Gold-Coated Nanoelectrospray Emitters Fabricated by Gravity

Nov 9, 2016 - Via gravity-assisted etching self-termination, the emitter with a tapered outer surface and a straight inner surface is prepared with go...
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Gold Coated Nanoelectrospray Emitters Fabricated by Gravity Assisted Etching Self-termination and Electroless Deposition Xudong Zhu, Yu Liang, Yejing Weng, Yuanbo Chen, Hao Jiang, Lihua Zhang, Zhen Liang, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03422 • Publication Date (Web): 09 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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Analytical Chemistry

Gold Coated Nanoelectrospray Emitters Fabricated by Gravity Assisted Etching Self-termination and Electroless Deposition Xudong Zhu1,2,#, Yu Liang1,#, Yejing Weng1,2, Yuanbo Chen1,2, Hao Jiang1,2, Lihua Zhang*1, Zhen Liang1, and Yukui Zhang1 1

Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, 116023, China 2

University of Chinese Academy of Sciences, Beijing 100039, China

ABSTRACT: To improve the stability and sensitivity of nanoelectrospray for LC-MS analysis, we presented a new method to fabricate gold coated emitters. By gravity assisted etching self-termination, the emitter with tapered outer surface and straight inner surface was prepared with good reproducibility, without the need of fluid introduced to protect internal surface during etching. Followed by electroless deposition, the emitter was further coated with gold film homogeneously, by which the RSD value of total ion current in 160 h was below 5%, showing good stability. Compared to that obtained by a commercial emitter, the identified protein number from 2 µg HeLa cell digests was increased over 10%, contributed by the stable electrospray and improved signal intensity of peptides. Furthermore, the integrated gold coated emitter was prepared at the end of the ultra-narrow bore packed column (25 µm i.d.), and 218 proteins were identified from 2 ng HeLa cell digests. All these results demonstrated the great promising of such emitters for ultrasensitive proteome analysis.

Nanoelectrospray ionization-mass spectrometry (nanoESIMS) has become a powerful tool in the sensitive detection of various samples1,2. The emitter is of great significance to ensure the stability and sensitivity of MS analysis by providing the stable Taylor cone3. Usually the emitter with small orifice is fabricated by the microelectrode puller, but with both the inner and outer surface tapered4,5, the emitter is prone to clogging, resulting in poor durability6,7. Although emitters without internal taper could be fabricated by physically grinding8,9, the stable spray could hardly be obtained at low flow rate due to the large outer diameter10. Chemical etching is an alternative favorite method to prepare the emitter with sharpened outer surface and straight inner surface of the capillary8. Smith’s group fabricated such emitters by etching capillaries with water pumped through to protect the interior, and stable picoelectrospray was achieved with 2-µm-i.d. emitters10,11, by which the signal-to-noise ratio of 10 could be obtained for 500 zmol of fibrinopeptide. However, in these works, the continuous fluid must be used to avoid etching the interior, including gas12, water11 and silicone oil13, and the etching procedure must be carefully monitored manually to obtain the desired wall thickness, which is difficult in ensuring the fabrication reproducibility of emitters. Furthermore, to generate stable electrospray, the high voltage must be applied to the emitter with the MS inlet as the counterelectrode, achieved either by a liquid junction provided by an electrode in contact with the solution using a union or tee14,15, or applying voltage directly to the conductive coating of the emitter16. In comparison, by the latter one, besides that the electrical resistance of the long column (if used) generated by the solution and the dead-volumes caused by union could be avoided17, the signal intensity of samples could be improved7. The coated emitter was firstly fabricated by the vacuum deposition of gold onto the surface of the silica emitter18,19, but the lifetime was only 15 min to 3 h. Its durability could be further prolonged to ~100 h

by surface modification with mercapto group20 or chromium layer21 at high temperature18, but the special apparatus is needed, and the operation is difficult. Consequently, new coating techniques should be developed to prepare emitters with good conductivity and durability.

