MS Method Providing Improved Sensitivity: Electrospray

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A New LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet Madeline A. Fenner, Shubhashis Chakrabarty, Beixi Wang, Vincent S. Pagnotti, Khoa Hoang, Sarah Trimpin, and Charles N McEwen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b05172 • Publication Date (Web): 07 Apr 2017 Downloaded from http://pubs.acs.org on April 8, 2017

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Analytical Chemistry

A New LC/MS Method Providing Improved Sensitivity: Electrospray Ionization Inlet Madeline A. Fenner,† Shubhashis Chakrabarty,ⱡ Beixi Wang,ⱡ Vincent S. Pagnotti,† Khoa Hoang,† Sarah Trimpin*,ⱡ and Charles N. McEwen *,† †

Department of Chemistry & Biochemistry, University of the Sciences, Philadelphia, PA 19104, ⱡ

Department of Chemistry, Wayne State University, Detroit, MI 48202 MSTM, LLC, Newark, DE, 19711

*Corresponding authors: Charles N McEwen, University of the Sciences, Department of Chemistry & Biochemistry, 600 South 43rd Street, Philadelphia, PA 19104, Email: [email protected]. Sarah Trimpin, Wayne State University, Department of Chemistry, 5101 Cass Avenue, Detroit, MI, 48202. Email: [email protected]

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Abstract Electrospray Ionization Inlet (ESII) combines positive aspects of electrospray ionization (ESI) and solvent-assisted ionization (SAI).

Similar to SAI, the analyte

solution is directly introduced into a heated inlet tube linking atmospheric pressure and the initial vacuum stage of the mass spectrometer. However, unlike SAI, in ESII a voltage is applied to the solution through a metal union linking two sections of fused silica tubing through which solution flows into the inlet. Here we demonstrate liquid chromatography (LC) ESII/MS on two different mass spectrometers using a mixture of drugs, a peptide standard mixture and protein digests. This LC-ESII/MS approach has little dead volume and thus provides excellent chromatographic resolution at mobile phase flow rates from 1 to 55 µL min-1. Significant improvement in ion abundance and less chemical background ions were observed relative to ESI for all drugs and peptides tested at flow rates from 15 to 55 µmin-1. At low inlet tube temperature, ESII has similar ionization selectivity as ESI, but at higher inlet temperature appears to have the attributes of both ESI and SAI.


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Introduction Electrospray ionization (ESI) is notable for its ability to ionize proteins, [1] but at least as important is the interface it provides between liquid separation methods and mass spectrometry (MS). The power and limitations of liquid chromatography (LC) ESIMS has been well documented [2-4]. LC/ESI-MS has progressed over the years from 4.2 mm ID columns to smaller ID LC columns with lower flow rates in order to conserve solvent and improve sensitivity.

For the most demanding sensitivity requirements,

nano-ESI is the preferred method, operating at nL min-1 mobile phase flow rates, but requires more exacting chromatography to eliminate dead volume and reproducibility issues. Achieving improved sensitivity at higher flow rates than nano-ESI is thus an important goal for simplifying the chromatography, improving reproducibility, and decreasing elution times. Other solution based methods such as thermospray [5] and sonic spray ionization [6] have not gained favor as ionization methods for LC-MS, partly because of low sensitivity relative to ESI. Solvent-assisted ionization (SAI) has been shown to have sensitivity for some compounds that exceeds that of ESI, eliminates the need for high voltage and desolvation and nebulizing gases, and operates at solvent flow rates typically used with 1 mm and smaller inner diameter (ID) LC columns [7,8]. SAI has been successfully coupled to LC/MS [9-10]. In SAI, charge separation is initiated in a heated inlet tube linking atmospheric pressure and the first vacuum region of the mass analyzer [11, 12].

