solutions was incubated for at least two hours in room temperature before mass spectrometric measurement.

Results and discussion

Fig. 1(a–c) show the scanning electron microscopy images of three different ESI probes used in this study: etched tungsten needle (a), stainless steel (b) and titanium wire (c). The corresponding mass spectra of gramicidin S (in 1 M NaCl) and myoglobin (in 150 mM NaCl solution) obtained with these PESI probes are depicted in Fig. 1(d–f) and (g–i) respectively. It is found that tungsten needle has better performance than acupuncture needle and titanium wire for the detection of gramicidin S from NaCl solutions (Fig. 1(d–f)). Alternatively titanium wire showed better performance than acupuncture needle and tungsten needle for detection of myoglobin from NaCl (Fig. 1(g–i)). Similar results were also observed for the samples in phosphate buffer solutions. However, in the case of urea solution, these three types of PESI probes produced nearly equivalent results for both myoglobin and gramicidin S. Thus in the following experiments, except for the case of urea solution, titanium wire and tungsten needle were used for myoglobin and gramicidin S/enkephalin detection, respectively, from NaCl and phosphate buffer solutions.

Fig. 1 SEM images of (a) tungsten needle, (b) acupuncture needle (stainless steel) and (c) titanium wire. Positive ion PESI mass spectra of 10⁻⁵ M gramicidin S in the solution that contains 25% (v/v) MeOH, 1% (v/v) HAc and 1 M NaCl, obtained by tungsten needle (d), acupuncture needle (e) and titanium wire (f). Positive ion PESI mass spectra of 10⁻⁵ M myoglobin in the solution that contains 25% (v/v) MeOH, 1% (v/v) HAc and 150 mM NaCl, obtained by tungsten needle (g), acupuncture needle (h) and titanium wire (i).

Fig. 2 shows the comparison of PESI-MS (using stainless steel acupuncture needle) and nano-ESI-MS for the detection of myoglobin in different concentrations of urea. Mass spectra in Fig. 2(a–d) are obtained by PESI-MS and Fig. 2(e–h) are the corresponding nano-ESI mass spectra. Remarkably PESI can detect the ion signal of myoglobin from the solution with 4 M or higher concentrations of urea (see Fig. 2(d)), whereas the detection limit for nano-ESI is 2 M (see Fig. 2(g)) with slight breaking of the emitter's tip. Similar results were also obtained in the case of enkephalin and gramicidin S (data not shown).

Fig. 2 (a–d) Positive ion PESI mass spectra of 10⁻⁵ M myoglobin in different concentrations of urea. (e–h) Positive ion nano-ESI mass spectra of 10⁻⁵ M myoglobin in different concentrations of urea. Stainless steel acupuncture needle was used as the PESI probe. Peaks labeled with asterisk (*) are those from holo-myoglobin. In (h), no mass spectrum could be obtained using nano-ESI-MS due to the severe plugging of the emitter's tip.

Fig. 3 depicts the positive ion mass spectra of myoglobin with varying amounts of NaCl obtained by PESI-MS (Fig. 3(a–d)) and nano-ESI-MS (Fig. 3(e–h)). As the concentration of NaCl increases, intensities of sodium adduct signals also increase for both PESI-MS and nano-ESI-MS. As shown in Fig. 3(c), PESI could detect the ion signal of myoglobin from the NaCl solution up to 250 mM or higher. When the NaCl concentration exceeded 50 mM, the nano-ESI produces only salt cluster signals without any peaks from myoglobin (Fig. 3(g)). The reason could be that while nano-ESI sprays whatever it has inside the capillary, PESI that uses a solid needle probe can selectively sample and detect the analyte from the concentrated salt buffers.

Fig. 3 (a–d) Positive ion PESI mass spectra (using titanium wire) of 10⁻⁵ M myoglobin in different concentrations of NaCl. (e–h) Positive ion nano-ESI mass spectra of 10⁻⁵ M myoglobin in different concentrations of NaCl. Insets show the expanded mass spectra of (d) and (h).

