Desalting by Crystallization: Detection of Attomole Biomolecules in

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Desalting by Crystallization: Detection of Atto-mole Biomolecules in Pico-liter Buffers by Mass Spectrometry Xiaoyun Gong, Xingchuang Xiong, Song Wang, Yanyan Li, Sichun Zhang, Xiang Fang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b01877 • Publication Date (Web): 27 Aug 2015 Downloaded from http://pubs.acs.org on September 1, 2015

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Analytical Chemistry

Desalting by Crystallization: Detection of Atto-mole Biomolecules in Pico-liter Buffers by Mass Spectrometry Xiaoyun Gong†,‡, Xingchuang Xiong‡, Song Wang†, Yanyan Li† , Sichun Zhang†, Xiang Fang*,‡ and Xinrong Zhang*,† †

Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing, China ‡

National institute of Metrology, Beijing, China

ABSTRACT: Sensitive detection of biomolecules in small-volume samples by mass spectrometry is, in many cases, challenging due to the use of buffers to maintain the biological activities of proteins and cells. Here, we report a highly effective desalting method for pico-liter samples. It was based on the spontaneous separation of biomolecules from salts during crystallization of the salts. After desalting, the biomolecules were deposited in the tip of the quartz pipette due to the evaporation of the solvent. Subsequent detection of the separated biomolecules was achieved using solvent assisted electric field induced desorption/ionization (SAEFIDI) coupled with mass spectrometry. It allowed for direct desorption/ionization of the biomolecules in situ from the tip of the pipette. The organic component in the assistant solvent inhibited the desorption/ionization of salts, thus assured successful detection of biomolecules. Proteins and peptides down to 50 amol were successfully detected using our method even if there were 3×105 folds more amount of salts in the sample. The concentration and ion species of the salts had little influence on the detection results.

Biological researches often involve analysis of samples with extremely small volumes.1-4 For instance, the analysis of special physiological fluids, or even single cells,5-8 have to deal with samples down to nano-liter, or even pico-liter level.1 Nano-ESI has been widely used in such applications due to its low sample consumption9 and high sensitivity10-12. However, successful detection of biomolecules in small volume samples is still challenging in many cases due to the high concentrations of salts added in the solutions during pretreatment. These salts are used to maintain the pH value and osmotic pressure of the aqueous solutions so that cells could keep alive. Salts are also added in solutions to maintain the structure and biological activities of proteins.13-15 As a consequence, the sensitivity of MS is significantly depressed due to the presence of salts. Even low milli-molar concentrations of some metal ion salts can cause severe ion suppression and peak broadening due to the formation of clusters and adducts.16-20 Several methods have been developed so far to separate targeted biomolecules from salts. Liquid chromatography21-23 and capillary electrophoresis24-27 based methods suffer from the diffusion of analytes in the liquid phase, which reduces the sensitivity.28 Ziptip-C1829, surface-functionalized nano-particles24-28 and surface modified MALDI plates30-36 have also been used for the fractionation and detection of biological samples (mostly for peptides). The multiple handling steps of these methods lead to inevitable sample loss and potential contaminants.20,35,37 In addition, these methods are not suitable for samples with ultra-small volume down to pico-liter level. We have previously reported a desalting method for small-volume samples based on step voltage nanoelectrospray.28 This method dealt with samples with a volume of about 0.1 µL, which was still too large for sin-

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gle-cell analysis. Till now, no method has achieved effective desalting of pico-liter biological samples and sensitive detection of atto-mole biomolecules in biological buffers. Here, we propose a desalting method based on the spontaneous separation of biomolecules from salts during crystallization of the salts, and coupled it with solvent assisted electric field induced desorption/ionization (SAEFIDI) for MS detection. As the desalting process was accomplished spontaneously within the tip of a quartz pipette, potential sample loss, dilution and contaminants were avoided. After desalting, the biomolecules were deposited in the tip of the pipette due to the evaporation of the solvent. Subsequent detection of the separated biomolecules was achieved using SAEFIDI coupled with MS. It allowed for direct desorption/ionization of the biomolecules in situ from the tip of the pipette.

