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Detecting Low-abundance Molecules at Single-cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer Xingyu Si, Xingchuang Xiong, Sichun Zhang, Xiang Fang, and Xinrong Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03390 • Publication Date (Web): 17 Jan 2017 Downloaded from http://pubs.acs.org on January 21, 2017
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Analytical Chemistry
Detecting Low-abundance Molecules at Single-cell Level by Repeated Ion Accumulation in Ion Trap Mass Spectrometer Xingyu Si, Xingchuang Xiong,† Sichun Zhang, Xiang Fang,*,† and Xinrong Zhang* Beijing Key Laboratory for Microanalytical Methods, Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China ABSTRACT: Low-abundance metabolites or proteins in single-cell sample were usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated to repeat cycle number. When repeatedly accumulated by 25 cycles, the sensitivity of adenosine triphosphate was increased by 22 fold within 1.8 seconds. Our technique could stably detect low-abundance ions, especially MSn ions, at single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ~0.2 MCF7 cell. The strategy presented in this study offers a possibility to aid single-cell analysis by enhancing MS detection sensitivity.
Life science researches have entered the era of single cell.1,2 Therefore, analytical methodologies capable of quantifying metabolites, transcripts, and proteins at single-cell level are necessary to broaden our horizon from cell populations to an individual cell.1,2 Among the analytical techniques, mass spectrometry (MS) was a widely used technique in single-cell analysis due to its label-free property and the capability to detect multiple components simultaneously,3-5 including electrospray ionization (ESI)-based MS,6-18 laser desorption ionization-based MS,19-23 secondary ion MS.19,24,25 Although
some MS techniques, such as laser ablation electrospray ionization (LAESI),21,22 probe ESI,10-13 and direct ESI,17,18 allowed the direct analysis of single cells without any dilution, many techniques required significant dilution of a single cell (~10 pL for a typical somatic animal cell) to a large volume (nL~μL) prior to MS analysis. Such sample dilution would lower the concentration of each analyte, calling for a need to further enhance the sensitivity of contemporary MS for their detection. If more ions could be obtained and transferred to mass analyzer, it would be possible to detect low-abundance molecules consistently by MS.26 Many researches have been conducted to enhance MS sensitivity by increasing the total amount of analyte ions directly or indirectly. For instance, laser-induced postionization could improve sensitivity of lipids, liposoluble vitamins, and saccharides in MALDI MS by increasing ion yield.27 Radio-frequency ionization of volatile and semivolatile organic molecules in ion cyclotron resonance MS could generate ions with high efficiency.28 Utilizing report-molecule-tagged gold microparticles to capture target protein, MS could achieve attomolar detection of target proteins.29 In addition, previous works indicated that ion trap has the potential to capture and accumulate selected ions for long period, thus increasing the MS sensitivity.30,31 These results reminded us of developing an ion trap-based technique to accumulate analyte ions in MS and achieving the detection
of low-abundance component at single-cell level. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is based on the capability to accumulate ions by linear ion trap. Our method could lead to 3-22-fold increase of the sensitivity of various molecules by repeatedly accumulating selected ions in linear ion trap for 25 cycles within 1.8 seconds. The technique developed in this study offers a possibility to aid single-cell analysis by enhancing MS detection sensitivity.
EXPERIMENTAL SECTION Materials. Reserpine, 5-methylcytosine·HCl, angiotensin II, malic acid, adenosine triphosphate (ATP), adenine, cytosine (Cyt) standards, methanol, formic acid, ammonium acetate were all bought from Sigma-Aldrich. Caffeine standard was purchased from CHEM SERVICE. Imatinib standard was purchased from J&K Scientific. The purified water was obtained from a filtration system (ThermoFisher Scientific). The capillary used as the tubing of flow injection and sample loop was purchased from SGE Analytical Science (tubing of flow injection: ID = 100 μm, OD = 363 μm; sample loop: ID = 25 μm, OD = 150 μm) (Figure S1). The nanospray emitter was purchased from SilacaTip with a tip diameter of 10 ± 1 μm (FS360-75-10-N-20-C12). MCF7 cells were purchased from ABGENT. Cell-culturing-related reagents and materials were bought from Corning (NY, USA).
