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ESI MS for Microsized Bioparticles Abdil Ozdemir, Jung-Lee Lin, Mustafa Gulfen, Szu-Hsueh Lai, Chun-Jen Hsiao, Nelson Gee-Con Chen, and Chung-Hsuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02937 • Publication Date (Web): 21 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017
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Analytical Chemistry
ESI MS for Microsized Bioparticles Abdil Ozdemir2, Jung-Lee Lin1, Mustafa Gulfen2, Szu-Hsueh Lai1, Chun-Jen Hsiao1, Nelson G. Chen3, Chung-Hsuan Chen1* 1
Genomics Research Center, Academia Sinica, Taipei, Taiwan Department of Chemistry, Faculty of Arts and Sciences, Sakarya University, 54187 Esentepe, Sakarya, Turkey 3 Department of Electrical and Computer Engineering, National Chiao Tung University, 1001 Da Xue Rd., Hsinchu, 30010, Taiwan 2
*
All correspondence should be addressed to
[email protected]
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ABSTRACT An ESI ion trap mass spectrometer was designed for high-throughput and rapid mass
analysis of large bioparticles. Mass calibration of the instrument was performed using commercially available polystyrene (PS) microparticles with a size comparable to cancer cells. Different sizes of MCF-7 breast cancer cells (8 to 15 µm) were used in this study. The masses of different cancer cells were measured. This system allows for the analysis of all types of particles.
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INTRODUCTION Recent discoveries and research have shown that nano- and microparticles play important
roles in our daily lives1. Particles with a size of ~2.5 microns have become a major environmental concern worldwide1. Analysis of microbioparticles is important for monitoring human health. Although there are different high-mass measurement methods, such as flow cytometry, gravimetric determination, cantilever transducers2-5 quartz crystal microbalances6-9 and chargereduced electrospray size spectrometry10, their precision is limited. Moreover, they often require complex sample preparation. Recently, there has been an increasing interest in using a mass spectrometer (MS) to measure the masses of microparticles, which include polymeric particles, human cells, bacteria and viruses11-20. CyTOF21 has also been used for measurements of biomarkers on each cell, but it cannot be used to measure the cell mass. Mass measurements of particles face some challenges, such as ionization of particles, sending particles inside the MS and their detection. The first particle ionization study was pursued inside an MS. Wuerker et al.22 used an electric powder injector that was placed right next to the ion trap. Another approach is called laser-induced acoustic desorption (LIAD)23-26. It desorbs the particles inside the ion trap MS. For LIAD, the particles are placed on a Si wafer and inserted inside MS chamber with a probe. A laser is fired onto the backside of the Si wafer. LIAD allows the ionization of nano- and microparticles, viruses and cells without using a matrix27-28. Visible MALDI cell mass spectrometry29 has a visible light source and matrix. Open-system MS, such as the Electrospray (ESI) MS, provides continuous sampling and faster data collection. Xiong et al.30 developed a semi-open ionization system and aerodynamic desorption (AD) for microsized particle sampling and ionization. One challenge in particle MS is the physical orientation of MS. Because all particles are under gravitational force, the RF trapping power and gravitational force compete with each other. If the mass analyzer is parallel to the gravitation force, heavy particles can fall before reaching the ion trap. The other challenge is particle detection. Different types of detectors have been developed30-33. The charge of a single particle can be measured directly by a charge detector. Since the speed of a microparticle (below 20 m/s) is slower than that of a small molecule, the charge collection time is in milliseconds, whereas Faraday disks begin to sense the particle 1 cm away from the disk. Thus, we need a charge detector with a long discharge time constant.
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A pioneering ESI study of megadalton proteins was performed by Robinson et al., who used a conventional ESI time-of-flight (TOF) MS
34
. Their study was in the nanometer region.