Experimental section Chemicals. Gentian violet, insulin, myoglobin, cytochrome c, acetic acid (HAc), methanol (HPLC grade) and acetonitrile (HPLC grade) were purchased from Sigma Aldrich. Polynucleotide was purchased from Sangon Inc. (Shanghai, China). NaCl, NaBr, NaHCO3, KBr and CaCl2 were purchased from Beijing Chemical Work (Beijing, China). Ultrapure H2O was obtained from a Millipore water purification system (resistance ≥ 18 MΩ•cm−1, Barnstead Nanopure, Thermo Scientific). Sampling. Samples solutions were sucked into the tips of the pipettes by spontaneous capillarity. The sampling volume was controlled by fixing the sampling time. It could be increased by extending the sampling time. The inner diameter of the tips was 1~2 µm. The sampling volume was typically 20~100 pL. For nano-ESI, the sampling volume was 3~5 µL. The pipettes were filled with samples. Desalting (Crystallization). After sampling, the pipettes were placed in the open air at room temperature (20~25 ℃) to let the solvents evaporate naturally. A lower relative humidity of the air would facilitate the desalting process. Commonly, the desalting process would only take a few minutes. For samples with certain proportion of organic solvents, the process was even faster. Direct Desorption/ionization (SAEFIDI). Direct desorption/ionization of the separated biomolecules from the pipette tip was achieved by solvent assisted electric field induced desorption/ionization (SAEFIDI) for MS detection (Figure 1). The strategy of direct desorption/ionization of the analytes from the tip of the sample holder has previously been used by our group for the detection of metabolites in single Allium Cepa cells.38 It successfully avoided sample dilution, and thus ensured sensitive detection of ultra-trace biomolecules. After desalting, the pipette was placed in front of the MS inlet with a distance of 5 mm. A copper wire was inserted into the pipette from end until the tip of the wire reached the tip of the pipette. The distance from the tip of the wire to the tip of the pipette (Figure 1b) was about 1.5 mm. Next, a high D.C. voltage of 1750 V (+1750 V for positive mode, and -1750 V for negative mode) was applied to the copper wire. Then a droplet of assistant solvent was used to wet the tip of the pipette (Actually, the purpose was to wet the biological sample in the tip, Figure 1b). The volume of the solvent droplet was controlled using a pipette. For each desorption/ionization process, a solvent droplet with a volume of 15 µL was placed at the nano-tip to wet it. Solvent was then sucked into the tip via spontaneous capillarity. The droplet was kept at the tip for a fixed time, commonly 1 s in our work. Then the droplet was moved away to allow subsequent desorption/ionization. Upon the contact of the biomolecules and the solvent, the biomolecules were dissolved into the assistant solvent and immediately motivated by the electric field to spray out from the tip (Figure 1c).39,40 Subsequent MS detection was then achieved. Similar ionization methods could be found in literatures.41,42 Safety Considerations. When experiments were performed, high D.C. voltage was applied to the copper wire. Actions for the insulation of both the operators and the MS from the high voltage source should be taken to avoid danger. ACS Paragon Plus Environment

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Mass spectrometry. All the experiments were accomplished on a LTQ MS (Thermo Scientific, San Jose, CA). The parameters were listed as follow. Capillary temperature: 275 ℃, capillary voltage: 9 V, tube lens voltage: 100 V, maximum inject time: 100 ms, micro-scans: 1. The commercial ionization source of ESI was removed ahead of our experiments.

Figure 1. Schematic setup of our method. a) Sampling. Samples were sucked into the tip via spontaneous capillarity. b) Wetting of the tip. A droplet of assistant solvent was placed at the tip for a fixed time to wet it. c) Desorption/ionization process. After wetting, the biomolecules were dissolved into the assistant solvent and immediately motivated by the electric field to spray out from the tip into MS. d) The obtained mass spectrum of insulin by our method in positive mode. The sample contained 500 nM insulin and 15 mM NaCl. The sampling volume was 100 pL. The assistant solvent used here was methanol/water = 1:1, with 0.1% HAc. e) The obtained mass spectrum of insulin by conventional nano-ESI. The spray voltage was +1500 V.