Figure 1. Scheme of the fabrication of gold coated emitters. a. Designed device for chemically etching, including perforated plate, and HF storage reservoir with the same orifice (i.d. 360 µm); b. etching procedure; c. gold film coated onto the emitter; d. photograph of silica and gold coated emitters.

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Herein, a new method was proposed to fabricate gold coated emitters. Based on the gravity assisted etching self-termination, the silica emitters with tapered outer surface and straight inner surface were prepared with good reproducibility without any fluid introduced into the capillary to protect the internal surface, and further coated with gold film homogeneously by electroless deposition technique at room temperature. The gold adhesion to the substrate could be enhanced by the adhesive reagents with –NH2 groups, and the homogeneity of gold film was further improved by the addition of ethanol. Such gold coated emitters were successfully used for proteome analysis, and showed advantages of high sensitivity and excellent stability.

Application in proteome analysis. The gold coated or commercial emitters (FS360-10-5-CE-10-C20, New Objective) were respectively connected to the 50 cm×75 µm C18 packed column for the analysis of 2 µg HeLa tryptic digests by nanoRPLC-MS. The mobile phase was 2% ACN with 0.1% FA (phase A) and 98% ACN with 0.1% FA (phase B). At the flow rate of 300 nL/min, nanoRPLC separation was performed by the gradient as follows: 0-1 min, B% from 2 to 5%, 5-210 min, B% from 5 to 25%, 210240 min, B% from 25 to 40%. LTQ orbitrap velos was operated in positive mode. All MS and MS/MS spectra were obtained in data-dependent mode with one MS full-scan ranging from m/z 300 to 1800 followed by 10 MS/MS scans.

EXPERIMENT SECTION

Furthermore, the 20 cm×25 µm C18 packed capillary column with an integrated gold coated emitter was used for the analysis of 2 ng tryptic peptides from HeLa cells by nanoRPLC-MS. A 100min linear gradient (7-35% phase B) was established at the flow rate of 20 nL/min. Q Exactive MS was performed with one MS full-scan followed by 20 MS/MS scans.

Chemically Etching. As shown in Figure 1a, the home-designed apparatus was used for chemical etching, including the perforated plate for fixing the capillary and the HF reservoir (i.d. 5±0.1 mm, wt 45±2 mg)(n=6) with 360 µm orifice for etching. Firstly, the polyimide coating located at ca. 0.5 - 1.5 cm from the capillary end was scraped off by the thin blade. Secondly, the capillary was fixed onto the perforated plate vertically (Figure 1a). Thirdly, the capillary was passed through the hole of the HF reservoir, and the uncoated part was stucked tightly in the HF reservoir (Figure 1b). Finally, HF was added into the reservoir, and the etching was continued until the vessel was dropped by gravity. Electroless Deposition. As shown in Figure 1c, the emitter was immersed in 1 M NaOH, water, 1 M HCl, water and methanol successively for 15 min. Subsequently, the emitter was immersed in a 10% (v/v) solution of 3-aminopropyl trimethoxysilane (APTMS) in methanol for 12 h. After rinsed with methanol and water, the resulting APTMS-modified emitter was immersed in the colloidal gold solution at room temperature for 6 h to assemble gold nanoparticles as preferential nucleation sites. Finally, to deposit gold film on the surface, the emitter was immersed in the solution containing 0.1 mL ethanol, 4.9 mL water, 0.7 mg HAuCl4 and 300 µL 30% H2O2 under magnetic stirring for 15 min, and then rinsed with water. Dozens of silica emitters could be coated simultaneously. Narrow-bore Packed Capillary Column with Integrated Gold Coated Emitter. Firstly, the poly(BMA-co-EDMA) monolithic frit was prepared by in-situ photo-polymerization. Briefly, a solution containing 5 mg of DMPA, 0.15 g of BeMA, 0.15g of EDMA, 0.34 g of 1-propanol and 0.26 g of 1,4-butanediol was sonicated for 10 min, and then filled into the 25-µm i.d. capillary, with the polyimide coating located at ca. 0.5 - 1.5 cm from the end removed. With both ends sealed by silicon rubbers, the capillary was exposed to ultraviolet with 365 nm in the XL-1500 UV (Spectronics, Westbury, USA) for 15 min, followed by flushing with methanol for 30 min. Thus the monolithic material was prepared in the uncoated part of the capillary. Subsequently, C18 silica particles were packed into the capillary with the monolithic material as the frit. Finally, the uncoated part was etched with the designed device and coated with gold film by electroless deposition. Evaluation on Nanoelectrospray Performance. To evaluate the ESI stability, electrospray was maintained with the voltage applied directly on the gold film. The consecutive MS scans from m/z 300 to 1800 were real-time monitored for 2 min and the RSD of the total ion intensity was calculated to evaluate ESI stability. The stability was characterized at different flow rate ranging from 20 to 300 nL/min. And the conductive emitter sprayed for a long period of time to evaluate its durability in 2% ACN with 0.1% FA at 100 nL/min.