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scan mode and repeated accumulation mode could be switched easily in the software without damaging the hardware or software of MS.
Figure 1. (a) The normal procedure for MS2 scan in ion trap MS and (b) the procedure of repeated ion accumulation by ion trap MS employed in our method.
Mass Spectrometry Analysis. All the mass spectra were recorded by an ion trap mass spectrometer (Orbitrap Elite, Thermo Scientific). Only the ion trap analyzer was used in this research. Nanospray voltage: +1.80 kV for positive ion mode, or -1.80 kV for negative ion mode; Capillary temperature: 300 °C; S-lens RF level: 65 %; microscans: 1. Automatic gain control was always off. A commercial nanospray ionization source was used. The distance between the tip of the nanospray emitter and the MS inlet was ~5 mm. Samples were injected by a syringe pump with a constant flow rate of 1 μL·min-1. Modifying Scan Function of MS. Since the scan function of ion trap MS was so complicated with many parameters optimized systemically, that the best strategy to accomplish repeated ion accumulation was to modify the scan function as simply as possible without modifying the value of each parameter. Actually, only additional steps of ion injection and isolation with original parameters were inserted into the scan function repeatedly before collision-induced dissociation (CID), accumulating the low-abundance ions gradually in the ion trap. We developed a software tool based on ITCL (Ion Trap Control Language), which regarded the scan function of original MSn scan of each ion as scan templates (Figure S2). When conducting repeated ion accumulation, the scan functions of ion initialization, ion injection, and ion isolation of original parameters (16 steps of scan function) were copied and inserted into scan sequence of MS for multiple times. Every 16 steps of scan function inserted into scan sequence meant precursor ions accumulated by one more cycle. Subsequent scan function of CID and mass analysis remained unchanged. Therefore, the repeated accumulation of ions was achieved in ion trap MS. The scan function could be repeated by 25 cycles at most, which was limited by the internal memory of the commercial mass spectrometer. The normal
Figure 2. (a) The MS spectra of [5mCyt+H]+ with ion accumulated of 1, 5, 10, 15, 20, and 25 cycles (r1-r25) when measuring standard solution of 20 nmol∙L-1 5mCyt. (b) The relationship between MS intensity and cycle number for [5mCyt+H]+, [5mCytNH3+H]+ in MS2, and [5mCyt-NH3-HCN+H]+ in MS3. Repeated accumulation mode of 1 cycle (r1) = normal mode.
Optimization of Ion Injection Time. Ion injection time was a crucial parameter to maximize the intensity of analyte ion under repeated accumulation mode. For instance, the optimized ion injection time for 50 nmol·L-1 5-methylcytosine (5mCyt) under normal scan mode was 15 milliseconds, whereas the optimized ion injection time under repeated accumulation mode of 25 cycles was 5 milliseconds (Figure S3). All the data presented in this paper was obtained at the optimized ion injection time. Comparison between Normal Scan Mode and Repeated Accumulation Mode. Standard solutions of reserpine, 5mCyt, caffeine, angiotensin II, adenine and Cyt were prepared in methanol/water (v/v = 1:1) supplemented with 0.1% formic acid, while the solutions of malic acid and adenosine triphosphate were supplemented with 1 mmol·L-1 ammonium acetate. Analyte ions were detected at optimized ion injection time, NCE and activation Q (Table S1). Under repeated accumulation mode, all the ions were accumulated by 25 cycles.
