Enhancing the Mass Spectrometry Sensitivity for Oligonucleotide

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Enhancing the mass spectrometry sensitivity for oligonucleotides detection by organic vapor assisted electrospray Guofeng Weng, Zheyi Liu, Jin Chen, Fangjun Wang, Yuanjiang Pan, and YuKui Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01695 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 6, 2017

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Analytical Chemistry

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Enhancing the mass spectrometry sensitivity for oligonucleotides

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detection by organic vapor assisted electrospray

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Guofeng Weng1,2, Zheyi Liu2,3, Jin Chen2,3, Fangjun Wang2*, Yuanjiang Pan1*, Yukui Zhang2

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Department of Chemistry, Zhejiang University, Hangzhou, 310027, China.

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CAS Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of Chemical

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Physics, Chinese Academy of Sciences, Dalian, 116023, China.

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* Correspondence and requests for materials should be addressed to F.W. (email: [email protected])

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and Y.P. (email: [email protected]).

University of Chinese Academy of Sciences, Beijing, 100049, China.

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ABSTRACT

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There are two challenges in oligonucleotides detection by liquid chromatography coupled with mass

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spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low

14

detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were

15

introduced into an enclosed electrospray ionization chamber and oligonucleotides could be well

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detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as ion-pair

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reagent. The MS signal intensity was improved 600 folds (for standard oligonucleotide dT15) by the

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isopropanol vapor assisted electrospray and effective ion-pair LC separation was feasibly coupled with

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high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS

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with little adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced

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more than 10 folds on average compared with adding acids into the electrospray solution. Finally,

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oligonucleotides and peptides or histones were simultaneously detected in MS with little interference to

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each other. Our strategy provides a useful alternative for investigating the biological functions of

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oligonucleotides.

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INTRODUCTION

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Nucleic acids (DNA and RNA) sequencing technologies have been greatly developed in recent years

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and even single cell sequencing has been realized1. Comparing with the nucleic acids sequencing

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technologies coupled with polymerase chain reaction (PCR), mass spectrometry (MS) can provide rich

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information of post-modifications on nucleotides, which play essential roles in biological processes2,3.

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Further, oligonucleotides gain increasing attention due to their extensive usages in many biological and

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therapeutic applications such as small RNA interference (siRNA)4. Therefore, new MS-based strategies

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are still needed for high-sensitive, high-accurate and high-throughput characterization of

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oligonucleotides. There are two challenges existed in MS detection of oligonucleotides. The first one is

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the serious MS signal suppression effect of ion-pair reagents which makes the commonly utilized

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ion-pair reversed-phase liquid chromatography (IP-RPLC) incompatible to MS detection. The second

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one is the low sensitivity of oligonucleotides in positive mode MS detection due to their highly

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negative backbone with abundant phosphodiester groups. Thus, it is difficult to simultaneously detect

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oligonucleotides with other biomolecules that usually detected in positive mode MS, such as peptides

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and proteins.

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Triethylammonium acetate (TEAA) is one of the most popular ion-pair reagents in

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oligonucleotides separation due to its high efficiency in improving the separation capability of RPLC5-7.

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Oberacher et al. achieved single nucleotide resolution oligonucleotides separation by using the TEAA

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containing mobile phase8. Dickman et al. demonstrated that IP-RPLC with TEAA enabled

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size-dependent separation of duplex DNA up to 458bp9,10. Although commonly utilized 100 mM TEAA

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could significantly improve the IP-RPLC separation resolution, it will seriously suppress MS signals at

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the same time11. Recently, Gong et al. demonstrated that 30 mM TEAA delivered an optimal balance

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between IP-RPLC separation efficiency and MS detection sensitivity after sophisticated optimization of

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the LC separation conditions12. Other strategies for alleviating the MS signal suppression effect of

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TEAA include removing TEAA ions by micro-dialysis before the mobile phase enters into ESI-MS13,

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post-column addition of organic solvent as sheath liquid to improve MS signal14,15, and utilizing

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hexafluoroisopropanol (HFIP) coupled with triethylamine (TEA) to instead of TEAA buffer11. However,

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these strategies will either introduce post-column dead volume and dilute the sample concentration or

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diminish the LC separation resolution especially for large oligonucleotides14,16.