The raw files obtained with LTQ orbitrap velos were uploaded into Proteome Discoverer (PD, version 1.4.1.14) with Mascot (2.3.2) and searched against the UniProtKB human complete proteome sequence database (release 2015_04, 42,121 entries) to show the intensity of peptides identified. The parameters for searching were set as follows: enzyme, trypsin; missed cleavages, two; fixed modifications, carboxyamidomethylation (C); variable modifications, oxidation (M) and acetylation protein N-termini. The peptide tolerance and MS/MS tolerance were 10 ppm and 0.5 Da respectively. The mass spectrometric raw files obtained with Q Exactive were converted to *.mgf with pXtract v1.036 and searched using MASCOT server version 2.4.0. against the database SP_human_1406. The peptide tolerance and MS/MS tolerance were 20 ppm and 0.05 Da respectively. The false discovery rate (FDR) for all the identified peptides was controlled less than 1%. Safety Tips. HF is extremely hazardous and has strong corrosion effect on the skin. Extreme care must be taken when operated. All the etching procedure should be operated in a ventilated hood with appropriate protective equipment. And the falling reservoirs were collected in the plastic container with 1 M NaOH. RESULTS AND DISCUSSION Herein, a gravity assisted etching self-termination strategy was proposed to fabricate silica emitters with precise geometry (Figure 1a). The capillary was passed through the HF storage vessel to avoid HF flowing into the inner channel. The etching began from the outer surface of the capillary with the coating removed, and stopped until the reservoir dropped by gravity. Therefore, the internal surface could be protected intact even without fluid introduced into the capillary (Figure S1). The outer surface of the emitter was etched to the cone-shape, owing to surface tension and the volatilization of HF. One of the most important factors relevant to the emitters’ geometry was the weight of HF reservoir and added HF. During etching, capillaries were kept vertical. Once the etched capillary couldn’t stand the gravity of the reservoir, the reservoir was dropped, and the emitter was formed. As long as the weight of HF reservoir and added HF was fixed, the generated emitters were of the same geometry. With the weight of the HF reservoir as 45.5±0.1 mg, and 20 µL HF added, for 10 µm i.d. capillary, the wall thickness of the emitter was measured by optical microscope as 3.72±0.13 µm (n=6) with the RSD only 3.59% (Figure S2). Compared to other reported etching approaches11,13, by which etching must be real-time monitored to achieve a desired wall thickness, our proposed strategy sim-2

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plified the preparation procedure and improved controllability of precise geometry.

sity distribution of identified peptides in each run was shown in Figure 3C, and the median of Log10(Intensity) was transferred from 4.70 to 5.15, demonstrating the improved ionization efficiency. In the scatter pot, the pearson correlation coefficient were 0.914 and 0.646, respectively for gold coated and commercial emitters (Figure 3D), indicating improved reproducibility and electrospray stability for the gold coated emitter. All these results were mainly attributed to the fact that the excellent electrical conductivity and precise geometry of the emitter ensured the stable electrospray and high ionization efficiency. What’s more, the homogeneous gold coating on the emitter was beneficial for enhanced ionization efficiency7. Therefore, the identified protein number was increased over 10% (Figure 3E).