ESI MS studies of compounds with large molecular weight have been reported by different groups35-37. A pioneering study about large bioparticles has been conducted by Fuerstenau et al.15 They obtained the ESI spectra of intact viruses (40.5 MDa) via a charge-induction tube. ESI QIT MS for cell/microparticle measurements has higher analysis speed compared with that of LIAD MS27. In addition, solution samples can be directly analyzed with this approach but not for LIAD. For LIAD, cells must be dried on a silicon wafer surface, and the sample plate is delivered through a load-lock vacuum system. After a few laser shots, a fresh sample plate is loaded after the initial sample has been consumed. These experiments require a lengthy analysis time and large sample amounts. The major advantage of ESI QIT MS is continuous feeding without further sample preparation. Although cell mass can possibly be estimated by microscopy based on the volume and density. Nevertheless, some cells are with irregular shapes so that the volume is very difficult to be accurately measured with a microscope. Red blood cell (RBC) is one example. Therefore, we developed an ESI QIT MS for rapid mass analysis of microsized particles including cells. The instrument does not need a turbo pump and allows the analysis of dry or solution phase samples. It can analyze cells as large as 15 µm.
EXPERIMENTAL SECTION Figure 1 shows the experimental setup of our new ESI QIT MS. The system has an ESI
source for ionization. The ESI source comprises a silica capillary with an ID of 150 µm, an OD of 360 µm and a high voltage connector. A 3.0-kV DC was applied to the needle with the cone grounded during the spray process. The entrance was built in a cone shape, and a 0.5 mm ID stainless steel (SS) capillary was inserted inside the cone. Capillary heating did not have a positive effect on the ionization of cell particles and did not change the instrument sensitivity. Since cancer cell membrane is in general fragile; the SS capillary was not heated during the spray process. After passing through the entrance capillary, the ESI-generated particles reach the first desolvation region, and the capillary ends with a skimmer. The hole size of the skimmer is 0.5 mm. After the skimmer, the particles enter the second desolvation region. There is a quadrupole after the skimmer, but the quadrupoles are not connected to any power source. Application of the RF potential to the quadrupoles did not result in any improvement in the particle transmission. Because of the particle size, the applied RF potential does not change the path of the particles. 4 ACS Paragon Plus Environment
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The lengths of the quadrupoles are 12 cm, and there is an entrance lens with a hole of 0.75 mm right after the quadrupole. The pressure inside the MS was maintained only by mechanical pumps, and no turbo pumps were used. The orientation of the MS is perpendicular to the floor to reduce the gravitational effect. When the particles are sprayed into the MS, they travel straight into the trap. The samples were introduced in either solid or liquid form. The sensitivity should reach a single cell for any cell trapped in the ion trap. The quadrupole ion trap is shown in Figure 1A. The trap has a hyperbolic surface. The dimensions of the trap are zo=7.07 mm and ro=10 mm. Two identical end-caps are separated by two Teflon (30 x 10 mm) insulators from the center electrode. Four holes are on the ring electrode: two for the laser light and another two for the observation of light scattering from the trapped particles. The sample is introduced from the upper electrode and ejected to both sides but detected by a charge detector placed right under the ion trap. The RF trapping voltage was provided by a function generator (DS345, Stanford Research), amplified by a power amplifier (TREK PZD2000A).
Sample Preparation. Polystyrene microparticles of different sizes (3-20 µm) were purchased from Sigma-Aldrich and NIS for instrument calibration. To remove the impurities from microparticles, the polystyrene particles were washed with deionized water several times and then recovered via centrifugation. The particles were subsequently resuspended in a 70% ethyl alcohol solution. Breast cancer cells were obtained from Academia Sinica Genomic Research Center. Different sizes of cells of the same type were used. The cells were cultured at 37 °C under a 5% CO2 atmosphere in RPMI 1640 medium (Gibco Life Technologies, New York, NY) containing 2 mM L-glutamine, 10% fetal calf serum and 1% penicillin/streptomycin (10,000 IU/mL, all from Invitrogen, Carlsbad, CA). After growing enough cells, they were detached from the surface of the Petri dish using the enzyme, trypsin. After harvesting the cells, they were washed three times and suspended in Dulbecco PBS (Gibco BRL) buffer. The cell membranes were fixed with 4% formaldehyde in PBS for 15 min at room temperature. The cells were centrifuged and then washed three times with deionized water. In the final step, the cells were resuspended in a 70% ethanol solution at a concentration of ~1x107 cells/mL.