Fig. 4 depicts the positive ion mass spectra of myoglobin obtained by PESI-MS and nano-ESI-MS as a function of potassium phosphate concentration. Phosphate buffer is commonly used for the preparation of biological samples, however, to retrieve good analyte ion signals from the phosphate buffer without any sample purification, it is more challenging for both PESI-MS and nano-ESI-MS compared to the sample in urea and NaCl solutions. Ion suppression effect also seems to be more severe compared to other salts. Nevertheless, PESI-MS appeared to be more tolerant to the phosphate salts compared to nano-ESI, and the ion signal from heme and myoglobin remained detectable even when the concentration of phosphate salts was increased to 250 mM. In nano-ESI-MS when the concentration of phosphate buffer was increased higher than 50 mM, only complex salt clusters of [(K₂HO₄)_m(KH₂PO₄)_n + K]⁺, [(K₂HO₄)_m(KH₂PO₄)_n + H]⁺, [K₃PO₄ + K]⁺ and [K₃PO₄ + H]⁺ were detectable, but ions from myoglobin and heme were not observed. The heme of myoglobin is still observable with 500 mM of NaCl and phosphate buffer (Fig. 3d and 4d) in PESI-MS experiments.

Fig. 4 (a–d) Positive ion PESI mass spectra (using titanium wire) of 10⁻⁵ M myoglobin in varying concentrations of potassium phosphate buffer. (e–h) Positive ion nano-ESI mass spectra of 10⁻⁵ M myoglobin in varying concentrations of potassium phosphate buffer. Peaks labeled with asterisk (*) are originated from the phosphate buffer. Insets show the expanded mass spectra of (d) and (h).

Fig. 5 illustrates the results for 10⁻⁵ M gramicidin S in NaCl and phosphate buffer solutions obtained by PESI-MS using tungsten needle and nano-ESI-MS. In the case of pure gramicidin S, PESI-MS and nano-ESI-MS showed similar results as shown in Fig. 5(a) and (e). Similar to protein, for high concentration of NaCl and phosphate buffer, PESI produced better mass spectra with weaker ion signals originated from salt clusters. Compared to myoglobin, gramicidin S is more hydrophobic (i.e., surface-active), and thus, the analyte ions are relatively easy to be released from the electrosprayed charged droplets to the gas phase. Good PESI mass spectra could still be obtained even with the presence of 1 M of salts in the sample solutions (see Fig. 1(d–f) and 5(c)). In Fig. 5(g), no mass spectrum could be obtained using nano-ESI-MS because the tips of nano-ESI capillaries were plugged easily when 1 M phosphate solutions were dealt with.

Fig. 5 Positive ion PESI mass spectra (using tungsten needle) of 10⁻⁵ M gramicidin S in different concentrations of potassium phosphate buffer (a–c) and 250 mM of NaCl (d). Positive ion nano-ESI mass spectra of 10⁻⁵ M gramicidin S in different concentrations of potassium phosphate buffer (e–g) and 250 mM of NaCl (h). Peaks labeled with asterisk (*) depict the ions from the phosphate buffer. In (g), no mass spectrum could be obtained using nano-ESI-MS due to the severe plugging of the emitter's tip.

Conclusion

Because of the intrinsic plugging problem of nano-ESI experiments, patience and some skill are needed to take good spectra from salts or urea contaminated samples. In some cases when dealt with the buffer solution with >1 M concentrations, we were forced to break the emitter tips a little bit to reduce the frequent plugging problem. This could somehow affect the reproducibility of nano-ESI spectra because the spray tips and handing process are responsible for varying results obtained by nano-ESI.²⁴ The results obtained before and after breaking the tips would be astonishingly different because the initial droplets, current and field strength of the tip diameters would change.²⁵

On the other hand, we found that it is much easier to obtain better mass spectra from these “dirty” samples using PESI with good reproducibility. Because solid needles are easier to fabricate compare to delicate capillaries, it is also more cost effective to use the solid ESI emitter in the disposable basis.

In summary, PESI-MS offers some fundamental properties that favour its use for biomolecules analysis owing to its high tolerances to salt/urea buffers, the rapid analysis, and the small amount of sample consumption like nano-ESI. Optimization with different types of materials for the PESI emitters has also shown to be effective in improving the PESI mass spectra, possibly due to the improved inertness of the needle emitter surface toward the electrochemical process during the electrospray process. Potential applications of PESI include the direct analysis of protein from salts/urea contaminated solutions, the high throughput analysis of cell/tissue extraction and digestion products, etc.