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In this study, we demonstrated that the MS detection sensitivity for oligonucleotides could be

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significantly enhanced by organic vapors (alcohols and acids) assisted electrospray ionization within an

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enclosed ESI source (Figure 1 and S1). Briefly, the signal suppression effect of 100 mM TEAA was

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significantly alleviated by introducing concentrated alcohol vapor into the enclosed electrospray

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chamber, and effective ion-pair RPLC separation was directly coupled with high-sensitive MS


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detection without any other purification or dilution procedures. Then, high-sensitive oligonucleotides

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detection in positive mode MS was achieved by organic acid vapor assisted electrospray. More

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importantly, oligonucleotides and peptides or histones were simultaneously detected in positive mode

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MS with high sensitivity, which exhibited great potential in investigating the biological processes of

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gene regulation by related proteins.

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EXPERIMENTAL SECTION

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Chemicals, proteins and oligonucleotides samples.

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Chemical reagents of TEA, triethylammonium bicarbonate (TEAB), propanoic acid (PA), acetic acid

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(HAc), trifluoroacetic acid (TFA), formic acid (FA), trypsin (TPCK treated), bovine serum albumin

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(BSA) were obtained from Sigma (St. Louis, MO). HPLC grade of acetonitrile (ACN), methanol and

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isopropanol were from Merck (Darmstadt, Germany). Deionized water used in all experiments was

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purified with a Milli-Q system from Millipore (Milford, MA). Histone proteins were from Worthington

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(Lakewood, USA). All of the oligonucleotides samples were purchased from Sangon Biotech

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(Shanghai, China) (Table S1).

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The tryptic digest of BSA was prepared according to our previous work with minor modification17.

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The 2 mg of BSA was dissolved into 1 mL of denaturing buffer containing 8 M urea and 0.1 M

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ammonium bicarbonate. After addition of 20 µL DTT (1 M), the mixture was incubated at 37 ℃ for 2

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h. Then, 7.4 mg IAA was added, and the mixture was incubated at room temperature in dark for 40 min.

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Finally, the mixture was diluted 8 folds with 0.1 M ammonium bicarbonate buffer (pH 8.2) and

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digested at 37 ℃ for 20 h with trypsin at enzyme-substrate ratio of 1/25 (w/w). After digestion, the pH

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of digested solution was adjusted to 2.7 by 10% TFA aqueous solution, followed with purification by a

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homemade C18 solid-phase extraction (SPE) column. Finally, the eluted peptides was lyophilized and

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stored at -20 ℃ before usage.

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RPLC-MS analysis.

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According to the reported method18, TEAA buffer (100 mM, pH7) was prepared by mixing appropriate

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amounts of TEA and HAc in mobile phases A (5% ACN and 95% H2O) and B (45% ACN and 55%

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H2O) respectively. Briefly, for 200 mL of 100 mM TEAA buffer, 1.1 mL of glacial acetic acid was

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added into 180 mL mobile phase A or B, and then 2.8 mL of TEA was slowly added with stirring and

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ice bath cooling. The pH of the resulting solution was carefully adjusted to pH 7.0 by addition of either



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TEA or HAc.

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All of the LC-MS and direct infusion MS analyses were performed on a LTQ-Orbitrap XL MS

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equipped with Accela 600 HPLC system (Thermo, San Jose, CA). The LC separation system includes a

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4 cm C18 capillary trap column (200 µm i.d.) and a 20 cm C18 capillary separation column (75 µm i.d.)

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packed with C18 AQ beads (5 µm, 120Å)19,20. The oligonucleotides mixtures were firstly loaded onto

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C18 trap column and then separated by IP-RPLC with gradient elution. The flow rate after splitting was

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adjusted to about 200 nL/min. The RP gradient was developed as follows: 0% buffer B (100 mM

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TEAA, 45% ACN and 55% H2O, pH 7.0) for 5 min, from 0 to 30% buffer B for 25 min, from 30 to

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80% for 12 min. After elution with 80% buffer B for 8 min, the separation system was equilibrated by

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buffer A (100 mM TEAA, 5% ACN and 95% H2O, pH 7.0) for 10 min.

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A commercial open ambient nano-spray source (Thermo) and an enclosed electrospray source

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(CEESI, Haochuang, China)21-23 were utilized in MS experiments, respectively. Electrospray voltage

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between the spray tip and the MS interface was 2.0 kV, the temperature of ion transfer capillary was

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250 ℃, the max inject time was 100 ms, microscans 1 and the MS1 resolution was 30,000 (at m/z =

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400). System control and data collection were carried out by Xcalibur software version 2.1 (Thermo).

