New Ionization Method for Analysis on Atmospheric Pressure

Feb 2, 2013 - Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States. Anal. Chem. , 2013, 85 (4), pp 2005–2009 ... ...
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New Ionization Method for Analysis on Atmospheric Pressure Ionization Mass Spectrometers Requiring Only Vacuum and Matrix Assistance Sarah Trimpin* and Ellen D. Inutan Department of Chemistry, Wayne State University, Detroit, Michigan 48202, United States S Supporting Information *

ABSTRACT: Matrix assisted ionization vacuum (MAIV) is a new ionization method that does not require high voltages, a laser beam, or applied heat and depends only the proper matrix, 3-nitrobenzonitrile (3-NBN), and the vacuum of the mass spectrometer to initiate ionization. Analyte ions of volatile as well as nonvolatile compounds are formed by simply exposing the matrix−analyte to the vacuum of a mass spectrometer. The reduced pressure at the inlet of an atmospheric pressure ionization mass spectrometer suffices to produce analyte ions, but unlike the previously reported matrix assisted ionization inlet method, with MAIV, heating the inlet is not necessary. Singly and multiply charged ions are formed similar to electrospray ionization but from a surface. Mass spectrometers in which a heated inlet tube is not available can be used for ionization using the 3-NBN matrix. We demonstrate rapid, high-sensitivity analyses of drugs, peptides, and proteins in the low femtomole range. The potential for high-throughput analyses is shown using multiwell plates and paper strips.

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sublimation of this matrix.27 Producing charge on the droplets/ particles and the ability of the matrix to evaporate or sublime is hypothesized to be crucial for producing ions of analyte molecules in inlet and vacuum ionization31,32 and appears to also be crucial for MAIV.26,27 Three additional matrixes have been found applicable as MAIV matrixes, 2-nitrobenzonitrile, 3nitrobenzaldehyde, and coumarin.27 None come close to the performance of 3-NBN. Previous work26,27 demonstrated that closed vacuum systems of either an intermediate pressure vacuum MALDI source with the laser turned off or an ESI source closed to AP, achieved with minor instrument modifications, produce analyte ions with MAIV. MAIV is problematic on current commercial vacuum MALDI sources because only one sample can be placed into the vacuum at a time to avoid simultaneous ionization of multiple samples. Using these commercial sources,26,27 continuous ion formation is observed for up to 30 min. While this can be important for many mass spectrometric applications, e.g., high-performance fragmentation methods, it is a distinct disadvantage for rapid analyses. Here, we show that a source partially open to AP is sufficient for analyte ionization. This source configuration, in which samples can be manipulated directly from an AP environment, is mechanistically important and provides a simple and flexible means for rapid analyses. The potential for high-throughput analyses and the applicability to a variety of different compound

tmospheric pressure (AP) ionization mass spectrometers are in wide use partly because of the ease of sample manipulation and speed of analysis.1−21 In fundamental work related to ionization for mass spectrometry (MS), several new ionization methods operating from AP22−24 and vacuum25−27 were discovered. These methods are sensitive, widely applicable, operate with and without a laser, from solid or solution, all producing ions with charge states similar to electrospray ionization (ESI) and thus are equally applicable with mass spectrometers having advanced structural characterization features such as high mass resolution and mass measurement accuracy, ion mobility spectrometry (IMS) separation,26−29 and advanced fragmentation capabilities such as electron transfer dissociation (ETD).22,26,30,31 The commonality of inlet and vacuum ionization is that matrix−analyte droplets or particles are proposed to serve as the charge carrier for ionization of the incorporated analyte.31,32 Droplets have been shown to be produced by laser ablation during laserspray ionization of a solid matrix at AP.33 Zenobi and co-workers verified the observation of clusters with laserspray ionization inlet.34 Matrix assisted ionization vacuum (MAIV) using the matrix 3-nitrobenzonitrile (3-NBN) was recently reported.26,27 With this method, the only requirement is that the matrix−analyte mixture, prepared similar to matrix-assisted laser/desorption ionization (MALDI) sample preparation methods, be exposed to sub-AP conditions. 3-NBN is known to sublime35 and to triboluminescence36,37 upon crystal fracturing, and these characteristics may be important in producing bare analyte ions for analysis by MS. Particle formation is observed during © 2013 American Chemical Society

Received: December 20, 2012 Accepted: February 2, 2013 Published: February 2, 2013 2005