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RESULTS AND DISCUSSION This new system was validated using a protein to understand the suitability of the system
as a charge-monitoring MS. All key parameters were adjusted for cytochrome c molecules. For signal detection, a homemade power amplifier was used to apply a 250-kHz AC with the amplitude adjustable to 600 Vp-p. The system was pumped down using three rough pumps, and no turbo pump or buffer gas was used. The pressure was adjusted to the 20-mTorr region by adjusting the pump valves. A standard mass-selective instability mode was used to select the ions of interest [30]. The frequency was scanned to determine the m/z values of the particles. Before the collection of mass spectra, the charge detector was calibrated by using a one-millivolt test voltage and measuring the resulting signal to calculate the charge number for each millivolt. The electronic noise was less than 250 electrons. Figure 1B shows the cytochrome c ESI-MS spectrum. To improve the resolving power, the scan time was decreased to 0.5 ms, and the collected spectrum was averaged 10 times. The collected spectra accurately reflected the cytochrome c spectra with a reasonable resolving power (m/∆m=100 at m/Z=900). For high-mass particles, the mass spectrometer was also tested using microparticles. Figures 1C, D and E show three different trapped particle photos inside the trap. The first image was taken at high pressure (20 mTorr), and other two were taken at low pressure (10-5 torr). In Fig D and E, the particles were not stabilized and moved in different orbits and radii’. As shown in Figure 1A, the ESI spray was oriented right above the interface cone in the perpendicular position. The syringe pump was also placed in the perpendicular position to provide a smooth flow into the ESI spray. Figure 2 shows a typical mass spectrum of different polystyrene (PS) particles. The amplitude of the peak related to the charge number of particles helps to obtain the particle mass. Most mass spectrometers measure the m/z of particles; however, the charge (z) is not measured directly in most cases. Because we can measure the charge number of a particle, we can calculate the mass of that particle. The width of the mass distribution represents the synthesis purity of particles of the same size. The number of peaks depends on the number of trapped particles. The ESI interface plays an important role. Critical parameters include the flow rate, applied potential, gas flow and orientations of injector and syringe pump. The primary concern is the gravitational force. Heavy particles can easily precipitate in the syringe or the capillary. Therefore, all parts of ESI spray were in a perpendicular position. The clogging becomes severe 6 ACS Paragon Plus Environment
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for larger particles. Moreover, the flow rate should not be too low. Thus, the best way is to use a lower concentration and higher flow rates. The flow rates were mostly set as 10 µl/min. The range is between 5 and 12 µl/min. In all experiments, no gas flow was used. Apparently, applying gas pressure increases the kinetic energy of the particles, and most of the particles just pass through the trap. For cell experiments, the situation is different; cells have a soft outer tissue, and a higher kinetic energy forces the cells to collide with the chamber walls. Another issue during the spray process is particle desolvation. Application of a high gas flow does not provide enough time for desolvation of the particles before the trap. The applied voltage is another important parameter. The applied ESI voltage should be selected carefully. Although the number of trapped particles is low, the solid particles can be trapped without the application of a voltage; a gas flow alone will be enough for trapping. This situation is also true for apolar particles that repel the apolar solvent molecules around them. The cells have both a polar and an apolar outer layer. Although the range of applied ESI voltage could be changed from 1000 to 5000 V for the cancer cells, there was a 3000-V DC potential applied throughout the experiments. For instrument calibration, 6 different PS particles were used, and Figures 2A, B, C, D, E and F show the m/z spectra and related histograms with sizes of 2, 4, 6, 9, 11 and 15 µm. Each figure also shows the corresponding microscope image of the PS particles. All of the calibration solutions and final cell solutions were prepared under the same conditions to avoid any effect on mass calibrations. The number of trapped particles gradually decreases as the particle size increases. Smaller particles are easy to trap; however, multiple particle formation will significantly change the histogram profile. As in Figure 2A, the mass distribution becomes very wide. The solvent cluster formation is also an important parameter for measuring small particles. Solvent cluster formation cannot be avoided, and the solvent particle spectra should be obtained. For each particle size, the mass was measured by averaging several measurements. The trapping efficiency of small particles is very high, and more than one hundred particles can be trapped each time; however, this number will decrease as the particle size increases. The mass distribution of the particles can be obtained using m/z and the particle charge number. The calculated average masses shown in Table 1 corroborate the theoretical calculations for different particles. Although the labeled size distribution of the PS particles is small, the experimental results show a broader distribution due to the cluster formation of the particles. Figure 2A 7 ACS Paragon Plus Environment
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confirms this problem. This may also be due to particle synthesis. As shown in Figure 2E, the histogram shows a broad and different mass distribution.