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For the mixture of oligonucleotides and histone proteins detection, the max inject time was 250 ms and

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microscans 20 to further improve the MS resolution.

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Organic vapor introduction

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The CEESI source has an enclosed chamber created by seamlessly connecting to the heated ion transfer

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capillary of MS (Figure 1 and S1). The source has two interfaces, one of them is used to fix LC

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capillary column for sample introduction and the other one is used for organic vapor introduction by

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absorbing air through a removable gas-washing bottle containing different organic solvents with the

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help of the high vacuum of MS. Thus, the atmosphere in the enclosed electrospray chamber is

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concentrated organic vapor and can be adjusted through changing airflow rate and the type of organic

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solvents in the gas-washing bottle. Moreover, this source can also work in open ambient condition after

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removing the gas-washing bottle.

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RESULTS AND DISCUSSION

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Alleviating MS signal suppression effect of TEAA on oligonucleotides detection

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Firstly, signal intensity (sum of the peak intensity of all individual charge states) of 10 µM


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oligonucleotide dT15 (in 45% ACN) with TEAA concentration varying from 10 to 100 mM was

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investigated in direct ESI-MS detection (negative mode) with an open ambient nanoESI source (Figure

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2 and S2). Along with the increase of TEAA from 0 to 100 mM, the charge-state distribution of

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oligonucleotide dT15 shifted to lower charge states and its MS signal intensity was significantly

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decreased due to the ion suppression effect of TEAA (Figure S3). Oligonucleotide signal was nearly

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disappeared when TEAA reached to 50 mM in sample solution. Then a variety of concentrated organic

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vapors including isopropanol, ethanol, methanol and acetonitrile were doped into the enclosed ESI

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chamber, respectively, to investigate the related influences on MS detection signals of 10 µM

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oligonucleotide with 100 mM TEAA. Obviously, all of these four organic vapors could significantly

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improve the signal intensity of oligonucleotide and the MS signal was disappeared as soon as the

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organic vapor was removed (Figure 3a). Among them, isopropanol and ethanol vapors exhibited higher

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efficiency in improving oligonucleotide signal intensity (Figure S4). The good reproducibility of

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isopropanol vapor assisted electrospray was demonstrated through direct infusion MS detection with

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five consecutive connecting and disconnecting the isopropanol vapor introduction flow path every 30

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seconds (Figure 3b). Then, we systematically investigated the performance of isopropanol vapor

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(consumption rate, 142 µL(liquid)/min) on enhancing the oligonucleotide signal intensity with TEAA

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concentration varying from 10 to 100 mM. Concentrated isopropanol vapor assisted electrospray could

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efficiently alleviate the signal suppression caused by TEAA. The signal enhancement ratio

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(Iisopropanol/Iambient) was increased from 3.5 to 50.0 along with the TEAA concentration increasing from

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10 to 100 mM (Figure 2). We further investigated the signal intensity of homologous oligonucleotides

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ranging in size from 11 to 30 nucleotides with 100 mM TEAA and compared the signal enhancing

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performance of different organic vapors (Figure S5). Isopropanol and ethanol vapors could enhance the

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MS signal intensity of oligonucleotides regardless of their length while methanol and ACN vapors

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exhibited lower efficiency in improving the signal intensity of large oligonucleotides (> 15mer

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nucleotides).

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Organic solvent concentration in the electrospray solution seemed to have a significant impact on

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ionization efficiency in open ambient electrospray and the signal intensity of oligonucleotide exhibited

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20-folds improvement during the ACN was increased from 5 to 45% (Figure 4a). The increase of signal

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intensity at higher concentrations of organic modifier could be attributed to the lower surface tension

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and faster evaporation of solvent from the electrospray droplets16. However, we observed organic



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solvent concentration has little influence on oligonucleotides signal intensity in concentrated

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isopropanol vapor assisted electrospray (Figure 4b), which might be helpful in improving the

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reproducibility of oligonucleotides intensity in quantitative analyses by gradient IP-RPLC-MS. In

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general, all of the oligonucleotides samples could be eluted by a mobile phase containing 20% ACN in

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IP-RPLC separation. Comparing with the MS signal intensity of oligonucleotide in 20% ACN

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electrospray solution (with 100 mM TEAA), isopropanol vapor assisted electrospray could improve the

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signal intensity about 600 folds (Figure 4), which further demonstrated the concentrated isopropanol

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vapor could significantly alleviate the signal suppression caused by TEAA.