SAI is in its infancy, and to achieve its full sensitivity potential

requires optimization of a number of parameters including the inlet tube temperature, the outer diameter of the fused silica tube relative to the ID of the mass spectrometer inlet tube, and the positioning of the fused silica tube within the inlet, thus limiting its appeal at this stage of development as an alternative to ESI for high sensitivity LC/MS analyses. Variants of SAI have been described for uses other than LC/MS [8,13-16]. Application of a voltage onto a metal union linking tubing from a solvent flow system, such as an LC, with a section of fused silica tubing inserted into the heated inlet tube of the mass spectrometer, similar to SAI, has been reported to provide superior ion abundance relative to ESI when using infusion for solution introduction [17]. 3 ACS Paragon Plus Environment

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method has been termed electrospray ionization inlet (ESII) and voltage SAI (vSAI). Up to 100X increase in analyte ion abundance was reported relative to ESI with the solvent being 100% water. ESII is easy to implement because optimization of parameters is less critical than with SAI. No nebulizing gas is required, the method operates efficiently at similar flow rates as SAI, and the voltage necessary to achieve maximum sensitivity is less than that of ESI. Adjusting the voltage applied to the metal union to zero allows SAI mass spectra to be obtained, although not at optimum sensitivity without at least adjusting the position of the fused silica capillary within the inlet. ESII and SAI have different selectivity as was demonstrated with a mixture of polyethylene glycol oligomers and a peptide [17]. With ESII, the multiply-charged peptide ions were the major peaks observed, while MH+ ions of PEG oligomers were the dominant peaks for SAI. The only report of LC/ESII-MS was a conference proceeding which showed a 3 – 10X sensitivity improvement using a limited number of analytes [18]. Here, we expand on these studies using a variety of analytes and flow rates ranging from 1 to 55 µL min-1 in comparison to results obtained at the same flow rates on the commercial ESI sources of Thermo Orbitrap Exactive and LTQ Velos mass spectrometers. Experimental Small molecule analytes: buspirone, clozapine, codeine, oxycodone, and prazosin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). HPLC grade water, acetonitrile, formic acid, verapamil, and angiotensin II were purchased from Sigma Aldrich (St. Louis, MO). MassPREP enolase digestion standard was purchased from Waters (Milford, MA). BSA tryptic digest was purchased from Anaspec, Inc (Freemont, CA). The mass spectrometers used in this study were an Orbitrap Exactive (Thermo Fisher, Bremen, Germany), and a LTQ Velos (Thermo Fisher, San Jose, CA). A Waters nanoAcquity (LC) was interfaced with the LTQ Velos mass spectrometer, while a Michrome Bioresources Paradigm A4 (Auburn, CA) LC and a nanoAcquity LC were interfaced with the Orbitrap Exactive. The MSTM (Newark, DE) multifunctional ionization


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platform with the SAI/vSAI/ESI module was used for operation in the SAI and ESII modes. Water/acetonitrile/1% formic acid gradients were used for all analyses. In ESII, voltage is placed on a stainless steel union linking tubing from an LC or infusion pump with a section of fused silica tubing, the exit end of which is placed into the heated inlet tube of a mass spectrometer. Typically, for ESII, less than 2.5 kV is placed on the metal union. In particular, black PEEK tubing, purchased from Cole Palmer (Vernon Hills, IL) (1/16 outer diameter and 1/100 inter diameter (Vernon Hills, IL), connected the exit of the LC column with a metal union (Upchurch Scientific, 1/16, 10-32, through hole 0.01” ID) to which the voltage normally applied to the ESI emitter was connected using an MSTM multifunctional ionization platform. A fused silica tube ca. 8 cm in length and 75 mm ID X 360 mm OD (SGE Analytical Science, Australia) was connected to the union with its exit end inserted into the mass spectrometer inlet. The MSTM platform replaces the Ion Max source, prevents contact with high voltage and allows ready positioning of the fused silica tubing inside the inlet tube. For the experiment using a flow rate of 1 µL min-1, a Waters BEH130C18 (1/µm, 100µm x 100 mm) LC column was connected to a 20 µm ID x 360 µm OD fused silica tubing using a VICI (Houston, TX) C360UFS2 union with a 50 µm thru-hole. For experiments reported here, the inlet tube temperature ranged from 250 to 325 ºC unless otherwise noted.