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Analytical Chemistry
Table 1. MS intensity of different ions under normal mode and repeated accumulation mode of 25 cycles. MSn
Precursor or product ion
m/z
[5mCyt+H]+
126.0
[5mCyt-NH3+H]+
109.0
MS2
[5mCyt-NH3-HCN+H]+
82.0
MS3
[Reserpine+H]+
609.3
[Reserpine-C10H11
ON+H]+
[Reserine-C10H12O5+H]+
Intensity (n = 5)
Increased folds of intensity
normal mode
r25 mode[a]
(1.16 ± 0.08) × 103
(3.25 ± 0.43) × 103
2.8
0.901 ± 0.126
7.72 ± 1.41
8.6
0.222 ± 0.272
2.04 ± 0.54
9.2
(1.79 ± 0.18) × 102
(1.12 ± 0.02) × 103
6.3
448.3
MS2
0.464 ± 0.315
3.39 ± 1.52
7.3
397.3
MS2
0.583 ± 0.538
4.23 ± 1.45
7.3
[Angiotensin+H]+
1046.8
49.5 ± 2.67
(3.06 ± 0.18) ×
[Angiotensin-NH3+H]+
1029.7
MS2
0.231 ± 0.250
2.25 ± 0.99
9.7
[Angiotensin-Asp+H]+
931.7
MS2
0.664 ± 0.556
3.83 ± 1.64
5.8
[Angiotensin-Phe+H]+
899.7
MS2
0.151 ± 0.202
1.53 ± 0.77
10.1
784.6
2
0.651 ± 0.409
5.77 ± 0.74
8.9
(3.22 ± 0.41) × 102
(2.04 ± 0.14) × 103
6.3
2
6.2 6.3
[Angiotensin-Phe-Pro+H]
+ [b]
[Angiotensin+2H]2+ [Angiotensin-Phe-Pro+H]
MS
523.9 + [c]
[Pro-Phe+H]+ +
102
784.7
2
MS
76.0 ± 19.3
(4.75 ± 0.29) × 10
263.3
MS2
37.2 ± 10.0
(2.33 ± 0.17) × 102
[Caffeine+H]
195.0
[Caffeine-CH3CNO+H]+
138.0
MS2
[Caffeine-CH3CNO-CO+H]+
110.0
MS3
[Malic acid-H]-
133.0
[Malic acid-H2O-H]-
115.0
[ATP-H]-
506.3
[ATP-H3PO4-H]-
408.1
(1.46 ± 0.08) × 10
MS2 MS2
2
0.511 ± 0.245
(7.77 ± 0.23) × 10 2.77 ± 0.73
2
6.2
5.3 5.4
0.0782 ± 0.1428
1.30 ± 0.44
16.7
3.78 ± 0.47
21.1 ± 1.8
5.6
0.521 ± 0.037
5.97 ± 0.44
11.5
0.788 ± 0.033
10.9 ± 0.9
13.8
0.314 ± 0.035
7.11 ± 1.10
22.6
[a] repeated accumulation mode of 25 cycles. [b] dissociated from [Angiotensin+H]+. [c] dissociated from [Angiotensin+2H]2+.