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Then, electrospray solution pH was adjusted from 9.2 to 6.1 through adding appropriate acetic

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acid. The pH of the electrospray solution also exhibited little influence on the signal intensity of

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oligonucleotide under the isopropanol vapor assisted electrospray (Figure S6). We investigated the

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influence of different organic vapor on ESI-MS detection with different ion-pair reagents in

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electrospray solution (Figure S7). Briefly, alcohols atmospheres could greatly improve the signal

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intensity in all of the three ion-pair reagents while the effect of ACN was negligible. Note that

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methanol vapor introduced a special signal enhancing effect for TEAB containing sample solution

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comparing to TEAA and TEAF while isopropanol and ethanol exhibited similar and relative high

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efficiency in alleviating the ion suppression effects of all three ion-pair reagents. The plot of signal

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intensity vs injected oligonucleotide concentration (within 100 mM TEAA) showed good linearity

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(R2=0.999) over more than 2 orders of magnitudes (0.1 µM - 10 µM) in isopropanol vapor assisted

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ESI-MS detection (Figure S8 and S9). For traditional ESI-MS without organic vapor, the signal

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intensity of oligonucleotide in 100 mM TEAA solution was much lower and only two visible data

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points were detected in the same concentration range with detection limit at near 5 µΜ (Figure S9).

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IP-RPLC-MS for oligonucleotides analysis.

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When coupling IP-RPLC with negative mode ESI-MS for analyzing complex oligonucleotides samples,

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traditional strategies usually reduce the concentration of TEAA to make a balance between optimum

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LC separation resolution and MS detection sensitivity2,7,12,24,25. As we have demonstrated that

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concentrated alcohol vapor could significantly alleviate the signal suppression effect caused by TEAA,

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IP-RPLC eluted mobile phase containing 100 mM TEAA could be directly injected into ESI-MS

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without any other purification or dilution procedures. Here, the mixture of 7 oligonucleotides with


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similar lengths and different sequences was analyzed by IP-RPLC-MS with mobile phase containing

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100 mM TEAA (Figure 5). All of the oligonucleotides were well separated and high MS signal

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intensity was feasibly achieved. Adduction with alkali metal ions is always the major problem in

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negative ESI-MS detection of oligonucleotides due to the existence of phosphodiester groups26.

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However, few adduction peaks were present in the MS signals obtained by concentrated isopropanol

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vapor assisted ESI-MS. Further, oligonucleotides with different lengths ranging from 11 to 25

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nucleotides could be effectively separated and high MS signal intensity was also obtained (Figure S10).

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We also compared the MS results in ambient electrospray condition and couldn’t find any stable MS

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signals of targeted oligonucleotides as previous reported11.

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Improving detection sensitivity of positive mode MS for oligonucleotides

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Oligonucleotides tend to form negative ions in ESI-MS detection due to its backbone phosphodiester

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groups (pKa < 1), and most of the oligonucleotides analyses including molecular weights determination

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and structural elucidation were performed in negative mode MS27-32. Meanwhile, a handful of ESI-MS

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studies were carried out on positively charged oligonucleotides33-37, while most authors conceded that

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negative mode MS was more suitable for oligonucleotides detection with higher sensitivity. Despite the

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generally accepted benefits of negative mode MS for oligonucleotide detection, being able to detect

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oligonucleotides in positive mode MS may have some practical advantages. For example, other

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biomolecules such as proteins and peptides are usually ionized and characterized in positive mode MS

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with higher efficiency38,39, which makes simultaneous detection of oligonucleotides, peptides and

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proteins in only one MS experiment impossible. However, the regulations of gene activation or

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inhibition are usually related with functional proteins, such as histones40,41.