Red Blood Cells. The most significant challenge in performing ESI QIT MS of particles comes from the solvent particles that are formed during the evaporation process. A fast solvent evaporation obtains a significant amount of energy from the droplets, and the droplets freeze before complete evaporation. This process creates charged particles, which may interfere with the mass spectra. Solvent particle interference is dominant in the small m/z region; however, in the higher mass region, there is almost no solvent effect. Therefore, it is important to primarily use low-polarity organic solvents to avoid the formation of larger solvent particles. Figure 3A shows the mass spectra of the PS particles alone and the related histogram for calibration. PS particles are not very pure and give two peaks because of the impurity inside the solution. Figure 3B shows the m/z and histogram spectra of the 70% ethanol-water system. Red blood cells are our first choice for testing our newly designed ESI QIT MS. The sizes of the red blood cells were approximately 6 µm; however, they did not have a spherical structure. Before the mass measurements of the RBC, the cell membrane was fixed using formaldehyde. Figure 3C shows the mixture of the 7-µm PS and fixed RBC. Using identical instrumental parameters, only the solvent particle mass spectrum was collected and compared with the cell mass histogram and the histogram of the 7-µm PS particles. Figure 3D shows the mass spectra of the RBC and related histogram. The mass of the solvent particles (4.0 x 1013 Da) almost overlaps with the RBC mass (5.0 x 1013 Da). Although the mass of the solvent particles is close to the cell mass, as shown in Figures 3B and D, the numbers of the solvent particles and RBCs are very different. Therefore, it is not difficult to distinguish the RBC particles from the solvent particles. The number of cells in the solution is an important parameter. If the concentration of particles is not sufficient inside the solution, it will be difficult to distinguish the solvent particles from the sample particles.
Cancer Cell Studies. The most significant challenge for cell MS is the size of the cells. The bigger the size, the lower the number of particles that can be trapped; for an informative histogram, more experimental results are needed. For cancer cell studies, different sizes of MCF7 cancer cells were used. Before the measurements, all of the cancer cells were treated in the same way, and in the final step, they were suspended in an ethanol/water (70/30) solvent mixture. 8 ACS Paragon Plus Environment
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If the cancer cells are immersed directly in a 70% ethanol solution, they will explode, and no intact cell will remain in solution. Instead of directly immersing the cells in 70% ethanol solution, the cells were transferred into a 3.7% formaldehyde solution to crosslink the cell walls. After washing and checking the cells under a microscope, the cells were transferred into a 70% ethanol solution. After crosslinking, all cells were dead, but their membranes were strong enough to withstand the osmotic pressure difference. The related mass region was calibrated using standard PS particles from 11 µm to 15 µm, and PS standards were also suspended in the same solvent system. The mass distribution of the MCF-7 cells shows a large variation. Figure 4A shows a microscope image of the suspended MCF-7 cells inside the culture medium. The size variation can be easily seen. The size distribution for the same cells can be observed in the histogram in Figure 4A. The black counts are live cells, and the red counts are dead cells. If we assume that all cells are uniform and that the average size of the cells is 14.1 µm, the masses of the cells should be close to the mass of the 15-µm PS particles. Three different MCF-7 cell sizes were utilized in this study. Figure 4B shows the 14.1-µm MCF-7 cell mass spectrum, histogram and suspended fixed cells with 15-µm PS particles inside the solvent system. The histogram spectrum shows the mass distribution of cancer cells from 1.0x1014 to 15.0 x 1014 Da. The histogram has three fitted peak regions or three different mass regions. If we assume that all cells have the same size, the histogram spectrum should be similar to the histogram of the 15-µm PS particles. Most particles were populated in the 2-6 x 1014 Da mass region, which corresponds to a size of 9 to 13 µm for the cells. It is possible that not all of the larger cancer cells survive during the spray process. Figure 4C shows the 13.1-µm MCF-7 cancer cells. By using the size of cells, the calculated average mass of cells should be approximately 6 x 1014 Da. The mass peak is approximately 4.5 x 1014 Da, and in the higher mass region, another small population