Cell Culture and Hydrolysis. MCF7 cells were grown in Dulbecco’s modification of Eagle’s medium with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in a humidified incubator containing 5% CO2. To harvest the cells, added 1 mL trypsin/EDTA in a 6-cm cell plate containing ~106 adherent MCF7 cells at room temperature, and incubated the cells for 3 minutes until the cells were suspended. Next, transferred 1 mL cell suspension into a 1.5-mL tube, centrifuged at 2000 rpm for 10 minutes and removed the supernatant. Resuspended the harvested MCF7 cells using 1 mL 0.9% NaCl water solution and counted the cells. Transferred approximately 1.0 × 106 cells to a new tube, centrifuged at 2000 rpm for 10 minutes and removed the supernatant. Afterwards, added 1 mL formic acid into the tube to suspend the cells and then transferred the whole suspension into a 1.5 mL glass vial, heated the vial at 140 °C for 1.5 hours. Evaporated the residual formic acid in a vacuum drier at 60 °C until the cell hydrolysate was totally dried. Finally, 1 mL methanol was added into the vial to resolve adenine, Cyt, 5mCyt, and other components in the lysate. Next the solution was diluted by 500 times in methanol/water supplemented with 0.1% formic acid to the final concentration of 2000 cells per mL. Single-Cell Level Analysis by Mass Spectrometry. In order to inject small-volume samples at single-cell level, a length of 20-centimeter long capillary was cut, serving as the sample loop of six-way valve of MS. The inner volume of the
sample loop was estimated to be ~100 nL (Figure S1). In our case, 500 nL solution contained the hydrolyzed components equivalent to ~1 single cell, and full sample loop of solution contained the hydrolyzed components equivalent to ~0.2 single cell, since we prepared the samples as follows: 106 MCF7 cells were directly hydrolyzed and diluted to the final concentration of 2000 cells·mL-1 in methanol/water (v/v=1:1) supplemented with 0.1% formic acid. Typically, the volume of a single MCF7 cell was estimated to be ~4 pL, therefore each cell was diluted by ~105 times. The MS3 conditions for detection at single-cell level of adenine, Cyt and 5mCyt were optimized using standard solution (Table S2). For analysis at single cell level, ~100 nL diluted cell lysate was injected into MS through six-way valve with the homemade sample loop. For recovery experiments, imatinib standard solutions of 0.5, 5, 50, 500, 2000, and 5000 nmol·L-1 were analyzed with 100 nmol·L-1 imatinib-d8 added as isotope internal standard to make a calibration curve. The isolation width of MS2 was set as 12 to let both [imatinib+H]+ (m/z=494.4) and [imatinibd8+H]+ (m/z=502.4) being isolated in the ion trap. Finally, 300 nmol·L-1 imatinib and 100 nmol·L-1 imatinib-d8 were added into cell lysate to calculated the recovery of imatinib.
RESULTS AND DISSCUSION Ion Accumulated by Multiple Cycles in Ion Trap. In a normal process for MS2 scan in ion trap tandem MS, all the ions were injected into high-pressure ion trap through octopole
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ion guide (Figure 1a). Precursor ions of interest were isolated from other matrix ion by ion trap. CID of precursor ions occurred, generating product ions in the high-pressure ion trap. Next, product ions were transferred to another low-pressure ion trap which acted as a mass analyzer. But in our technique (Figure 1b), we modified the scan function of tandem MS, making the isolated precursor ions stored in the high-pressure ion trap without CID. Subsequently, another batch of ions were injected and isolated from matrix ions in the same highpressure ion trap again. After repeating ion injection and isolation for several cycles, larger amount of precursor ions was accumulated in the ion trap. Finally, product ions generated by CID were analyzed and a stronger signal would be obtained by our method. To demonstrate the accumulation of low-abundance ions resulted from increasing cycle number of ion injection and isolation, we prepared standard solutions of 20 nmol·L-1 5mCyt and 10 pmol·L-1 reserpine in methanol/water (v/v=1:1) supplemented with 0.1% formic acid. Both samples were analyzed under repeated accumulation mode of different cycle number. Figure 2a, S4a showed the MS spectra of [5mCyt+H]+ and [Reserpine+H]+ with ion accumulated for 1, 5, 10, 15, 20, and 25 cycles, respectively. Obviously, the intensities of both precursor ions were positively correlated with the cycle number (Figure 2b, S4d). Similarly, the intensities of product ions were also gradually increased as the cycle number of ion accumulation increased (Figure S4b-d, S5a, S5b). Averaged by 5 scans, the MS signal of [5mCyt+H]+ was improved by 2.8 fold under