Supporting Information s16

Supporting Information

Induced Dual-Nanospray: A Novel Internal Calibration Method for Convenient and Accurate

Mass Measurement

Yafeng Li1, Ning Zhang1, Yueming Zhou1, Jianing Wang1, Yiming Zhang2, Caiqiao Xiong1,

Suming Chen1, and Zongxiu Nie1,3*

1. Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry

Chinese Academy of Sciences, Beijing 100190, China; Beijing National Laboratory

for Molecular Sciences, Beijing 100190, Beijing, China.

2. Jiangsu Skyray Instrument Co., Ltd, 1888, West Zhonghuayuan Rd., Yushan, Kunshan,

Jiangsu Province, China.

3.Beijing Center for Mass Spectrometry, Beijing 100190, China.

* Corresponding author. E-mail addresses: [email protected] (Z.X. Nie). Experimental Details Supplementary

Flow Rate Detection and Comparative Experiments. Flow rate detection experiments were done repeatedly in the conditions of 2.0 kV, 400 Hz, sine wave on Q-TOF MS. The comparative experiments were done as follows: PEG-400 (10-4 g/L) and peptide GPRP (10-4 mol/L) (shown in Scheme S1 E) were mixed by 1:1 (v:v) and then ionized by induced nanospray using one sprayer. After that, PEG-400 and the peptide were both diluted half and the induced dual-nanospray internal calibration device was used. All these were completed in the condition of sine wave, 2.0 kV, 400 Hz on LTQ MS.

Accurate Mass Measurement. The Q-TOF MS was used to do the accurate mass measurements. The concentrations of both the reference compound and the analytes were an order of magnitude lower than that used on LTQ MS while detecting in positive ion mode.

When the detection mode was turned to the negative ion mode, the concentrations of the analytes were a little higher. Calibration calculation was done by Masslynx 4.0 software.

The reference used in negative ion mode was PEG-400 sulfate. PEG sulfates have previously been reported as an excellent negative ion reference.22 But as PEG-400 sulfate was not available in the market, it was synthesized in lab.

Determination of the Targeted Compound in Urine. In this part, urine was directly used as a test sample without any pretreatments. As there was no appropriate PEG or other standard reference compounds could be used in this low mass range, two amino acids, serine and valine, were chosen to act as references. The two amino acids were dissolved in 1:1 (v:v) acetonitrile/water to get 10-2 M solutions respectively. Then 1% acetic acid was added to each solution followed by mixing the serine and valine by 5:2 (v:v) to make up a mixed-reference.

Synthesis of PEG-400 sulfate. 1 mL of acetonitrile as solvent, 100 μL of PEG-400 and 20 μL concentrated sulfuric acid was added in succession. Then the solution was reacted for 4 h at room temperatures and pressures. And after that, water was added quickly to stop the reaction. There were two notes of caution: first, the amount of concentrated sulfuric acid must be strictly controlled; second, after 4 h of reaction, certain amount of water must be added into the solution to stop the reaction. The purpose of doing so was to cut down side reactions and guarantee the mass spectrum of the product was neat enough to be used as a reference. The product solution must be diluted by 1:1 (v:v) acetonitrile /water to about 200 times while doing calibration.

Characterization of the Source. A series of studies were performed for the best characterization of the fundamentals of this reported novel source related to behavior and performance. In this section, both PEG-600 and reserpine were used as reference and sample, respectively. In each experiment, the position of the ion source and the relative positions of the two sprayers were primely adjusted to get the best calibration signals. The sprayers were renewed while the experimental conditions were changed. For each study, there were at least three replicate detections at each condition.

To characterize the effects of frequency of the alternating current on the calibration performance, experiments were carried out in the frequency range of 1–2000 Hz (with the frequencies under 100 Hz, only 1 Hz, 5 Hz, 10 Hz, 50 Hz were chose to do the detection; with the frequencies above 100 Hz, detections were done at every 100 Hz increment) with the sine wave as the induced voltage type.

While studying the influences of waveforms, three typical waveforms (sine wave, square wave, as well as triangular wave) were chosen. Under each waveform, a series of experiments with regularly changing frequencies (the frequency interval was 200 Hz) were carried out.

An induced voltage of 2.0 kV was selected to carry out all the experiments due to the narrow changeable voltage range and the negligible influences.

External calibration experiment on Q-TOF. 10-5 M PEG-400 and peptide GPRP were used with DC nanoESI method. Firstly, PEG-400 was used to calibrate the instrument; then, the peptide was detected and recorded for 40 min. this process was repeated for three times and the best result was chosen.