Results and discussion Desalting and detection capability As a proof of concept, an aqueous solution of insulin was used as the sample. The concentration of insulin was 500 nM. Additional 15 mM NaCl was added into the sample. The sampling volume was 100 pL, corresponding to 50 amol insulin. As was shown in Figure 1d, the 50 amol insulin was successfully detected using our method. In comparison, conventional nano-ESI failed in the detection (Figure 1e) due to the presence of 15 mM NaCl. The desalting and detection capability of our method was further demonstrated by increasing the concentrations of the salts in samples (Figure 2). The concentration of NaCl was ranged from 0 to 150 mM (The concentration of physiological saline), while the concentration of insulin was kept at 500 nM. In other words, the concentration of NaCl was 0~3×105 folds greater than that of insulin in the samples. The sampling volume was 100 pL ACS Paragon Plus Environment

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for each time, corresponding to 50 amol insulin. According to the obtained mass spectra in Figure 2, successful detection of insulin could be achieved regardless of the increasing concentrations of NaCl. Furthermore, it should be noted that although the concentration of NaCl increased on a wide range of five orders of magnitude, the obtained signal intensity of insulin only reduced within 3 folds compared to the sample without NaCl. In comparison, conventional nano-ESI was also used to detect insulin in these samples (Figure S1 of the Supporting Information). Insulin was detected by conventional nano-ESI when the concentration of NaCl was only 1.5 mM (Figure S1b of the Supporting Information). However, obvious sodium adduct peaks of insulin could be seen in the spectrum. The signal to noise ratio was greatly lowered. The detection of insulin failed when the concentration of NaCl reached 15 mM (Figure S1c of the Supporting Information). Adding 0.1% HAc to the solution could not make any improvement (Figure S1d of the Supporting Information), either.

Figure 2. Detection results of insulin aqueous solutions with different concentrations of NaCl using our method. The concentration of insulin was 500 nM in all the samples. The sampling volume was 100 pL for each time, corresponding to 50 amol insulin. The assistant solvent was methanol/water = 1:1, with 0.1% HAc. The mass spectra were obtained in positive mode. a) The concentration of NaCl was 0. b) The concentration of NaCl was 1.5 mM. c) The concentration of NaCl was 15 mM. d) The concentration of NaCl was 150 mM.

In addition to insulin, we further investigated two proteins including myoglobin (Figure 3) and cytochrome c (Figure S2 of the Supporting Information). Both of the two species were successfully detected under different concentrations of NaCl using our method. In comparison, the detection results of conventional nano-ESI were obviously affected when the concentration of NaCl reached 1.5 mM. Sodium adducts were observed in the mass spectra. Specially, we mixed insulin, myoglobin and cytochrome c in one sample and added 30 mM NaCl to investigate the detection ability of our method for the multi-species. The results were shown in Figure 4. Insulin, myoglobin and cytochrome c were successfully detected simultaneously (Figure 4e). According to the extracted ion chromatogram (EIC), the analytes and NaCl were separated in time order. We hypothesized that the analytes were solubilized more readily than NaCl, so their signals were obtained before NaCl. Besides, other biomolecule species were also investigated, including a peptide (Figure S3 of the Supporting Information) and a polynucleotide (Figure S4 of the Supporting Information). Both the peptide and the polynucleotide were successfully detected by our method under different concentrations of NaCl. In comparison, the detection results of nano-ESI were greatly degenerated due to the presence of NaCl. It should be noted that successful detection of biomolecules by our method required suitable assistant solvent. This assistant solvent should be able to dissolve the targeted biomolecules readily, so that the desorpACS Paragon Plus Environment

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tion/ionization process of biomolecules and salts could be separated in time order. Otherwise, the detection results might be degenerated.

Figure 3. Comparison of the detection results of myoglobin samples using our method and conventional nano-ESI. All the mass spectra were obtained in positive mode. The concentration of myoglobin was 10 µM in all the samples. In our method, the sampling volume was 100 pL, corresponding to 1 fmol myoglobin. The assistant solvent was methanol/water = 1:1, with 0.1% HAc. a) Mass spectrum obtained using our method when the concentration of NaCl was 1.5 mM. b) Amplified mass spectrum of that in a). c) Mass spectrum obtained using our method when the concentration of NaCl was saturated. d) Amplified mass spectrum of that in c). e) Mass spectrum obtained using conventional nano-ESI when the concentration of NaCl was 1.5 mM. The spray voltage was +1500 V. f) Amplified mass spectrum of that in e).