Acknowledgements

The authors acknowledge the financial supports for this work by the Grants-in-Aid for Scientific Research (S) and Development of System and Technology for Advanced Measurement and Analysis Program (SENTAN) from Japan Science and Technology Agency (JST).

References

1. J. Fenn, M. Mann, C. Meng, S. Wong and C. Whitehouse, Science, 1989, 246, 64–71.
2. J. B. Fenn, Int. J. Mass Spectrom., 2000, 200, 459–478.
3. T. Liu, A. M. Martin, A. P. Sinai and B. C. Lynn, J. Proteome Res., 2008, 7, 4256–4265.
4. P. Tantipaiboonwong, S. Sinchaikul, S. Sriyam, S. Phutrakul and S. Chen, Proteomics, 2005, 5, 1140–1149.
5. J. Cavanagh, L. M. Benson, R. Thompson and S. Naylor, Anal. Chem., 2003, 75, 3281–3286.
6. K. Shrivas and H. Wu, Anal. Chem., 2008, 80, 2583–2589.
7. C. Liu, S. A. Hofstadler, J. A. Bresson, H. R. Udseth, T. Tsukuda, R. D. Smith and A. P. Snyder, Anal. Chem., 1998, 70, 1797–1801.
8. L. Wang and W. Colón, Protein Sci., 2005, 14, 1811–1817.
9. N. Nagaraj, A. Lu, M. Mann and J. R. Wiśniewski, J. Proteome Res., 2008, 7, 5028–5032.
10. D. Chang, C. Lee and J. Shiea, Anal. Chem., 2002, 74, 2465–2469.
11. H. Chen, A. Venter and R. G. Cooks, Chem. Commun., 2006, 2042.
12. H. Chen, S. Yang, M. Li, B. Hu, J. Li and J. Wang, Angew. Chem., Int. Ed., 2010, 49, 3053–3056.
13. M. R. Emmett and R. M. Caprioli, J. Am. Soc. Mass Spectrom., 1994, 5, 605–613.
14. M. S. Wilm and M. Mann, Int. J. Mass Spectrom. Ion Processes, 1994, 136, 167–180.
15. M. Wilm and M. Mann, Anal. Chem., 1996, 68, 1–8.
16. R. Juraschek, T. Dülcks and M. Karas, J. Am. Soc. Mass Spectrom., 1999, 10, 300–308.
17. K. Hiraoka, K. Nishidate, K. Mori, D. Asakawa and S. Suzuki, Rapid Commun. Mass Spectrom., 2007, 21, 3139–3144.
18. L. C. Chen, K. Nishidate, Y. Saito, K. Mori, D. Asakawa, S. Takeda, T. Kubota, H. Hori and K. Hiraoka, J. Phys. Chem. B, 2008, 112, 11164–11170.
19. L. C. Chen, K. Yoshimura, Z. Yu, R. Iwata, H. Ito, H. Suzuki, K. Mori, O. Ariyada, S. Takeda, T. Kubota and K. Hiraoka, J. Mass Spectrom., 2009, 44, 1469–1477.
20. C. Hong, C. Lee, Y. Lee, C. Kuo, C. Yuan and J. Shiea, Rapid Commun. Mass Spectrom., 1999, 13, 21–25.
21. C. Kuo, C. Yuan and J. Shiea, J. Am. Soc. Mass Spectrom., 2000, 11, 464–467.
22. J. Jeng and J. Shiea, Rapid Commun. Mass Spectrom., 2003, 17, 1709–1713.
23. J. Jeng, C. Lin and J. Shiea, Anal. Chem., 2005, 77, 8170–8173.
24. I. V. Chernushevich, U. Bahr and M. Karas, Rapid Commun. Mass Spectrom., 2004, 18, 2479–2485.
25. A. Schmidt, M. Karas and T. Dülcks, J. Am. Soc. Mass Spectrom., 2003, 14, 492–500.

---