Result and discussion supplementary

Frequency Dependence. Figure S3 shows the frequency dependence trend of calibration signal intensities in the calibration process. Generally, the signal intensities of the sample and the reference compound should be comparative to get a good calibration result. So each data point here was a selected best calibration signal result (this means that the signals of the sample and the reference compound match with each other very well, and that the selection was done by adjusting the positions of the two sprayers) of several detections. From 100 Hz to 500 Hz, the calibration signal intensities experienced a sudden increase, but they decreased rapidly after 600 Hz. In the region of 800 Hz-1500 Hz, the calibration signal intensities were little changed, but a sudden decrease happened again when the induced frequency reached to

1600 Hz. The signal of 1700 Hz was roughly the same as 1600 Hz. When the frequency increased to 1800 Hz or higher, getting relatively good calibration signals became very difficult. In the very low frequency region like 1 Hz or 10 Hz, signals were discrete and could not be used for calibration. The signals became continuous as the frequency was further increased (about 50 Hz), but because the intensities was very low (results were not shown here), this frequency region was not used while doing calibration. Therefore, the best frequency region for doing accurate mass measurements is between 400 Hz to 600 Hz. The reasons for this tendency still need further study.

To choose a suitable frequency in this region, the relative stability must also be taken into consideration. Although 500 Hz and 600 Hz have the highest signal intensities, they are followed by a sudden intensity decrease, and this leads to relatively poor repeatability of the signal intensities. The results of multiple detections all showed this point. Therefore, 400 Hz is the best choice because it has relatively high and stable signal intensity. Effects of Waveforms. A series of experiments have been done to investigate the influences of alternating voltage type on the calibration signals. We have studied three typical types of waveform, they are the sine wave, the square wave as well as the triangular wave. Using either the sine wave or the square wave, the intensities of the calibration signals were on the same level, without any significant advantages and disadvantages observed. However, when the wave type was changed to the triangular wave, a distinct decrease of signal intensities was appeared. Actually, this result could be foreseen because for the same voltage level the effective voltage of triangular waveform is lower than the other two forms. As a result, the intensity of the total ion current is lower. Besides, how the intensities vary with frequency is roughly the same under these three waveforms.

Result of Flow Rate Detection. To know the exact flow rate of the induced nanospray ionization method, three repeated experiments were done: the first and the second time, 2.0

μL of 10-5 mol/L reserpine was injected into the sprayers, and the signals lasted for 45 min and

43 min respectively. The third time, 2.5 μL reserpine was used and it lasted for 55 min. Thus the flow rate of this ion source is calculated to be 44 nL/min, 47 nL/min and 45 nL/min.

Although the evaporation consumption in this flow rate detection experiment is neglected, this result is able to partly explain the low interference between the reference ions and the analyte ions.

External calibration experiment on Q-TOF.

Figure S8 was the result of this external calibration. Each data point was the average of two minutes’ detection. Mass errors were stabilized at 38.5 ppm in the first twenty minutes. Later, the mass errors floated to 58.5 ppm and almost stabilized there. This result makes the advantages of this internal calibration method very clear.

Determination of the Molecular Formula of the Targeted Compound in Urine. As a component of urine, the compound which gave the signal of m/z 114 was chosen to be calibrated by this new method to further test the practical applicability. Five repeated experiments were done and the calibration results are shown in Table S1. There was no Na+ or

K+ found in the ion of targeted compound according to the preliminary calculation results, indicating that it must be a [M+H]+. The mass of H+ was then subtracted before further calculation. For a compound with such low molecular mass (around 100), the error might be a little higher. So the weight tolerance was set at 7 ppm. As is listed in Table 2, four-fifths of the calculation results only had one compound found and they were perfectly consistent. For the third result, although there were two compounds found and the second had a relatively low error, it could be easily eliminated based on the nitrogen rule. After preliminary and further molecular formula calculations, it can be confirmed that the targeted compound is C4H7N3O. Figure S1. Structure pictures of the induced dual-nanospray internal calibration ion source.

The red wire was used for connection convenience and a resistor was to protect the high voltage amplifier. The screw was used to connect the metal tubes and the wire. Two nanosprayers are inserted into two mental tubes respectively. Figure S2. Dual DC nanoESI result of PEG-700 and rhodamine. Sprays could not happen simultaneously and the signals were very low and unstable. The ions were severely suppressed, and signals of PEG-700 or rhodamine could only be seen intermittently, as can be seen in (a) and (b).

Scheme S1. Structures of the seven compounds that are used as calibration samplesa.

aSample A-E were calibrated in positive ion mode, and sample F and G were in negative ion mode. Figure S3. The frequency dependence trend of calibration signal intensities in the calibration process. The two dotted lines (red and black) connect all the experimental data points

(reserpine and PEG) respectively while the two solid lines (red and black) indicate the variation tendency respectively. Figure S4. This is the induced nanospray Q-TOF negative ion mass spectrum of synthetized

PEG 400 sulfate. The inset is the enlarged view of the low mass region. m/z 195 was the signal of sulfuric acid dimer, others are the signals of double-esterified PEG which are ionized as [M−2H]2-. Compared with single-esterified PEG signals in the high mass region, these undesired signals are so low that will not affect the calibration process, as is shown in Figure

S5. Figure S5. This is the single-acquisition mass spectrum of PEG 400 sulfate and sample G.