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At first, we tried to detect oligonucleotides in positive mode MS by adding different kinds and

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concentration (0.1% or 1%) of organic acids in the electrospray solution (Figure S11). Obviously, the

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MS signal of oligonucleotides obtained in this way always carried with a lot of metal adducts, which

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interfered the accurate characterization of targeted oligonucleotide and caused signal attenuation. Then

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we tried to improve the oligonucleotides signals in positive mode MS by doping different organic acid

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vapor into the enclosed electrospray chamber. It was observed that the peak adducts were almost

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disappeared and the corresponding MS signal intensity of oligonucleotides was highly enhanced

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compared to direct changing the electrospray solution pH42. Generally, the propanoic acid vapor



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(consumption rate, 21.8 µL(liquid)/min) gave the highest MS signal enhancement efficiency among all

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investigated organic acid vapors and the enhancement ratio was more than 10 folds for oligonucleotide

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N-9 (15mer) (Figure S11). Then we compared the MS detection results of a mixed oligonucleotide

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sample containing 14 different oligonucleotides ranging from 11mer to 29mer with identical

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concentration in both positive and negative modes MS (Figure S12). Interestingly, the signal intensity

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exhibits no dependence on the length of oligonucleotides and similar peak intensity was obtained for

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these oligonucleotides in positive mode MS. While oligonucleotides signal within pure water solution

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in negative mode MS was not so stable and we used a common buffer (400 mM HFIP, 16.3 mM TEA,

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pH 8.5) to stabilize and improve the signals. Even so, the oligonucleotides signal intensity in positive

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mode MS by using propanoic acid vapor assisted electrospray strategy was higher than that in negative

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mode MS (Figure S12). Furthermore, the plot of signal intensity (sum of the peak intensity of all

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individual charge states) vs injected concentration exhibited good linearity (R2=0.999) over more than

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2 orders of magnitudes (0.05 µM - 5 µM dT15) in the propanoic acid assisted ESI-MS (Figure S13).

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The detection limit was near 0.01 µΜ (Figure S14b) with propanoic acid vapor-assistance, which was

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much better than the detection limit at about 0.5 µΜ in the traditional ambient ESI-MS detection

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without organic acid vapor (Figure S14c).

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The MS/MS characterizations of the oligonucleotides N-1 (Figure 6), N-3 (Figure S15a) and N-4

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(Figure S15b) were performed in both negative and positive mode MS, respectively. Although similar

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types of fragment ions were obtained, the MS/MS fragment ions exhibited good complementary

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between positive and negative modes. For example, some fragment ions of oligonucleotide N-1, such

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as a9-B (1312.73, z=2), was more abundant in positive mode MS while others like a3-B (705.13, z=1)

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and a5-B (1338.24, z=1) were easier to be observed in negative MS signals. The fragment ions could be

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easily recognized because of the same fragment ion had a fixed mass difference as 2 Da between

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negative and positive modes. Thus sequencing an unknown oligonucleotide could be simplified if we

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excluded the interfering fragment ions by comparing the MS/MS spectra between negative and positive

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modes MS.

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Furthermore, simultaneous detection of oligonucleotides with peptides or proteins could be

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realized in the present study. Firstly, 14 oligonucleotides (Table S3) were mixed together with tryptic

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digest of BSA at the ratio 100/1 (w/w). Both oligonucleotides and peptides were well detected in

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propanoic acid vapor assistant ESI-MS analysis. Then we increased the addictive amount of peptides


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10 times, and the signal intensity of 14 oligonucleotides ranging from 11mer to 29mer exhibited little

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change (Figure S16). Control experiments were also performed with open ambient ESI-MS in negative

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mode MS (Figure S17, Table S4). Although the simultaneously detection of oligonucleotides and

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peptides could be obtained in alkali solution (400 mM HFIP, 16.3 mM TEA, pH 8.5), the signal

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intensity of peptides decreased seriously with -1 charge state. On the other hand, the signal intensity of

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BSA tryptic peptides was improved 2-3 times after introducing propanoic acid vapor into the enclosed

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ESI source (Figure S18), which was consisted to previous work by Li et al43. The peptides charge states

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were increased after the acidic vapor introduction (Figure S18a and S18b). For example, the major

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charge state of the peptide KVPQVSTPTLVEVSR moved from +2 (m/z 820) to +3 (m/z 547).

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Furthermore, the MS signals of BSA tryptic peptides was nearly not influenced after mixed together

250

with oligonucleotides and simultaneously detected under acid vapor assisted ESI-MS (Figure S18b and

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S18d). As histones contribute to dynamic maintenance and regulation of chromatin structure, to gene

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activation, DNA repair and many other processes in cell nucleus, it makes sense if we could

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simultaneously detect histone proteins along with complex oligonucleotides25,44. Thus, the 14

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oligonucleotides were mixed together with histones at the ratio 1/4, 1/40 (w/w) and analyzed by

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propanoic acid vapor assisted ESI-MS (Figure 7 and S19). Briefly, the composition of histone proteins

256

were successfully attributed to H2A, H2B, H4 and their accompanying post-translational modifications

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such as acetylation (42 Da, Figure S20)45,46, and the 14 targeted oligonucleotides were also feasible

258

detected and the signal intensity was comparable to the experiments without mixing histones. Therefore,

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simultaneously detect oligonucleotides and peptides or proteins in positive mode MS were realized in

260

our study and the signals have little interferences to each other.