arises, which may be due to dimers and trimers of cells. Similarly, the mass spectrum of the 11.1 µm MCF-7 cells was taken and is shown in Figure 4D. The theoretical mass should give approximately 3.85 x 1014 Da. The experimental results showed that the particles were distributed between 1 and 4 x 1014 Da, which represented the 11.1 µm MCF-7 cells. The mass shift from the theoretical calculations is most likely due to the cell membrane fixing process. During this process, all conditions should be adjusted very carefully to protect the osmotic pressure and keep it equal both inside and outside of the cell. Any difference will cause the cell to shrink or explode. During the cell wall crosslinking with formaldehyde, the process should be continuously monitored under the 9 ACS Paragon Plus Environment
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microscope. Otherwise, most cells can aggregate and form larger clusters. All m/z values and related charge numbers of the cells can reveal the approximate mass of the particles. A comparison of the charge number of PS particles reveals an approximate linear relationship between the mass and charge number of the particles. Similarly, the cell particles carry the same amount of charge depending on their size. The mass calculation requires the charge number of the available particles. There is an important static electricity property that should be considered regarding the charge of the particles. Static electricity is a localized charge, and it is not certain that the charge distribution is even on the surface of particles. Table 1 shows the mass distribution of all of the PS particles and MCF-7 cells. The particle mass distribution of the PS particles should exhibit a narrow mass distribution depending on the uniformity of the particles. Although the mass of the particles is uniform, they may form dimers, trimers or larger clusters. For small particles, this is important for distinguishing the particle clusters due to the small mass shift and low resolving power; however, for larger masses, multiple particle clusters can be distinguished easily because of the large mass shift. For cell mass spectrometry, the particles exhibit a larger mass distribution due to the lack of uniformity. This should be analyzed very carefully to understand the mass spectra of particles. As shown above, although cancer cells have a very broad mass distribution because of their continuous growth until division and the formation of smaller cells after division, RBCs show narrow distributions. The ESI QIT MS we built for cell measurements is a more practical tool to reduce the analysis time to determine red blood cell distributions associated with the diseases compared with LIAD measurements performed in the past or microscope examination. Since RBC is not even spherical (dish shape), it is more difficult to estimate the size. When many red blood cells need to be analyzed by microscope, it is very time-consuming. It is well known that cancer cells sometimes have both small and giant cells. The percentage of larger cells is related to the cancer prognosis38. Our technique can be used to distinguish among different types of cancer cells easily. Quantitative estimation of the cell size can also be obtained. The distinction of cancer cells versus normal cells relies on cancer biomarkers. A typical approach is an antibody which is specific to a special kind of cancer cell to attached onto a magnetic nanoparticle so that these specific cancer cells can be pulled out by a magnetic field. For example, circulating tumor cells (CTC) are often pulled out by anti-EpCAM-functionalized (EpCAM: epithelial cancer marker) supported lipid bilayer39. Our technique can be used to measure the mass distribution of 10 ACS Paragon Plus Environment
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cancer cells after they are pulled out from the biomarkers. The percentage of cancer cells compared to the total cells can also be estimated. If giant cells are observed in the removed tissue of a brain tumor, the possibility of this brain tumor is malignant is high.38
CONCLUSIONS An ESI-QIT-MS was built for rapid mass analysis of large particles and cells in their
native environment. This new system does not require the application of different voltages to the interface lenses. The only voltages applied to the whole system were the DC voltage and RF trapping voltage to the ESI source and ion trap, respectively. No nebulizing gas was applied to the spray source. The masses of cancer cells and their mass distributions were measured successfully. In comparison with other laser-based methods, the sample preparation is much simpler and takes less time. This method can easily provide information for the measurement of solution-based particles without drying.