repeated accumulation mode of 25 cycles, while that of [Reserpine+H]+ was improved by 6.3 fold (Table 1). The signal of product ions [5mCyt-NH3+H]+, [Reserpine-C10H11ON+H]+, and [ReserpineC10H12O5+H]+ were improved by 8.6, 7.3 and 7.3 fold, respectively (Table 1). Illustrated by the changing trend of the intensity, if more cycles of accumulation could be conducted, the intensity might be increased by more times. When measuring high-concentration solutions, space charge effect of ion trap would be observed which was demonstrated in our following experiment. We prepared a standard 5mCyt solution of 10 μmol·L-1. In both normal mode and repeated accumulation mode, the intensity of precursor ion [5mCyt+H]+ was around 6000 at an optimized ion injection time of 20 milliseconds (Figure 3). The half-peak width of [5mCyt+H]+ was 0.36 and 0.88 respectively (Figure 3). Correspondingly, the mass resolution was 350 and 140 for normal mode and repeated accumulation mode, respectively. This difference in mass resolution confirmed the space charge effect upon repeated ion accumulation when relatively high-concentration solutions were analyzed. Space charge effect was also observed in the MS/MS analysis of 5mCyt. Following CID, the mass resolution of [5mCyt-NH3+H]+ was 520 in normal scan mode and reduced to 420 following ion accumulation (Figure S6). Space charge effect under repeated accumulation mode also indicated that ions were accumulated by our technique. It also meant that, due to the shift in mass accuracy caused by space charge effect, analytes with a concentration beyond the dynamic range of the ion trap were difficult to be identified and quantified. As a result, either or both of the number of cycles of ion accumulation or ion injection time needs to be adjusted to reduce the number of ions accumulated in the trap.
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Figure 3. The MS spectra of [5mCyt+H]+ under (a) normal scan mode and (b) repeated accumulation mode of 25 cycles when measuring standard solution of 10 μmol∙L-1 5mCyt. Various Types of Ions Accumulated in Ion Trap. Our technique could increase the MS sensitivities of a diversity of molecules including large molecules and multiple-charged ions. A polypeptide, angiotensin II, was analyzed under repeated accumulation mode. The MS spectra of singlecharged ion and double-charged ion of 10 nmol·L-1 angiotensin II under repeated accumulation mode and normal mode were compared in Figure S7a, S7b. The MS2 spectra of both ions were showed in Figure S7c, S7d. The MS peaks of precursor ions and product ions were 6.2-10.1-fold stronger with ion accumulation repeated by 25 cycles (Table 1). We also investigated whether the signal of MS3 ions could be improved. For this experiment, a standard solution of 1 nmol·L-1 caffeine was tested. MS spectra illustrated that precursor ion [Caffeine+H]+, MS2 ion [CaffeineCH3CNO+H]+, and MS3 ion [Caffeine-CH3CNO-CO+H]+ became 5.3-16.7-fold stronger with ion accumulation repeated for 25 cycles (Figure S8a-c, Table 1). The results were consistent with our expectation. Usually, biological samples contain a huge number of metabolites,3,14 and multiple isobars or isomers may be mapped to a single mass to charge ratio in a MS spectrum. 3 Therefore, multiple-stage tandem MS is often necessary to identify a specific molecule. The increased intensities of MSn ions should help metabolite identification and quantification. Our method was also applicable to negative ions. Standard solutions of 100 nmol·L-1 malic acid and 100 nmol·L-1 ATP supplemented with 1 mmol·L-1 ammonium acetate were analyzed by MS. Results showed that the intensity of precursor ion [Malic acid-H]- and [ATP-H]- could be increased by 5.6 and 13.8 fold respectively, while that of product ion [Malic acid-H2O-H]- and [ATP-H3PO4-H]- could be increased by 11.5 and 22.6 fold respectively (Figure S9a-d, Table 1). All the above results illustrated that repeated ion accumulation by ion trap MS was a universal strategy to improve the intensity of different ions.
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Analytical Chemistry
Figure 5. (a) The EIC curve of [Cyt-NH3-HCN+H]+ from sample equivalent to ~0.2 MCF7 cell under normal scan mode and repeated accumulation mode of 25 cycles with 3 parallel injections. (b) The MS3 spectra of product ions of Cyt under normal scan mode and repeated accumulation mode of 25 cycles. +
Figure 4. (a) The EIC curve of [Caffeine-CH3CNO-CO+H] with ion accumulated by 1, 3, 5, 10, 15, 20, and 25 cycles. (b) The percentage of detected scan and MS scan speed under ion accumulation mode of 1, 3, 5, 10, 15, 20, and 25 cycles respectively. Repeated accumulation mode of 1 cycle = normal mode.