Figure 2. Scanning electron images of the surface of emitters coated by electroless deposition with different amount of ethanol as additive. A. 0 µL; B. 100 µL; C. 300 µL; D. 500 µL; E. 700 µL; F. 900 µL. The scale bar was 1 µm. The silica emitter was further coated by electroless deposition (Figure 1c). To improve the stability of gold film, firstly, APTMS as the adhesive reagent was silanized onto silica surface, followed by the attachment of gold nanoparticles served as preferential nucleation sites via high affinity between amino groups and gold. Then Au3+ ions from the HAuCl4 precursor were reduced by H2O2 to gold atoms, and deposited onto the modified surface22,23, . During the deposition, the ethanol in the coating solution had an important effect on the homogeneity of gold film (Figure 2). Without ethanol added (Figure 2A), the gold on the surface was dispersive, resulting in large resistance. As Shin24 stated, pure water hampered the deposition of metal film, resulting in the formation of nanoparticles in the solution. However, with only 100 µL of ethanol added (Figure 2B), the Au clusters slowly self-assembled on emitters, and homogeneous gold film could be coated with the resistance less than 200 Ω. With more ethanol added (Figure 2C-F), the stability of gold colloids was destroyed, resulting in a rough gold film. Herein, the addition of 100 µL ethanol was used for gold coating. By our proposed method, five gold coated open tubular emitters (i.d. 10 µm) were prepared simultaneously, by which the consecutive MS scans were acquired for 2 min respectively. As shown in Figure S3A, the RSD values of TIC were all less than 3%, indicating the good stability of the electrospray and the excellent preparation reproducibility of the emitters. What’s more, the open tubular emitter could spray stably with RSD of TIC less than 5% at different flow rates ranging from 20 to 300 nL/min even for mobile phases with different composition (Figure S3B, C). Even after 160-h usage, the gold coated emitter still supplied stable electrospray with the RSD of TIC less than 1.33% (Figure S3D). The excellent durability was attributed to precise dimension without easily clogged internal taper and good stability of homogeneous gold film with APTMS as the adhesive reagent. The performance of gold coated emitter was compared with the commercial conductive one. The mixture of five standard peptides was directly infused to MS and the intensity ratios of five peptides obtained by the gold coated emitter and the commercial emitters were 1.28, 1.31, 1.11, 2.15, 2.06 respectively (Figure S4), showing the improved MS response of the gold coated emitters. Furthermore, they were used for the analysis of tryptic digests from HeLa cells by coupling with a 50 cm-length capillary column respectively. As shown in Figure 3A, the mass spectrometric signal intensity was increased by ca. 2.8-fold using the gold coated emitter. With the in-depth investigation of all the identified peptides in three runs, we found that the signal intensity of most peptides were increased (Figure 3B). The violin plot of the inten-

Figure 3. Gold coated emitter improved the performance for analysis of 2 µg tryptic peptides from HeLa cells by nanoRPLC-MS. A. TIC of gold coated (yellow) and commercial emitters (blue). B. Intensity distribution of identified peptides in three run. C. The violin plot of the intensity distribution of peptides for each run. The box plots show span from 25th to 75th percentage. D. Scatter plots of peptide intensities between both runs. E. Number of proteins identified in three technical replicate.