Tuning is accomplished by infusing a 100 nM 1:1 water:

acetonitrile (1% formic acid) solution of angiotensin II at a flow rate of 25 µL min-1 and optimizing on the doubly charged peptide ions.

A tune file is created using the

automatic tune feature of the Thermo Scientific X-Caliber software. For ESI analysis, the HESI probe was optimized for temperature and spray position using the same angiotensin II solution and flow rate with an ESI created tune file. Results and Discussion Most LC/MS analyses are performed with small molecule compounds, and therefore a mixture containing 100 ppb each of the drugs buspirone, clozapine, codeine, oxycodone, prazosin, and verapamil was used to determine the sensitivity of ESII 5 ACS Paragon Plus Environment


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relative to the commercial ESI probe on a LTQ Velos mass spectrometer. The elution gradient for the Waters nanoAcquity LC with an Acquity UPLC HSS T3 1.8µm 1.0 x 100mm column was used for the analysis injecting 4 µL at a flow rate of 50 µL min-1 with the elution gradient shown in Table S1. The results applying 4 kV to the ESI probe are shown in Figure 1A and applying 1 kV to the ESII metal union used with ESII is shown in Figure 1B. For the drugs obtained with ESII, the base peak chromatograms ion abundances are, on average, 3X greater than obtained from the extracted ion chromatogram obtained from ESI. However, the ion abundance obtained by extracting the MH+ ion of verapamil, for example, is 25X more abundant using the ESII method (Figure 1). Besides the sensitivity gain, ESII is characterized by lower background ions. This is in contrast to another recently introduced ionization method, voltage obstructive ESI or UniSpray, which provides a 3-10x increase in analyte ion abundance for most compounds, but also increases the background ions by a similar amount using a high flow of nebulizing gas [19-21].

An additional drawback of atmospheric pressure

ionization methods, including ESI, is that contaminants within the ESI source housing are often observed as background ions, possibly because of a DESI effect ionizing surface contaminants [22], or because of capture of volatile analytes by the charged spray droplets [23]. This was observed during LC/ESI-MS acquisitions of the drug mixture shown in Figure 1, where an unknown background peak at m/z 279 was more abundant than the ions from the eluting drugs (Supplemental Figure S1). Therefore, without first cleaning the source housing region, peaks corresponding to the drugs were not observed in the base peak or total ion chromatograms. The signals for the expected ions were only observed after extracting all ions between m/z 285 and m/z 500 (extracted ion chromatogram) in order to not include the m/z 279 contaminate ion signal. However, replacing the ESI housing with an MSTM platform and acquiring mass spectra using the ESII method, the m/z 279 ion, and other background ions, were well below the signal for the drugs. With ionization occurring within the heated inlet tube, ions from source contaminants are greatly reduced, which is expected to result in more reliable analyses. By reducing background ions, charge competition is also reduced which likely contributes to the improved sensitivity using the ESII method. The same 6 ACS Paragon Plus Environment

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mixture was acquired using the same column but after cleaning the ESI source housing and with the LC flow rate lowered to 15 µL min-1. Here, the ion abundance observed for ESII was on average ca.10X (ranging from 7X to 20X) higher than that obtained with ESI (Figure S2-S5). The base peak chromatograms, with reduced chemical background, for ESII and ESI show well separated peaks for all of the drugs. ESII was also acquired at a flow rate of 1 µL min-1 using a Waters nanoAcquity LC interfaced with a Thermo LTQ Velos with the fused silica outlet of the column connected directly to the low dead volume metal union. On the inlet side of the union a smaller inner diameter fused silica was used relative to that at higher flow rates. Comparison with ESI using the standard Thermo IonMax ESI probe was not realistic because of the higher dead volume inherent with the commercial probe which was designed to accommodate higher flow rates.