Detection of Low-abundance Molecules. Repeated ion accumulation not only amplified the MS signal of analyte ions, but also achieved the stable detection of MS1 and MSn ions from low-abundance molecules, which were undetectable under normal scan mode. We prepared two low-concentration solutions: one was 10 pmol·L-1 caffeine supplemented with 0.1% formic acid, the other one was 10 nmol·L-1 ATP supplemented with 1 mmol·L-1 ammonium acetate. According to the extracted ion chromatograms (EIC) of MS3 ion of caffeine, not each MS scan could detect the signal of [Caffeine-CH3CNOCO+H]+ under normal scan mode (Figure 4a, r1). We analyzed 1463 MS scans completed in 3 minutes under normal scan mode and found that caffeine was undetected in 703 scans (Table S3). In other words, only about half of MS scans could detect the target ion. But, as the cycle number of ion accumulation increased, the percentage of detected scan increased dramatically (Figure 4b). With ion accumulation repeated for 25 cycles, although only 107 scans could be completed in 3 minutes, the peak of [Caffeine-CH3CNOCO+H]+ could be found in each MS scan. Besides, other product ions in MS3 spectra could also be detected stably with cycle number increasing, such as m/z 69.0, 83.1, 109.0, and 111.1 (data not shown). Similar phenomenon was observed in the case of ATP. Under normal scan mode, the signal of [ATP-H3PO4-H]- was detected in only 4 out of 1623 MS scans completed in 3 minutes (Figure S10a, Table S4). On the contrary, with ion accumulation repeated for 25 cycles, although the scan speed was decreased, the target ion was found in each scan of 102 scans during 3-minute analysis. According to MS spectra, the signal of [ATP-H3PO4-H]- could be detected with cycle number more than 5, while that of [ATP-H2O-H]- could be detected with cycle number more than 15 (Figure S10b).
Analysis at Single-cell Level. Single-cell samples are usually composed of an enormous number of compounds.3,14 Hundreds of molecules of different elemental composition or isomers may correspond to a single mass to charge ratio in MS spectrum.3 Therefore, MS2, or even MS3, is often necessary to identify and quantify a specific component. For a diluted single-cell sample, the MS2 or MS3 ions of low-abundance components were usually undetectable. The capability of our method to increase the intensity of product ions can help identify or quantify these low-abundance molecules. With optimized conditions, our method was applied to analyzing samples at single-cell level. In our case, only hydrolysate equivalent to ~0.2 MCF7 cell was analyzed for each sample injection. With three parallel sample injections, the MS signals of adenine, Cyt and 5mCyt hydrolyzed from nucleic acids equivalent to ~0.2 MCF7 cell were recorded. The EICs of [Adenine-NH3-HCN+H]+, [Cyt-NH3-HCN+H]+, and [5mCytNH3-HCN+H]+ in MS3 were presented in Figure S11a, 5a, S12a respectively, while the averaged MS spectra were presented in Figure S11b, 5b, S12b. The EIC peak area of [Adenine-NH3-HCN+H]+ was 1106 ± 127 counts under normal scan mode and 6937 ± 621 counts under repeated accumulation mode of 25 cycles (n=3). The EIC peak area of [Cyt-NH3-HCN+H]+ was 102 ± 8 counts under normal scan mode and 590 ± 68 counts under repeated accumulation mode of 25 cycles (n=3). In terms of 5mCyt, it was hard to recognize the EIC peak of [5mCyt-NH3-HCN+H]+ under normal scan mode, so the peak area could not be calculated. However, the EIC peak was recognized easily under repeated accumulation mode of 25 cycles. The peak area was 52 ± 9 counts (n=3). The above data demonstrated that our technique not only increased the intensity of analyte ion at single-cell level by several times, but also enhanced the capability of MS to detect low-abundance molecules. In order to test the recovery of samples analyzed by our method, imatinib, a kind of anticancer drug, was spiked into MCF7 cell lysate solution and quantified with imatinib-d8 as isotope internal standard. In this experiment, the product ions 217.2 and 225.2 were utilized to quantify the concentration of imatinib (Figure 6a). Figure 6b showed the calibration curve of imatinib analyzed under repeated accumulation mode of 25
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cycles, indicating that the relative intensity of imatinib had a fine linear relationship with its concentration from 0.5 nmol∙L1 to 5000 nmol∙L-1 in our method (R2 = 0.9839). For recovery experiments, 300 nmol·L-1 imatinib was added into the MCF7 cell lysate solution. Next, the concentration of added imatinib was determined according to the calibration curve. Consequently, the recovery was calculated to be 102.3% ± 4.8% (n=3). Results indicated that our method was capable to quantify molecules at single cell level, whose concentration was within the dynamic range of the ion trap.