Acknowledgement
We acknowledge grants from Ministry of Science and Technology (MOST 102-2113-M-001002-MY5) and National Health Research Institutes (NHRI-EX106-10603EI) in Taiwan and the Genomics Research Center, Academia Sinica. REFERENCES (1) (2) (3)
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Chem. Int. Ed. 2001, 40, 982. Tito, M. A.; Tars, K.; Valegard, K.; Hajdu, J.; Robinson, C.V. J. Am. Chem. Soc. 2000, 122, 3550–3551. Schriemer, D. C.; Li, L. Anal. Chem. 1996, 68, 2721–2725. Wenzel, R. J.; Matter, U.; Schultheis, L.; Zenobi, R. Anal. Chem. 2005, 77, 4329–4337. Fuerstenau, S. D.; Benner, W. H. Rapid Commun. Mass Sp.1995, 9, 1528–1538. Chang, H.C. Annu. Rev. Anal. Chem. 2009, 2, 169. Wuerker, R. F.; Shelton, H.; Langmuir, R.V. J., Appl. Phys. 1959, 30, 342–349. Cahill, J. F.; Darlington, T. K.; Fitzgerald, C.; Schoepp, N. G.; Beld, J.; Burkart, M. D.; Prather, K. A. Anal. Chem. 2015, 87, 8039-8046. Cahill, J. F.; Fei, H. ; Cohen, S. M.; Prather, K. A. Analyst. 2015, 140:1510-1515. Keifer, D. Z.; Shinholt, D. L.; Jarrold, M. F. Anal. Chem. 2015, 87, 10330-10337 Bandura, D .R.; Baranov, V. I.; Ornatsky, O. I.; Antonov, A.; Kinach, R.; Lou, X.; Pavlov, S.; Vorobiev, S.; Dick, J. E.; Tanner, S. D. (2009). Anal. Chem. 2009, 81, 6813– 6822. Golovlev, V.V.; Allman, S. L.; Garrett, W. R.; Chen, C. H. Appl. Phys. Lett.1997, 71, 24702–3266. Golovlev, V. V.; Allman, S. L.; Garrett, W. R.; Taranenko, N. I.; Chen, C. H. Int. J. Mass Spectrom. 1997, 169, 69–78. Pérez, J.; Ramírez-Arizmendi, L. E.; Petzold, C. J.; Guler, L. P.; Nelson, E. D.; Kenttämaa, H. I. Int. J. Mass Spectrom. 2000, 198, 173–188. Shea, R. C.; Petzold, C. J.; Campbell, J. L.; Li, S.; Aaserud, D. J.; Kenttamaa, H. I. Anal., Chem. 2007, 79, 2688–2694. Schlemmer, S.; Wellert, S.; Windisch, F.; Grimm, M.; Barth, S.; Gerlich, D. Appl. Phys. AMater. 2004, 78, 629–636. Peng, W.; Yang, Y.; Kang, M.; Lee, Y. T.; Chang, H. J. Am. Chem. Soc. 2004, 126, 11766– 11767. Schlemmer, S.; Illemann, J.; Wellert, S.; Gerlich, D. J. Appl. Phys. 2001, 90, 5410–5418. Twerenbold, D. Rep. Prog. Phys. 1999, 59, 349–426. Xiong, C.; Zhou, X.; He, Q.; Huang, X.; Wang, J.; Peng, W.-P.; Chang, H.-C.; Nie, Z. Anal. Chem. 2016, 88, 11913–11918. Bahr, U.; Lautz, C.; Strupat, K.; Schtirenberg, M.; Hillenkap, F. Inter. J. Mass Spectrom. 1996, 153, 9–21. Aksenov, A. A.; Bier, M. E. Journal of the American Society for Mass Spectrometry 2008, 19 (2), 219–230. Keifer, D. Z.; Shinholt D. L. ; Jarrold, M. F., Anal. Chem., 2015, 87 (20), pp 10330–10337. Benesch, J. L. P.; Ruotolo, B. T.; Simmons, D. A; Robinson, C.V. Chem. Rev. 2007, 107, 3544–3567. Wong, S. F.; Meng, C. K.; Fenn, J. B., J. Phys. Chem. 1988, 92, 546-550. Nohmi, T.; Fenn, J. J. Am. Chem. Soc. 1992, 114, 3241–3246. Schwartz, B. L.; Rockwood, A. L.; Smith, R. D., Rapid Commun. Mass Sp., 1995, 9, 1552-1555. Kozak, K. R.; Moody, J. S.; Neuro. Oncol. 2009; 11, 833-841 Tsai, W.; Chen, J.;Shao, H.; Wu, J.; Lai, J.; Lu, S.; Hung, T.; Chiu, Y.; You, J.; Hsieh,P.; Yeh, C.; Hung, H. ;Chiang, S.; Lin, G. ;Tang, R. ; Chang, Y. Sci. Rep. 2016, 6, 24517 12 ACS Paragon Plus Environment