The chromatogram for a 30 min

acquisition shows excellent peak resolution and good sensitivity (Figure S6). While these results demonstrate the potential of ESII for low flow rate analyses, at this flow rate, there seems to be little advantage relative to nano-ESI. Improved results were also obtained using ESII at a flow rate of 35 µL min-1 when a peptide standard mixture, which contained a dipeptide, tripeptide, angiotensin II, leuenkephalin, and met-enkephalin, was analyzed using a Waters nanoAcquity UPLC interfaced to an Orbitrap Exactive. One microliter of a 2 ppm solution was injected onto a Restek Viva C18(5 µm, 100 x 1 mm) column and analyzed using ESI (Figure S7A) as well as ESII (Figure S7B) with the inlet tube at 250 ºC. For these compounds, ESII on average produced >5X more ion abundance than ESI.

As can be seen from the

chromatograms, part of the sensitivity gain is due to lower dead volume and sharper chromatographic peaks using the ESII method, which we partly attribute to the use of the larger internal diameter commercial ESI emitter tubing which can operate at flow rates exceeding 1 mL min-1. This same mixture was also analyzed by turning the voltage used with ESII to zero (unoptimized SAI). Comparison of these results (Figure S8) with those obtained with ESII, show the mass spectra obtained by ESII, as well as the relative ion abundances of the peptides, is more similar to ESI than to SAI indicating that the term 7 ACS Paragon Plus Environment


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ESII better reflects the ionization family tree than vSAI. Interestingly, as the inlet tube temperature is increased, late eluting compounds most prominent in the SAI chromatogram increase in ion abundance in ESII but not ESI. Thus, for these results, at the lower inlet tube temperature (350 ºC, compounds that are better ionized by SAI become more prominent in the chromatogram produced using ESII.

In general, we observe that high inlet tube

temperature favors more inclusive ionization using ESII than is obtained with ESI. In order to test if the gain in ion abundance is common for peptides using ESII, LC/MS of protein tryptic digests were acquired using ESII and ESI under identical chromatographic conditions. Figure 2 shows the base peak chromatograms for a 1 µL injection of a 200 fmol µL-1 solution of a BSA digest using the Orbitrap Exactive and a mobile phase flow rate of 35 µL min-1 for ESI (Figure 2A) and ESII (Figure 2B). On average a 3X increase in signal is achieved for this sample with ESII relative to ESI. Several peaks are observed in the chromatogram obtained using ESII that are not above the background noise of the base peak chromatogram using ESI (Table 1). By decreasing the voltage applied to the metal union to zero, unoptimized SAI is achieved for this sample. The SAI results are shown in Table 1. ESI and SAI show selectivity to different peptides in the mixture, but ESII detects all peptides observed with ESI and SAI. The simplicity of the ESII setup, eliminating the need for desolvation or nebulizing gases, and the increased signal with decreased chemical noise demonstrate the utility of LC/ESII-MS for peptide and bottom-up protein analysis approaches. Similar results were observed with injection of a 100 fmol µl-1 solution of enolase digest. Overall ESII was able to detect more peptides expected for the enolase tryptic digest than ESI (Table S2). As with all tested analyses, ESII produced improved results in terms of ion abundance and low background relative to the commercial ESI source.

The low

background might be expected to reduce false hits in proteomic studies. While ESII has distinct advantages over ESI, a potential disadvantage is that the entire solvent flow enters the inlet of the mass spectrometer, while in ESI, most of the 8 ACS Paragon Plus Environment

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solvent sprays onto surfaces around the inlet aperture. Solvent flow of at least 100 µL min-1 are readily handled by modern mass spectrometer pumping systems, and thus when this entire flow enters the inlet, the potential for contamination must be considered. Nonvolatile salts could pose a source of inlet contamination during LC/MS, therefore diversion of the solvent front, commonly performed automatically in LC-MS, should eliminate this issue. The potential for contamination was previously explored by introducing a solution of acetonitrile/water 0.1% formic acid continuously into the inlet of an Orbitrap mass spectrometer for 5 hours with minimal loss of sensitivity for SAI [7]. As the flow rate is reduced by using smaller ID LC columns, any contamination approaches that of nano-ESI where almost all of the LC effluent enters the inlet to the mass spectrometer. Finally, instrument contamination issues have not been observed after many hours of operation using ESII on two different mass spectrometers, and three mass spectrometers have been operated in sum for 20 years using various inlet ionization methods without any unusual contamination or operational issues.