261

In the present study, the MS ion suppression effect of high concentration TEAA was significantly

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alleviated by highly concentrated alcohol vapor assisted electrospray (Figure S21). Actually, there are

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two main reasons for the MS signal suppression effect of TEAA on oligonucleotides detection6. Firstly,

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TEA has a lower boiling point than acetic acid (89℃ vs. 118℃) and evaporates more quickly than

265

acetic acid during the ionization process. The enrichment of acetic acid decreases the pH in the

266

electrospray droplet, which then induces the ionization competition between the acetic acid and

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oligonucleotides and decreases the ionization efficiency of the latter one. On the other hand, TEAA is

268

also less hydrophobic than other ion-pair reagents such as HFIP-TEA. Thus, the surface tension of the

269

droplets is higher and would decrease the ionization efficiency of oligonucleotides43,47. The exposure of



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TEAA containing charged droplets to concentrated alcohol (ethanol and isopropanol) vapors could

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improve the ion evaporation rates of both TEA and acetic ions through reducing their activation energy

272

for ion evaporation compared to the ambient condition48,49. In our experiments, the MS signals of both

273

TEA and acetic acid from 100 mM TEAA solution exhibited significant improvement after introducing

274

concentrated alcohol vapors (Figure S22 and S23). We also observed the MS signal suppression effect

275

of 100 mM acetic acid solution could be effectively alleviated with isopropanol vapor assisted

276

electrospray (Figure S24). Furthermore, the alcohols could engage in extensive hydrogen bonding on

277

the droplet surface and thereby reduce the surface tension through lowering the solvation energy48.

278

On the other hand, the MS signals of oligonucleotides were significantly enhanced and the metal

279

adducts signals were greatly reduced (Figure S11 and S14e) in the positive mode MS detection by

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acidic vapor (mainly propanoic acid) assisted electrospray. Unlike other biomolecules, such as protein

281

and peptide, oligonucleotide tends to form negative ions in ESI-MS detection due to its backbone

282

phosphodiester groups (pKa < 1). The nucleobases exposed in highly concentrated propanoic acid

283

atmosphere could get more chance to combine with dissociated protons and the concentrated acidic

284

vapor could also prevent the dissociation of protons from backbone phosphodiester groups37,50,51.

285

Therefore, the effect of acidic vapor observed in positive mode MS was more likely to be

286

improvements in both MS signal intensity and signal-to-noise through improving the oligonucleotide

287

ionization efficiency and reducing the metal counter adducts48.

288 289

CONCLUTION

290

In this study, the signal suppression effect of high concentration (100 mM) TEAA was significantly

291

alleviated by alcohol vapor (isopropanol or ethanol) assisted electrospray ionization strategy. Then,

292

effective IP-RPLC separation (with 100 mM TEAA) was feasibly coupled with high-sensitive MS

293

detection (negative mode) for oligonucleotides. The signal enhancement ratio was about 600 folds (for

294

sample dT15) in the mobile phase containing 100 mM TEAA and 20% ACN. Furthermore,

295

oligonucleotides detection in positive mode MS was realized with little adducts by using organic acid

296

(propanoic acid) vapor assisted electrospray and the average signal intensity enhancement ratio was

297

more than 10 folds compared to direct adding acids into the electrospray solution. As oligonucleotides

298

could be detected in positive mode with high performance, simultaneous detection of oligonucleotides

299

with complex peptides or histone proteins were feasibly realized with little influence to each other.


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300

Overall, we provideed a promising alternative for high-sensitive characterization of oligonucleotides by

301

MS, which might play important roles in elucidating the oligonucleotides biological functions in the

302

future.

303 304

FUNDING

305

This work was supported by the China State Key Research Grant [2016YFF0200504], the China State

306

Key Basic Research Program Grant [2013CB911203], the National Natural Science Foundation of

307

China [21675152 and 21235005], the Youth Innovation Promotion Association CAS [2014164] and

308

grant from DICP (ZZBS201603).