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FIGURES
Figure 1. A shows the schematic of the ESI QIT MS, B is the ESI MS spectrum of cytochrome c. The data were collected by using a charge detector, and the concentration of the cytochrome c solution was 1x10-6 M. C, D and E show different particle trapping profiles for the ion trap instrument.
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Figure 2. Mass spectra, mass distributions and images of PS particles; A) 2 µm, B) 4 µm, C) 6 µm, D) 9 µm, E) 11 µm, and F) 15 µm. The images of the PS particles were taken using a microscope with the same magnification. Red lines show the curve fitting for the histogram. 14 ACS Paragon Plus Environment
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Figure 3. A shows the mass spectrum for 7-µm PS particles and histogram, sprayed in a 70% ethanol solution, B shows the 70% ethanol solution mass spectrum and histogram. C shows a mixture of the 7-µm PS and fixed RBC in a 70% ethanol solution, and D is the mass spectrum and histogram of RBC.
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Figure 4. A shows an image of MCF-7 14.1-µm cancer cells and their size distribution histogram. The average mass is 14.1 µm, but it is populated around the 13 µm region. B, C and D show the mass spectra for MCF-7 14.1-, 13.1- and 11.1-µm cancer cells and their mass histograms.
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Table 1. Different sizes of PS particles and information of MCF-7 cancer cell molar mass. Particles 2 4 6 7 9 11 15
Blood Cell MCF-7 14.1 MCF-7 13.1
MCF-7 11
Theoretical Experimental MW (Da) MW (Da) 2.30E+12 1.85E+13 6.24E+13 9.91E+13 2.11E+14 3.85E+14 9.75E+14 -
2.17E+12 1.89E+13 5.80E+13 6.37E+13 1.95E+14 3.65E+14 9.20E+14 5.20E+13 2-6 E+14 4.50E+14 1-3.6E+14
Area*
Center*
Width*
W Error*
2.18E+14
2.18E+12
2.22E+12
9.53E+10
2.41E+14 2.88E+15 2.15E+14 1.33E+15 7.65E+15 2.47E+16 1.65E+12 4.95E+15 1.08E+15 1.70E+15
1.89E+13 5.80E+13 6.37E+13 1.96E+14 3.66E+14 8.86E+14 1.65E+12 5.85E+14 4.50E+14 3.44E+14
5.21E+12 1.27E+13 1.88E+13 2.96E+13 9.54E+13 5.15E+14 1.65E+12 1.70E+14 3.50E+14 6.37E+13
9.94E+10 2.14E+11 1.65E+12 5.95E+11 3.65E+12 7.43E+12 1.65E+12 2.06E+13 2.50E+13 5.07E+12
*
Area refers to the area under the fitting curve. *Center refers to the center point of the fitted curve (red line). *Width is the width of fitted curve and *W Error is the width calculation error.
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