Conclusion ESII is shown to give substantial improvement in ion abundance and reduced signal-to-background ions relative to ESI for peptides and small molecule drugs on mass spectrometers having a heated inlet tube. Background ions in ESI associated with ion source contamination are reduced or even eliminated. ESII is shown to be applicable with LC columns with ID’s between 0.1 and 1 mm, provides excellent chromatographic resolution because of low dead volume, eliminates the need for desolvation and nebulizing gas, and reduces complexity relative to commercial ESI ion sources. Nebulization of the mobile phase is assisted by the gas flow through, and the heat applied to, the inlet tube resulting in a voltage requirement similar to nano-ESI and potentially smaller droplet formation relative to ESI. At inlet temperatures below 300 ºC, ESII provides similar selectivity to ESI, but increasing the inlet temperature to > 350 ºC provides a combination of ESI and SAI like ionization and is thus more universal. Studies are underway to identify which compound types benefit from the use of a higher inlet tube temperature. 9 ACS Paragon Plus Environment


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Acknowledgements This material is based in part upon work supported by the National Science Foundation under Grant Numbers NSF CHE-1112289 to CNM, NSF CHE 1411376 to ST, and NSF STTR Phase I 1417124 and Phase II 1556043 to MSTM, LLC.

Any

opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.

CNM gratefully acknowledges funding through the Richard Houghton

endowment from the University of the Sciences.

Figure Captions

Figure 1: (A) ESI extracted ion chromatograph (m/z 285-500) and (B) ESII total ion current chromatogram for a 4 µL injection of a small molecule mixture of drugs containing 100 ppb of each drug at a mobile phase flow rate of 50 µl min-1 on nanoAcquity UPLC column and acquired using a Thermo Fisher LTQ Velos mass spectrometer. Chromatographic conditions are shown in Table S1. Figure 2. Base peak chromatogram of a BSA tryptic digest injecting 200 fmol at a flowrate of 35 µL min-1 on a nanoAcquity LC using a Restek Viva C18 column (5 µm , 100 x 1 mm) interfaced with an Orbitrap Exactive mass spectrometer. (A) ESI vs. (B) ESII showing improved ion abundance and lower background.


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Figure 1:



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Figure 2.


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Table Caption Table 1. Peptide coverage comparison between ESI, ESII, and SAI for a 200 fmol injection of a Waters BSA tryptic digest using a nanoAcquity LC with a mobile phase flow rate of 35 µL min-1 onto a Restek Viva C18 column (5 µm , 100 x 1 mm) interfaced with an Orbitrap Exactive mass spectrometer.

Table 1.

Isotopic Masses (m/z)

Peptide Sequence

Elution Times

Ionization Method

395.24, 789.47 461.75, 922.49

LVTDLTK AEFVEVTK

4.78 5.26 5.29

ESII ESII, ESI

636.98, 954.97

LFTFHADICTLPDTEK

432.24 740.40 1014.62 1479.80

VGTR LGEYGFQNALIVR

5.34 5.74

ESII, ESI ESII, ESI, SAI

464.25, 927.50

YLYEIAR

6.35

ESII, ESI

501.80, 1002.58

LVVSTQTALA

6.17

ESII, SAI

653.36

HLVDEPQNLIK

7.40

ESII

507.81, 1014.62

QTALVELLK

8.05

ESII, ESI, SAI

8.83 8.97 10.11 10.36

ESII ESII, ESI, SAI ESII, SAI ESII

978. 48 DAIPENLPPLTADFAEDK 700.35, 1399.70 TVMENFVAFVDK 784.37 DAFLGSFLYEYSR 352.18 VLASSAR

ESII, ESI, SAI



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Table of Content Graphic


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MS Method Providing Improved Sensitivity: Electrospray

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