309 310

REFERENCES

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338

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Page 12 of 19

Page 13 of 19

382

Open state Ambient ESI source

6

2

1

Enclosed state

a

7

CEESI source 3

b 5

4

383 384

Figure 1. Instrumental configuration illustration of the (a) ambient ESI source and (b) enclosed CEESI

385

source (Haochuang, China). 1, high voltage; 2, C18 capillary separation column; 3, air flow; 4, organic

386

solvent; 5, rubber ring; 6, heated ion transfer capillary; 7, MS high vacuum.

387 388 107

Isopropanol Ambient 106

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

105

b

104

0

389

20

40

60

80

100

TEAA (mM)

390

Figure 2. Influence of different TEAA concentration on the dT15 signal intensity in isopropanol vapor

391

assisted (a) or open ambient (b) ESI-MS detection, respectively. Conditions: TEAA concentration,

392

varying from 10 to 100 mM; 45% ACN; sample, 10 µM dT15; negative mode MS detection; sample



393

injection rate, 3 µL/min.

394 395 100

a Am bie nt

Ac eto nit ril e Am bie nt Iso pr op an ol

Am bie nt

20

Et ha no l

40

Iso pr op an ol Am bie nt

60

Me tha no l Am bie nt

80

Am bie nt

Relative Abundance

0 100 80

b

0 0.0

396

0.5

1.0

1.5

2.0

2.5

3.0 3.5 Time (min)

4.0

4.5

5.0

Am bie nt

20

Iso

40

pr op an ol Am bie nt Iso pr op an ol Am bie nt Iso pr op an ol Am bie nt Iso pr op an ol Am bie nt Iso pr op an ol

60

Am bie nt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 19

5.5

6.0

397

Figure 3. (a) Influences of different alcohol vapor (isopropanol, ethanol, methanol and acetonitrile) on

398

the MS signal intensity of dT11. (b) The reproducibility of dT11 MS signals in isopropanol vapor

399

assisted electrospray. Conditions: TEAA concentration, 100 mM; 5% ACN; sample, 10 µM dT11;

400

negative mode MS detection; sample injection rate, 3 µL/min.

401 402 403 404 405 406


Page 15 of 19

Ambient Isopropanol

106 2.63E+05

3.42E+05

2.85E+05

2.62E+05

3.66E+05 3.42E+05

a

105

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


600-fold

104

9.57E+03 6.28E+03 4.38E+03

b 1.16E+03

103

569

489

102 0

407

10

20

30

40

50

ACN%

408

Figure 4. Influence of acetonitrile in electrospray solution on the dT15 signal intensity in isopropanol

409

vapor assisted (a) or open ambient (b) ESI-MS detection, respectively. Conditions: TEAA

410

concentration, 100 mM; ACN percentage, varying from 5 to 45%; sample, 10 µM dT15; negative mode

411

MS detection; sample injection rate, 3 µL/min.

412 413 414 415 416 417



100

N-1

95

100

N-5 NL: 1.61E6 CCGCTCGAGGC

90 85

[M-3H]

N-6 [M-3H]

N-4 NL: 1.70E6 ATGCCTCAAGG

55 50 45

N-3 NL: 2.03E6 ATGGCGAGCTAT

40 35

dT11 NL: 1.81E6 TTTTTTTTTTT

30 25 20

dT12 NL: 1.68E6 TTTTTTTTTTTT

15 10

dT11

N-5

2-

[M-3H]

3-

[M-3H]

3-

[M-2H]

2-

50 Relative Abundance

60

[M-2H]

N-4

dT12

0 100

N-1 NL: 2.34E6 TTGTGAAGCTT

3-

50

70 65

N-3

2-

50

N-6 NL: 1.70E6 CCGCTCGAGGCA

75

3-

[M-2H] 0 100

80

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 19

0 100

[M-2H]

2-

50 0 100

[M-3H]

3-

[M-2H]

2-

50 0 100

[M-3H]

3-

[M-2H]

2-

50 0 100

[M-3H]

3-

[M-2H]

50

2-

0 1000

1200

1400

1600

1800

2000

m/z

5 0 6

8

10

12

14

16 Time (min)

18

20

22

24

26

418 419

Figure 5. IP-RPLC-MS analysis of oligonucleotides with 100 mM TEAA as ion-pair reagent and

