Direct Characterization of Bulk Samples by Internal Extractive Electrospray Ionization Mass Spectrometry
Hua Zhang, Haiwei Gu, Feiyan Yan, Nannan Wang, Yiping Wei, Jianjun Xu and Huanwen Chen*
H. Zhang, H. Gu, F. Yan, N. Wang, Prof. H. Chen
Jiangxi Key Laboratory for Mass Spectrometry and Instrumentation, East China Institute of Technology
Nanchang 330013 (China)
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Y. Wei, J. Xu
Department of Cardiothoracic Surgery
The Second Affiliated Hospital ofNanchangUniversity
Nanchang, 330006 (China)
[]This work is supported by National Natural Science Foundation of China (No. 21225522, No. 21175019).
Supplementary information
Samples and chemical reagents for the experiments
Strawberries, garlic leaves,garlic bulbs, radish taproots, Doublemintchewing gum(Kent®, USA) and pork meat were purchased from local supermarket. The Chlorophytum comosum leaves and the canna indica L. flowers were picked from campus of ECIT. All the raw samples were analyzed by iEESI-MS directly with no/minimum pretreatment. Methanol (HPLC Grade) was bought from ROE Scientific Inc. (Newark, USA), and TNT was purchased from Chem Service®, Inc. (PA, USA). C18 was bought from Agilent (USA)with bead sized of 5 m and the other chemicals were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai) with the highest purity grade available. The deionized water used for the experiments was provided by the chemistry facilities at ECIT.
The lung cancer tissue andpericarcinomatous tissue samples were clinically obtained from tencases of patients in the Second Affiliated Hospital of Nanchang University, with full consent of the volunteers. This research was approved by Hospital Institutional Review Board of the SecondAffiliatedHospital to NanchangUniversity. Inclusion criteria for patients enrolled into this study were as follows: (1) diagnosis of non-small cell lung cancer was pathologically confirmed; (2) no other malignancies were accompanied; (3) no other lung diseases were diagnosed, such as bronchiectasis or pulmonary infection; and (4) chemotherapy or radiation treatment was not applied previously. All samples included two matched pairs, namely one piece of tumor tissue and a separate piece of adjacent normal lung tissue (≥5cm away from tumor), were collected within 5 minutes by a professionally trained surgeon after the diseased tissue was resected, and immediatelypreserved at -80℃in a refrigerator. Special care was taken to ensure safety of the operators for performing lung cancer tissue measurements, with onsite guidance from doctors of the hospital.All the lung cancer tissue samples were stored in a ultra-low temperature refrigerator at-80℃ before the iEESI-MS experiments without any further treatment and used for iEESI-MS directly without treatment.
Mass spectrometry analysis
All mass spectra were recorded in the positive/negative ion detection mode using a commercial linear ion trap mass spectrometer (LTQ-XL, Finnigan, San Jose, CA).
To facilitate and standardize the sample manipulation, the sample tissues were cut into the same dimensions (with certain shape e.g., a small cuboid(10×20×2 mm3), the thickness was 2 mm, which was slightly larger than the diameter of the fused silicon capillary tubing). An ESI tip was inserted into the sample, allowing a gap of 2–5 mm between the ESI tip orifice and the edge of the apex of tissue sample. The small sample was held by the fused silicon capillary, so that the apex of the tissue intentionally pointed to the ion entrance of the mass spectrometer (Figure 1). The optimal distance between the tissue apex and the mass spectrometer inlet was 4–8 mm, in current experimental scenario. An extraction solvent (e.g., methanol) biased with high voltage was fed at a flow rate of 1~8 µL/min by a syringe pump (250 µL, Hamiltion, USA). The analytes were extracted by the solvent while the solvents were running through the tissue section, producing a spray of charged droplets carrying endogenous chemicals toward the adjacent mass spectrometer inlet. The optimized temperature of the heated capillary was set to be 150°C.Other parameters were set as default values of the instrument and no further optimization was performed.