Compared to nano-ESI, our method successfully inhibited the addition of metal ions to protein molecules during the desorption/ionization process (Figure 3a~d). For each charge state, mainly one ion species was observed in the mass spectra. Additional metal ion adducts with protein molecules was greatly inhibited, even if the salt was saturated in the sample (Figure 3c and d). Conventional nano-ESI barely achieved the detection of myoglobin when the concentration of NaCl was 1.5 mM (Figure 3e and f). However, serious adduction of sodium ions to the protein molecules was observed, which severely lowered the signal to noise ratio. Detection of myoglobin with higher concentrations of NaCl could hardly be achieved by nano-ESI. Similar phenomena were also observed in the detection of other proteins (Figures S2 and S5 of the Supporting Information, and Figure 5) and polynucleotide (Figure S4 of the Supporting Information).


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Figure 4. Detection capability of our method for multi-species. The concentrations of insulin, myoglobin and cytochrome c were all 10 µM. The concentration of NaCl was 30 mM. In our method, the sampling volume was 100 pL, corresponding to 1 fmol of each species. The assistant solvent was methanol/water = 1:1, with 0.1% HAc. a) The EIC of m/z = 1156, representing insulin. b) The EIC of m/z = 1529, representing cytochrome c. c) The EIC of m/z = 808, representing myoglobin. d) The EIC of m/z = 1308, representing NaCl. e) The mass spectrum obtained at the beginning of desorption/ionization process. f) The mass spectrum obtained at the end of the desorption/ionization process.

Despite NaCl, other salt species were also investigated. Na+ was changed into other cation species of Ca2+ and K+. As was shown in Figure 5, even though the salts were saturated, the replacement of Na+ by other cation species seemed to have little influence on the final detection results of our method, either. In comparison, the spectra of nano-ESI were dominated by adducts of Ca2+ and K+ with protein ions, although the concentrations of the salts were only 1.5 mM. The identity of anion species was also investigated, as was shown in Figure S5 of the Supporting Information. The replacement of Cl- by Br- and HCO3- had little influence on the final detection results of our method.


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Figure 5. Comparison of the detection results of cytochrome c under different salt cation species using our method and conventional nano-ESI. The concentration of cytochrome c was 10 µM. All the mass spectra were obtained in positive mode. In our method, the sampling volume was 100 pL, corresponding to 1 fmol cytochrome c. The assistant solvent was methanol/water = 1:1. The spray voltage of nano-ESI was +1500 V. a) Detection result of our method under saturated CaCl2. b) Detection result of nano-ESI under 1.5 mM CaCl2. c) Detection result of our method under saturated KBr. d) Detection result of nano-ESI under 1.5 mM KBr. e) Detection result of our method under saturated NaCl. f) Detection result of nano-ESI under 1.5 mM NaCl.

Desalting and desorption/ionization mechanism To elucidate the desalting mechanism, an aqueous solution of gentian violet was used as the sample (Figure 6). The concentration of gentian violet was 500 nM. Additional 1.5 mM NaCl was added into the sample. The sampling volume was 30 pL, as was calculated according to the picture obtained from a microscope (Figure 6a). Thus the sampled gentian violet in the pipette was 15 amol. After sampling, the sample was let dry under room temperature to allow the salt crystallize. Afterwards, the separated gentian violet was directly desorbed/ionized from the tip of the pipette using SAEFIDI for MS detection. As was shown in Figure 6f, the 15 amol gentian violet was successfully detected using our method. In comparison, conventional nano-ESI failed in the detection. The separation of gentian violet from the salts during crystallization was confirmed using a microscope (Figure S6 of the Supporting Information). Those organic gentian violet molecules did not dope into the crystals of salts during crystallization. Instead, they deposited on the surfaces of the salt crystals and the walls of the container (Figure S6a and c of the Supporting Information). Furthermore, the deposited gentian violet could easily be washed away by organic solvents without observable damage to the salt crystals (Figure S6b and d of the Supporting Information). This indicated that gentian violet and the salts were actually separated. In the desorption/ionization process, the organic assistant solvent (Acetonitrile) quickly dissolved the gentian violet molecules surrounding the NaCl crystals. The dissolved gentian violet molecules were then motivated by the electric field to spray out from the tip and detected by MS. After detection, the NaCl crystals were checked again (Figure 6d). Regardless of the desorption/ionization procedure of gentian violet, the NaCl crystals remained almost unchanged (compare Figure 6c and d). This confirmed that gentian violet was actually separated from the salts during crystallization, and could be desorbed/ionized individually without salt. ACS Paragon Plus Environment