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such as microfluidics, nano-LC and capillary electrophoresis, our method can help identify and quantify more metabolites, peptides or proteins in MS-based single cell analysis.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The homemade sample loop, the user interface of the software, optimization of ion injection time for 5mCyt, optimized ion injection time, NCE and activation Q of different ions for standard solutions, optimized ion injection time, NCE and activation Q of different components at single-cell level, the relationship between MS intensity and the cycle number for reserpine and 5mCyt, the MS2 spectra of 5mCyt when measuring high-concentration standard solution. the MS and MS2 spectra of angiotensin II. The MS, MS2, and MS3 spectra of caffeine, the MS and MS2 spectra of malic acid and ATP, the EIC curve of low-concentration ATP with ion accumulated by different cycle numbers, the detected scan of low-concentration caffeine and ATP completed in 3 minutes with ion accumulated by different cycle number, the EIC curve of adenine and 5mCyt from sample equivalent to ~0.2 MCF7 cell under normal scan mode and repeated accumulation mode of 25 cycles with 3 parallel injections (PDF) Available software used for modifying the MS scan function and corresponding step-by-step user instructions (ZIP)
AUTHOR INFORMATION Corresponding Author *E-mail:
[email protected] *E-mail:
[email protected] Present Addresses †National Institute of Metrology, Beijing 100029, P. R.
China Author Contributions MS2
spectrum of MCF7 cell lysate solution Figure 6. (a) The supplemented with 300 nmol·L-1 imatinib and 100 nmol·L-1 imatinib-d8. (b) The calibration curve of 0.5-5000 nmol·L-1 imatinib analyzed under repeated accumulation mode of 25 cycles, indicating that the relative intensity of imatinib had a fine linear relationship with its concentration in our method.
X. Zhang and X. Fang supervised the project. X. Si and X. Xiong designed and performed the experiments. X. Zhang, S. Zhang and X. Si analyzed the data and wrote the paper. All authors discussed the results and reviewed the manuscript.
Notes The authors declare no competing financial interests.
CONCLUSIONS
ACKNOWLEDGMENT
In conclusion, a technique capable of increasing the MS intensity of various molecules by accumulating ions in ion trap for multiple cycles was developed in this research. The intensity increase was positively correlated to the repeat cycle number. Our method significantly improved the capability of MS to analyze low-abundance molecules at single-cell level. This technique was better suited for targeted analysis of low-
This work was supported by the National Natural Science Foundation of China (Grants 21390410, 21621003 and 21575132), the 973 program (Grant 2013CB933800), and the Ministry of Science and Technology of China (Grants 2011YQ6008402).
concentration analytes whose concentration were within the dynamic range of the ion trap. The repeated ion accumulation of
a specific ion may decrease the duty cycle of MSn events for other molecular signals. Although a limitation of our technique, the duration of each MS scan was no more than 2 seconds, which was not a very long time to conduct sequential analysis of other molecules. Therefore, the current method can be coupled to LC. Coupled with other separation techniques
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