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approaches for producing abundant multiply charged MAIV ions could be evaluated. For example, simply placing a KimWipe, with 1 μL of a matrix (3-NBN)−analyte sample deposited, against the inlet aperture opening to the mass spectrometer produces abundant analyte ions (Figure S1A in the Supporting Information). A strip of filter paper can also be used to obtain abundant ions when the pointed tip of the paper strip is aligned with the opening of the inner cone (Figure S1B in the Supporting Information). A pipet tip with the small end held against the inner skimmer cone opening on a SYNAPT G2 mass spectrometer provides another convenient means of ionization (Figure S1C in the Supporting Information). In all cases, the best results are obtained when there is gas flow to vacuum. We previously reported with vacuum tight MAIV that increasing the inner and outer skimmer cone openings on the Waters Z-spray ESI source relative to the commercial opening produced improved results.26 We thus evaluated three modified cone openings that have larger diameters than the commercial cone. Ionization was initiated by simply bringing the sample to the inlet (continuous mode), but with larger (∼1 and ∼3 mm) inlet openings, the vacuum isolation valve was closed when the sample was not in place against the inlet (discontinuous mode). Using the pipet tip and a medium modified inner cone opening, full scan detection of 10 fmol for clozapine and 25 fmol for ubiquitin were obtained using the discontinuous mode (Figure S2 in the Supporting Information). The sensitivity was similar to that reported for MAIV on a commercial intermediate pressure vacuum MALDI source (e.g., 5 fmol of clozapine and 50 fmol of ubiquitin).26,27 Negative or positive ions from drugs, lipids, fatty acids, peptides, and proteins are readily formed using this simple method (Figure 1A and Figure S3 in the Supporting Information). On the basis of MAIV using the vacuum seal approach on a modified ESI source, we expected the largest inlet opening (∼3 mm) using the discontinuous mode (Figure IB, inset in the Supporting Information) to be most sensitive. One means of creating the airflow for improved sensitivity with this larger opening is to provide an imperfect seal between the mass spectrometer inlet and a metal ferrule placed over the entrance of the inlet. When a glass microscope slide, onto which the matrix−analyte sample is deposited, was set tight against the ferrule, sufficient vacuum is achieved with the imperfect seal to produce abundant analyte ions. A number of surfaces were tested using this opening (e.g., filter paper, foil, PCR tube and cap; Figure S4 in the Supporting Information), all of which produced analyte ions from 3-NBN. Unlike with matrix assisted ionization inlet (MAII),23,31 the 3-NBN matrix does not require a heated inlet tube. In fact, heat decreases the ion abundance when the matrix−analyte is exposed to low subambient pressure conditions (Figure S5 in the Supporting Information). The ability of this matrix to lift large and nonvolatile compounds into the gas phase without the necessity of providing heat is crucial in producing analyte ions using this matrix in an intermediate pressure vacuum MALDI source without the necessity of a laser.26,27 The large opening inner cone was next evaluated for rapid analyses using multiwell plates (Figure S6 in the Supporting Information). In this experiment, 3-NBN solution was added to wells of either 96 or 384 well plates containing a variety of samples of different compound classes and concentrations. Exposing the matrix−analyte sample to vacuum simply by creating a low pressure at the bottom of a well using the inlet

classes is demonstrated using paper and paper strips as well as multiwell microtiter plates.