420

isopropanol assisted electrospray. Conditions: Column, AQ-C18, 5 µm, 20 cm × 75 µm i.d.; mobile

421

phase, 100 mM TEAA, pH 7.0, 5% acetonitrile (buffer A), 100mM TEAA, pH 7.0, 45% acetonitrile

422

(buffer B); linear gradient, 0% buffer B for 5 min, from 0 to 30% for 25 min; flow rate, 200 nL/min;

423

electrospray voltage, 2.0 kV; negative mode MS detection; sample, N-1, N-3, N-4, N-5, N-6, dT11 and

424

dT12, 2 pmol for each.

425 426


Page 17 of 19

w3 100 Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a3-B705.13 z=1

80 60

w2 -

40

625.10 z=1

20 0 100

401.02 z=?

a5-B1338.24 z=1

w62-

721.10 z=2 833.14 z=2

1313.23 z=2

a9-B2+

w2 +

60 609.10 z=1 483.06 z=1

305.05 z=1

w5 -

1436.23 1556.27 z=1 z=1

w8 a9-B2-

490.06 z=1

627.11 z=1

40

w fragment a-B fragment

934.16 w4 z=2 1243.21 989.67 z=1 z=2 2a8-B2a7-B 1146.20 z=2 2-

Positive mode

80

20

914.15 z=1

Negative mode

z=1

400

600

1869.34 z=1

.53 48 43 .38 .33 .27 .21 6 1 7 37 3. 4. 00 71 58 45 6.1 .1 .0 31 283 250 22 18 15 12 91 627 323

4 9 5 1 6 7 9 4 9 3.0 7.1 6.1 0.2 .2 .3 .4 .4 .4 40 70 103 134 16691982 229526242913

a3-B+ 1084.53 1237.71 z=3 w3 + a5-B+ 707.14 z=2 z=1 1340.24 916.16 2+ 785.39 z=1 2+ w6 2+ a8-B w82+ z=1 z=4 a -B 2+ w5

7

1586.27 z=2

a6-B+1760.28 z=1

0

427

w6-

a6-B- 1765.29

800

1000

1200

1400

1600

1800

a7-B+ 1982.34 z=1 2000

m/z

428

Figure 6. The MS/MS spectra of N-1 (11mer) in negative and positive modes ESI-MS detection,

429

respectively. Collision induced dissociation was applied in both cases.

430 431 432 433 434



Oligonucleotides

Histones

H2A

1133.21 z=4

N-7

100

984.69 N-5 z=4

90

N-4 80

908.68 z=4

701.16 z=20

60 50

N-1

N-6

70

N-3 1229.23 N-6z=3

N-8

778.90 z=18 637.46 z=22

N-12

Relative Abundance

N-9

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 19

100

H2B

80

689.74 z=20

701.16 z=20

698.45 z=19

60 40

20 1463.67 N-7 z=5 0 1312.58 690 z=3 N-10 N-13 1529.48 z=5 N-2 1613.54 z=4

H4

703.26 z=20

694.69 z=20

710.23 z=16

700

710

720

m/z

N-8 1413.93 z=3

N-3

N-9 N-12

40

1829.33 z=4

30

N-14

1778.32 z=5

20

1681.82 z=2

10

N-13 1911.59 z=4

0 600

800

1000

1200

1400

1600

1800

2000

m/z

435 436

Figure 7. The full MS spectrum of the mixture of oligonucleotides and histones in propanoic acid

437

vapor assisted positive ESI-MS detection. Conditions: electrospray solution, H2O; sample, 50 µg/mL

438

oligonucleotides (N-1 to N-14) and 200 µg/mL histone proteins; positive mode MS detection; sample

439

injection rate, 3 µL/min.

440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459


Page 19 of 19

460 461 462

TOC graphic Open state

a

Ambient ESI source

C18 capillary analysis column Enclosed state

high voltage

heated ion transfer tube high vacuum CEESI source

air

b rubber ring organic solvent

b

100

b

80

b

b

1.63

0.59

2.62

1.71

b

4.59

3.55

2.68

0.55

90

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


4.65

3.58

2.72

4.71

3.71 4.56

1.57

70 60 50 40

a

a

a

a

a

a

30 20 10 0 0.0

463 464 465

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

Ti me (min)


Enhancing the Mass Spectrometry Sensitivity for Oligonucleotide

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