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Figure 6. Desalting and desorption/ionization of gentian violet investigated by microscope. a) Gentian violet solution sucked in the tip. b) NaCl crystal formed in the tip after crystallization. c) Amplified image of the NaCl crystal formed in the tip. d) NaCl crystals remained in the tip after the desorption/ionization procedure. The assistant solvent used for desorption/ionization was pure acetonitrile. e) The obtained mass spectrum using conventional nano-ESI in positive mode. The spray voltage was +1500 V. f) The obtained mass spectrum using our method in positive mode. Gentian violet was represented by the peak of m/z = 372, losing a Cl- during ionization.

The assistant solvent used for the desorption/ionization of gentian violet was acetonitrile. Salts could hardly dissolve in acetonitrile. However, if the assistant solvent contained water, the situations might be different. To look into this issue, we investigated the desorption/ionization process of insulin. An aqueous solution of insulin (500 nM, 100 pL, corresponding to 50 amol insulin. Additional 150 mM NaCl was added in the solution) was used as the sample. The results were shown in Figure S7 of the Supporting Information. As the assistant solvent contained water, it caused the desorption/ionization of NaCl. However, the desorption/ionization of insulin and NaCl were separated in time (Also revealed by Figure 4). Insulin was desorbed/ionized before NaCl. Just like gentian violet, insulin was supposed to deposit on the surfaces of the salt crystals and the walls of the quartz pipette. Upon the addition of assistant solvent, insulin was dissolved faster than NaCl. Furthermore, the organic portion of the assistant solvent might inhibit the dissolution of salts. Thus, the signal of insulin was obtained before NaCl. McLuckey’s group has just reported about the phenomenon of metal ion removal from proteins and protein complexes upon the electrospray droplet exposure to organic vapors.43 It showed that the organic solvent could reduce the adduct formation of metal ions with protein molecules. However, the manipulation and desalting mechanism were different between their method and ours. In our method, the desalting process was accomplished in nano-tip pre-electrospray. The separation of protein molecules from salt was achieved during the crystallization process of salt. Subsequently, the separated protein molecules were desorbed/ionized ahead of salt for MS detection, as was revealed by Figure 4 and Figure S7 of the Supporting Information. The organic component of the assistant solvent was supposed to help inhibit the dissolution of salt and reduce metal ion adduction to protein molecules.44,45 Desalting could be achieved in both positive and negative mode. In comparison, in McLuckey’s method, the desalting process was accomplished post-electrospray. The generated nano-electrospray droplets were faced with organic vapors in the gas phase, where the desalting process occured. The mechanism was supposed to involve the removal of alkali metal ions via ion evaporation of alkali cation/organic clusters before the


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protein ions were formed via the charged residue mechanism. Desalting could only be achieved in positive polarity.

Conclusions In conclusion, we have demonstrated a novel desalting and detection method that allowed for sensitive detection of biomolecules at atto-mole level in pico-liter concentrated buffers. As the desalting process was spontaneous and no artificially imposed procedure was used, potential sample loss, dilution and contaminants were avoided. The concentration and ion species of the salts have little influence on the detection results. Our method was quite simple in device setup and manipulation, which made it more practical. We believe that our method might have a wide application in the analysis of samples with extremely small volumes. ASSOCIATED CONTENT Supporting Information Additional information as noted in text, including supplementary figures and discussions. The Supporting Information is available free of charge via the Internet.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. Present Addresses †

Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing, China ‡ National institute of Metrology, Beijing, China Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the Ministry of Science and Technology of China (Nos. 2011YQ090005, 2013CB933800 and 2012YQ12006003) and the National Natural Science Foundation of China (Nos. 21390410 and 21125525). We sincerely thank our teammates Shaoqing Cai, Zhenwei Wei, Xingyu Si and Yaoyao Zhao for their help in the experiments.

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