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EXPERIMENTAL SECTION Materials, Sample Preparation, and Analysis. Unless otherwise stated, chemicals were purchased from Sigma Aldrich Inc. (St. Louis, MO). Illicit drugs were purchased from Cerilliant (Round Rock, TX), clozapine from Santa Cruz Biotechnology (Santa Cruz, CA), and N-actylated fragment of myelin basic protein from Anaspec (Fremont, CA). Myoglobin was obtained from Waters Co. The following matrix preparations were developed: 3-NBN matrix was prepared by dissolving 5 mg in (I) 50 μL of acetonitrile with 0.1% formic acid, (II) 150 μL of 50:50 acetonitrile−water with 0.1% formic acid for positive mode, or (III) 150 μL of 50:50 acetonitrile− water with 1% ammonium hydroxide for negative mode measurements. These matrix preparation could also be used to precoat surfaces with matrix. For matrix−analyte preparation, 1:3 and 1:2 volume ratio were used for positive and negative measurements, respectively, and spotted on the sample holder (e.g., glass plate, KimWipe, foil, filter paper (Whatman grade no. 50), PCR tube, and their caps (Fisherbrand MCT) or mixed directly in the 96 or 384 well plates. Matrix−analyte spots were used dry or wet, and the mass spectrometers were operated as previously described.26−29 Source Modifications. Because no high voltage, heat, laser, or closed vacuum are required and vapors are not generated, operation without the source housing is deemed safe. The vacuum at the sample, however, must be sufficient to initiate analyte ionization. ESI-SYNAPT G2. The ESI source interlocks were overridden using a source adapter plug providing open access to the inlet. In addition to the commercial inlet cone, three larger inner cone openings were evaluated. The inner skimmer cone entrance aperture was widened in various increments (0.5, 1, and 3 mm), as was the outer cone (∼4.5 mm). The two smaller cone openings allowed the instrument to operate with the vacuum isolation valve continuously open (“continuous operation mode”), although the larger 1 mm opening was problematic, and the largest opening required that the isolation valve be closed except when the opening was covered by the sample holder (“discontinuous operation mode”). ESI-LTQ Velos. The ESI source was overridden as previously described,30,31 and an inlet tube was used with a diameter of 750 μm. With this arrangement, some airflow is required for good results. One means of achieving the necessary gas flow was by placing a metal ferrule over the inlet tube to make an imperfect seal, and another was by using air-permeable sample holders. Ionization is initiated by placing the matrix−analyte sample on a holder against or right to the vacuum opening.

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RESULTS AND DISCUSSION MAIV has been shown to operate from vacuum using either a MALDI source or a sealed ESI source.26,27 Because of the ease of sample introduction directly from an AP environment, we evaluated the potential for high sensitivity and rapid MAIV analyses with sample introduction from ambient conditions. A vacuum seal was discovered to not only be unnecessary but with the ESI source resulted in lower sensitivity, apparently because airflow is helpful in “carrying” ions through the field free regions to the ion optics for transport to the mass analyzer. Without the necessity for a vacuum tight seal, a number of 2006

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occasionally vented when the vacuum isolation valve was open and the sample was improperly in place. Smaller modified inlet openings were tested and found to produce almost equivalent results to the largest opening, so long as there was some airflow into the vacuum. The smaller modified inlets could be operated in continuous mode. With the success using the smaller modified inlet opening, the commercial, unmodified ESI skimmer assembly was tested and found to achieve good sensitivity provided the matrix−analyte sample is placed on an air-permeable surface capable of generating sufficient vacuum conditions to initiate ionization when held against the inlet as well as enough gas flow for good ion transmission. Thus, optimization with any of the skimmer inlet openings could be achieved by selecting the proper surface, but use of a cone with a sufficiently small opening not to vent the instrument allowed convenient use of continuous mode operation enabling rapid analyses. With the commercial cone arrangement, different sample supports (glass plate, filter paper, KimWipe, and pipet tip) onto which a matrix−analyte sample is applied and held against the outer cone on the SYNAPT G2 mass spectrometer produce excellent quality mass spectra (Figure S8 in the Supporting Information). Nothing more is required as long as the sample holder provides air flow and sufficient vacuum for the instrument to operate properly. The source temperature in these experiments is kept at 50 °C to prevent burns by inadvertently touching the source housing and to provide high ion abundance (Figure S8.I in the Supporting Information) as is shown by comparisons with a source temperature of 150 °C, as in MAII, and dislodging the sample through exposure to the vacuum (Figure S8.II in the Supporting Information) or through tapping (Figure S8.III in the Supporting Information). As with the larger cone opening, the matrix−analyte sample can be wet or dry (Figure S9 in the Supporting Information), and the analysis can be simplified by precoating the surface with either the matrix or the analyte (Figure S10 in the Supporting Information). In continuous mode, operation is straightforward, flexible, and robust. The ease and convenience of obtaining mass spectra from an air-permeable surface at AP without the need of a high voltage, a laser, or even sufficient heat to cause a safety issue, suggest a very simple approach to high-throughput analyses for small and large nonvolatile molecules. Such an approach would not need an ion source and could use rolls of ribbon or paper as the matrix−analyte substrate. As a proof of principle experiment, a 2 cm × 11 cm strip was cut from filter paper and 5 spots from a solution of various analytes with 3-NBN as matrix were placed at 2 cm intervals. The vacuum from the mass spectrometer held the strip against the inlet. By pulling the paper strip so that successive samples crossed the inlet, all 5 samples were analyzed in 25 s (Figure S11 in the Supporting Information). The filter paper provides sufficient vacuum for ionization and enough air flow for high sensitivity ion transfer. Samples ranging from small molecules to proteins (Figure 2) were analyzed using this approach; isotopic distributions are displayed in Figure S12 in the Supporting Information. As can be seen in the total ion current (TIC) display, the ionization event is sharp (Figure 2B) and cross contamination is not observed. Contrary to MALDI, the chemical background38,39 is minute and both low and high mass compounds can be observed with this high-performance instrument because the method produces multiply charged ions for high-mass compounds so that the mass-to-charge falls within the