The signals are almost perfect for doing calibration. Figure S6. Induced dual-nanospray mass spectrum of amino acids (serine and valine) mixture-reference and urine. The targeted ion (m/z114) is calibrated by serine (m/z 106) and valine (m/z 118). Figure S7. MS/MS spectra of the targeted ion (m/z 114) in urine sample. Figure S8. The external calibration result of peptide GPRP on Q-TOF. Each data point was the average of two minutes’ detection. Mass errors were stabilized at 38.5 ppm in the first twenty minutes. Later, the mass errors floated to 58.5 ppm and almost stabilized there. Table S1. Calibration and calculation results of the targeted compound (m/z 114) in urine.

Calibrated Error Final Number Molecular Formula Calculation Result (ppm) Result(ppm) Weight

Compounds found: 1 1 113.0594 4.3 C4H7N3O MW=113.0589092 dm=-4.3 ppm

Compounds found: 1 2 113.059 0.8 C4H7N3O MW=113.0589092 dm=-0.8 ppm

Compounds found: 2 3.1±2.5 3 113.0596 C4H7N3O MW=113.0589092 dm=-6.1 ppm 6.1 C6H9O2 MW=113.0602514 dm=5.8 ppm

Compounds found: 1 4 113.0589 0.1 C4H7N3O MW=113.0589092 dm=0.1 ppm

Compounds found: 1 5 113.0594 4.3 C4H7N3O MW=113.0589092 dm=-4.3 ppm 4 pep 10-4 single induce 400Hz_1 2013/3/7 10:37:42

4 pep 10-4 single induce 400Hz_1 #89-116 RT: 0.34-0.45 AV: 28 NL: 9.72E5 T: ITMS + p ESI Full ms [150.00-800.00] 426.25 100

70 e c n

a 60 d n u b

e v i t a

l 40 e R 30

20 274.33 10 213.67 318.33 448.25 195.08 218.17 346.33 393.33 468.25 536.08 576.08 598.17 654.17 675.25 710.08 763.25 795.17 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 4 pep+PEG10-4 dual induce 400Hz_2 2013/3/7 10:41:53 m/z

4 pep+PEG10-4 dual induce 400Hz_2 #197-201 RT: 0.79-0.81 AV: 5 NL: 2.05E5 T: ITMS + p ESI Full ms [150.00-800.00] 426.33 100 476.33 432.33 90

80 520.33 70 e

c 388.33 n

a 60 d n u

b 564.33

v 437.25 i t

a 481.25

l 40 e R 344.33 393.25 608.33 30 525.25

20 453.25 569.25 497.25 652.33 274.33 349.25 409.25 300.25 541.25 613.33 10 371.25 585.33 696.33 213.67 629.33 195.08 256.17 740.42 776.33 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z

Figure S9. Influences of reference spray on analyte spray. (a) Spectrum of peptide GPRP when only one sprayer is used with induced nanospray. The signal intensity is 9.72E5. (b)

Spectrum of peptide GPRP and PEG-400 when dual sprayers are used with induced nanospray. The signal intensity of peptide GPRP is 2.05E5. The concentration of PEG-400 and peptide GPRP are both about 10-4 M. The interferences between analyte signals and reference signals do exist but not very severe. And it decreases when the concentrations decreases. E:\研究生资料 \...\4Pep inano ESI 1_2 2013/3/1 17:29:05

4Pep inano ESI 1_2 #809-851 RT: 3.07-3.23 AV: 43 NL: 9.06E5 T: ITMS + p ESI Full ms [115.00-800.00] 426.25 100

70 e c n

a 60 d n u b

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10 279.17 195.00 301.17 136.00 217.08 330.33 381.33 462.00 483.08 536.08 557.00 610.08 631.00 684.00 711.00 739.08 766.92 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 E:\研究生资料 \...\4Pep induce 400Hz_1 2013/3/1 17:09:36 m/z

4Pep induce 400Hz_1 #482-508 RT: 1.98-2.08 AV: 27 NL: 5.05E5 T: ITMS + p ESI Full ms [115.00-800.00] 426.25 100

70 e c n

a 60 d n u b

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20 274.33

213.67 10 302.33 330.33 146.08 393.25 448.25 180.08 246.25 346.33 468.25 536.08 598.08 659.17 683.00 711.08 739.08 766.92 0 150 200 250 300 350 400 450 500 550 600 650 700 750 800 m/z

Figure S10. Comparison of DC nanoESI and induced nanospray. (a) Spectrum of peptide

GPRP when normal nanoESI is used. The signal intensity is 9.06E5. (b) Spectrum of peptide

GPRP induced nanospray is used. The signal intensity of peptide GPRP is 5.05E5. The concentration of peptide GPRP is about 5*10-5 M. The signal intensity of induced nanospray is almost half of the DC nanoESI, as the AC power has half cycle’s negative voltages.