By our proposed method, the inner surface and packed stationary phase can be well protected during etching from the outer surface of the capillary. Therefore, it is convenient to fabricate an integrated emitter at the end of the capillary columns, no matter for narrow-bore or ultra-long separation columns. Compared with the external emitter, for columns with the integrated emitter, the the dead volume between the unions could be avoided25. Herein, a 25 µm-i.d. packed capillary column with an integrated gold emitter was prepared, and used for the analysis of 2 ng tryptic digests from HeLa cells at the flow rate of 20 nL/min. As shown in Figure S4, 392 peptides and 218 proteins could be identified with the overlap of 45.4% in three runs, showing the great potential for the ultrasensitive analysis of proteomes. CONCLUSIONS

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In conclusion, we presented a novel method to prepare the gold coated emitters for nano-LC MS/MS based analysis. Based on the gravity assisted etching self-termination strategy, the silica emitter could be fabricated with precision geometry and excellent reproducibility. Furthermore, electroless deposition was used to coat the emitter with homogeneous and stable gold film under room temperature, ensuring the excellent electrical conductivity, which could provide stable electrospray for over a week. Furthermore, the signal intensity of peptides was enhanced and more peptides were identified compared with that obtained using the commercial emitter, contributed by the improved ionization efficiency. Finally, narrow-bore packed capillary columns with integrated gold coated emitters were prepared, and demonstrated the great potential to achieve the ultra-sensitive proteome analysis.

ASSOCIATED CONTENT Supporting Information Photographs of silica emitters with different i.d., and comparison of total ion chromatogram obtained with home-made and commercial emitters. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

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S. 2004, 13, 940-946. (14) Horie, K.; Kamakura, T.; Ikegami, T.; Wakabayashi, M.; Kato, T.; Tanaka, N.; Ishihama, Y. Anal. Chem. 2014, 86, 38173824. (15) Wilson, R. E.; Groskreutz, S. R.; Weber, S. G. Anal. Chem. 2016, 88, 5112-5121. (16) Forzano, A. V.; Becirovic, V.; Martin, R. S.; Edwards, J. L. Analytical Methods 2016, 8, 5152-5157. (17) Dietze, C.; Scholl, T.; Ohla, S.; Appun, J.; Schneider, C.; Belder, D. Anal. Bioanal. Chem. 2015, 407, 8735-8743. (18) Valaskovic, G. A.; McLafferty, F. W. Rapid Commun. Mass Spectrom. 1996, 10, 825-828. (19) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (20) Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67, 385-389. (21) Barnidge, D. R.; Nilsson, S.; Markides, K. E.; Rapp, H.; Hjort, K. Rapid Commun. Mass Spectrom. 1999, 13, 994-1002. (22) Petkov, N.; Stock, N.; Bein, T. J. Phys. Chem. B 2005, 109, 10737-10743. (23) Hu, J. D.; Wei, L. B.; Jian, C. B.; Zhang, X. H.; Zhao, X. Y. Surf. Coat. Technol. 2008, 202, 2922-2926. (24) Shin, K. S.; Cho, Y. K.; Kim, K. L.; Kim, K. Bull. Korean Chem. Soc. 2014, 35, 743-748. (25) Zhang, H. Y.; Ou, J. J.; Wei, Y. M.; Wang, H. W.; Liu, Z. S.; Zou, H. F. J. Chromatogr. A 2016, 1440, 66-73.

Corresponding Author *E-mail: [email protected].

Author contributes #XD.Z and Y. L. contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are grateful for financial support from National Natural Science Foundation (21235005, 21575139),National Basic Research Program of China (2012CB910604), and National Key Scientific Instrument and Equipment Development Projects (2012YQ120044-8).

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Herein, we presented gravity assisted etching self-termination strategy to fabricate nanoelectrospray emitters with precise geometry, without any fluid introduced to protect internal surface. Followed by electroless deposition, the silica emitter was coated with stable homogeneous gold film. Such emitter can spray stably for more than one week. Compared to the commercial emitter, the signal intensity of peptides was improved and the identified protein number was increased. All these results demonstrated the great promising of such emitters for ultrasensitive proteome analysis.

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