The comparison experiments of spray-MS and DESI-MS were performed in conventional way according ref1,2. The voltage was set at +4.5 kV in the leaf spray experiments under the positive ion detection mode during the detection of chewing gum with 8 µL methanol added per time. To detect the spiked TNT easily, negative ion detection mode was setup with a voltage of -4.5 kV applied on the chlorophytum comosum leaf. As to DESI-MS the voltage was set at +4.5 kV in the detection of chewing gum, the same voltage intensity as iEESI-MS and leaf spray-MS. Methanol as ESI extract solvent was infused at a flow rate of 8 µL/min and nebulized by a nitrogen sheath gas (0.8 MPa, measured at the outlet of the nitrogen tank) toward the chewing gum surface.
The full scan mass spectra were recorded under an average time of 2 minutes subtraction of the background. For tandem mass spectrometry,the precursor ions were isolated with a mass window width of 1.5 Da and the collision-induced dissociation (CID) was performed under collision energy of 15~30% (Thermo LTQ arbitrary unit). The tandem mass spectra were collected with enough scan time, usually a combination of 20 scans.
Separation of aniline and arginine loaded on C18 column
As the schematic illustration shown in Figure S9a,C18 (bead size 5 m, Agilent, USA) was filled in one end of the fused silicon capillary (ID 0.25 mm, OD 0.30 mm Agilent, USA) formed a C18 column with 8 cm length, and the other end of the fused silicon capillary was fed by a syringe pump (250 µL, Hamiltion, USA) at a flow rate of 1 µL/min. In the first place, 5 µL mixture of aniline-arginine (1/1,v/v) was loadedat the end of the capillary column as the sample for the separation experiment. The aniline-arginine mixture was pushed forward along the capillary by the ESI solvent (methanol), allowing the separation of the two analytes.
The ion chromatogram showed that the signal of aniline (m/z 94) and arginine (m/z 175) were totally separated by iEESI (FigureS9b), and aniline and arginine were identified by tandem mass spectrometry. The powerful separation capability of iEESI should account for the analyte’s polarity, its solubility in ESI solvent and the fluxion function of the injected solvent. Therefore,samples’ texture, ESI solvent, and the flow rate of the ESI solvent are important factors to iEESI-MS experiments.
Quantitative analyze salbutamol in pork meat
Quantitative analysis of bio-samples without pretreatment is a big challenge for mass analysis. Herein iEESI-MS was performed to analyze the salbutamol (a drug for the treatment of asthma) spiked in pork meat to demonstrate its merit of iEESI for potentially resolving this problem. In this experiment, seven sets of large pork meat tissues were cut with the same dimensions (322 cm) and each set was composed of 4 individual pork tissues. The seven sets of pork tissues were saturated in salbutamol solution (water as solvent, and with a series of concentration gradient (100ppt, 1ppb, 10ppb, 100ppb, 400ppb, 800ppb, 1ppm)) for 5 hours before the procession of iEESI-MS.The same dimension pork tissue sample was saturated in deionized water as blank control.
Salbutamol (MW 239) was detected as protonated molecules (m/z 240) from all the seven sample sets. The parent ions were caught for tandem mass spectrometry, getting the two major characteristic fragment of m/z222 and166 by the loss of H2O and CH2C(CH3)2 as the fragment ions (Figure S10). In the MS3tandem mass spectrometry, the precursor fragment ions of m/z 222 produce three major fragment ions of m/z 204, 166 and 148 by the loss of H2O, CH2C(CH3)2 and [H2O + CH2C(CH3)2], respectively (inset of Figure S10).The characteristic fragment of m/z 222 was chose as the signal of salbutamol for quantitative analysis. The average signal density of salbutamol from each set was found to a linear relationship with the spiked density of the pharmaceutical compound (Figure 5a).
Lung cancer tissues sample analysis
In the differential analysis of lung cancer tissue andpericarcinomatous tissues by iEESI-MS,ten cases of lung cancer volunteers from the Second Affiliated Hospital of Nanchang University took part in this experiment. Guaranteeing the safety of the volunteers, ten sets of lung cancer tissue samples andpericarcinomatous tissue samples were sampled from each patient,respectively. The ten sets of lung tissues(include lung cancer tissue andpericarcinomatous tissue) were detected by iEESI-MS over a relatively wide massrange (m/z 50-2000) in full mass scan without treatment.
Principal component analysis (PCA) of the mass spectral fingerprint data were performed using the Matlab (version 7.8.0 , Mathworks, Inc., Natick, MA, USA). The mass spectral fingerprint data of six sets of lung tissues (include lung cancer tissue andpericarcinomatous tissue) were exported into Microsoft Excel® and arranged using the m/z values as independent variables. The data in the Excels were further imported into Matlab and expressed by the relative abundance normalized to individual base peak. The PCA processing was based on the ‘princomp’ function in the ‘Matlab Toolbox’, and then the results of statistical analysis were graphically presented in the PCA score plots.
Video S1.The color of canna indica L. flowers was bleached as the anthocyanin inside the flower tissue was gradually extracted by the injected methanol solvent with a capillary (fused silicon, ID 0.25 mm, OD 0.30 mm) inserted in directly.
Table S1.Compounds detected from Doublemint chewing gum by iEESI-MS/MS.
Compounds (MW) / Observed ions / MS2 product ions m/zIonic species / m/z
Glucose/fructose (180) / [M + NH4]+ / 198 / 181, 177
Glucose/fructose (180) / [M + H2O +Na]+ / 221 / 203, 191
Glycerol (92) / [M +K]+ / 131 / 113, 74
Glycerol (92) / [M +Na]+ / 115 / 97, 70
Menthol (156) / [M + H]+ / 157 / 137, 111, 81
Menthone (154) / [M + H]+ / 155 / 137, 111, 81
Sucrose (342) / [M + K]+ / 381 / 291, 219, 201
Sucrose (342) / [M + NH4]+ / 360 / 342, 324, 288
Figure S1. The chemical ingredients such as glycerol, menthol, glucose and sucrose in the chewing gum were identified by iEESI-MS.
Figure S2. Distinguished mass spectral patterns recorded from the pork meat sample by different ionization techniques. a) iEESI; b) DESI; c) leaf spray.
Figure S3. Distinguished mass spectral patterns recorded from the garlic leaf sample by different ionization techniques. a) iEESI; b) DESI; c) leaf spray.
Figure S4. Distinguished mass spectral patterns recorded from the intact garlic bulb tissue sample by different ionization techniques. a) iEESI; b) DESI; c) leaf spray.
Figure S5. Detection of TNT under the negative ion detection mode using internal EESI-MS and leaf spray-MS: a) the spectra obtained by internal EESI-MS of chlorophytum comosum with 0.2 µL of TNT (100 ppm, in water) placed on center surface, the inset shows the MS/MS spectra of the radical anion of m/z 227; b) MS/MS spectra of m/z 227 obtained by internal EESI-MS when the flow rate of the extractive solvent turned higher than 12 µL/min resulting the methanol penetrated to the sample’s outer surface, and the loaded TNT was detected; c) MS/MS spectra of m/z 227 obtained by leaf spray-MS of chloroqhytum comosum with 0.2 µL of TNT (100 ppm, in water) placed on center surface.
Figure S6. iEESI-MS spectra of ripe strawberry under the positive ion detection mode, the inset shows the MS/MS spectra of pelargonidin-3-O-glucoside (m/z 433).
Figure S7. iEESI-MS spectra of chewing gum repeated under the conditions: high flow rates (4 µL/min), short distance (4 mm) and humid ambient air (25 ℃, 65 % R.H.). a) small molecules were detected in the spectrum recorded after 20 s; b) large polymers were also showed up in the spectrum recorded after 4 min.
Figure S8.Sequential detection of different metabolites of radish taproot sample by iEESI-MS. a) detection of choline within 15 min; b) detection of valine within 12 min; c) detection of glucose in 35~75 min.
Figure S9. Full separation of aniline and arginine using C18 column by iEESI. a) schematic illustration of the separation of aniline and arginine loaded on a C18 column by iEESI-MS; b) separation of aniline (retention time ~2.33 min) and arginine (retention time ~4.15 min) loaded on a C18 column by iEESI-MS with well-shaped ion chromatogram peaks with full width at half maximum (FWHM) about 20 s. The insets show the MS/MS spectra of protonated aniline (m/z 94) and arginine (m/z 175).
Figure S10. MS/MSiEESI mass spectra of the protonated salbutamol (m/z 240) from the pork meat, the inset shows the MS3 spectra of the major fragment ion (m/z 222) in MS/MS.
Figure S11. iEESI-MS spectra of the lung cancer and pericarcinomatous tissue samples: a) lung cancer tissue samples; b) pericarcinomatous tissue samples.
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