Figure 1. MAIV using 3-NBN matrix (preparation I) performed on a custom modified ESI source of a SYNAPT G2 mass spectrometer using an ∼1 mm inner cone opening of the skimmer at a source temperature of 50 °C: (A) 10 pmol of carbonic anhydrase (MW ∼29 kDa) using a pipet tip and (B) 5 pmol of illicit drug lysergic acid diethylamide (LSD) (MW 323 Da) using a 384 well plate and a nozzle consisting of a copper tube extension with a 90° bend. Insets show photographs of the source operation. Supporting information data shows the use of KimWipes, PCR tubes or their caps, 96-well plates, and paper strips (Figure S4 in the Supporting Information).

(vertical arrangement, Figure S6I in the Supporting Information) or connecting a well with the inlet using a nozzle tube (horizontal arrangement, Figure 1B) produces ions for analysis by MS. As noted above, AP ionization mass spectrometers are designed for gas flow aiding in ion transport and possibly desolvation of charged matrix−analyte particles. Improved results are obtained for single and multiwell plate containers, especially with the nozzle extension, using an imperfect seal to the well. With the well plate arrangements, acquisition of mass spectra from 6 samples is obtained in 55 s (vertical, Figure S6II,III in the Supporting Information). The ease of this approach suggests rapid analyses with more sophisticated engineering designs. Compounds in matrix solution placed on, e.g., filter paper or a KimWipe and subsequently in the well plate cavity can also be used, and the ion abundance is frequently improved (Figure S7 in the Supporting Information). The larger skimmer cone opening, however, was problematic because in some configurations higher background ions were observed and, most importantly, the instrument 2007

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sampled. This makes the MS and MS/MS analysis (Figure S13 in the Supporting Information) ultimately simple and portable. MAIV-IMS-MS using the Waters SYNAPT G2 allows efficient IMS gas-phase separation to be obtained. The separation is enhanced because of the formation of multiply charged ions (Figure S14 in the Supporting Information). MAIV has also been shown to operate on a Thermo Scientific LTQ Velos mass spectrometer with an inlet tube rather than a skimmer AP to vacuum interface. The inlet is heated to 50 °C for myoglobin using 3-NBN as the matrix (Figure S15A in the Supporting Information). ETD characterization of peptides from filter paper strips obtains good c- and z-ion sequence coverage (Figure S15B in the Supporting Information).

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CONCLUSION We demonstrate here several approaches for using the MAIV method to analyze samples from an AP environment without need of a voltage, laser, or even added heat. Simple and convenient proof of principle approaches for high-throughput analyses are presented. Other approaches can be envisioned for using this highly sensitive ionization method to rapidly observe and identify chemical composition changes related to, for example, drugs, lipids, peptides, and proteins. As with any new method, many aspects need to be evaluated and utility explored and expanded. Aspects include sensitivity, suppression effects,40 dynamic range, applicability to ultra high-resolution mass spectrometry, high-performance fragmentation technology, and surface imaging approaches. It can be expected that methods and technologies used with current ionization approaches, such as moving stages, will ensure rapid progress. With the flexibility of this source design, it is also expected that rapid progress in discovering new matrix compounds and best sample preparation conditions that limited previous MAIV developments will be forthcoming. This new method, which eliminates the need of a conventional ion source, may potentially be useful in many areas where simplicity, sensitivity, and throughput are important such as blood spot analyses, portable mass spectrometers, and production control.

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ASSOCIATED CONTENT

* Supporting Information S

Figure 2. MAIV with 3-NBN matrix (preparation I) performed on a custom modified ESI source using the ∼3 mm inner cone opening of the skimmer at source temperature of 50 °C and a filter paper strip spotted with matrix−analyte. The paper strip adheres to the inlet because of the mass spectrometer vacuum created when the vacuum isolation valve is opened. The strip is moved across the inlet to sequentially sample each spot. (A) Photograph, (B) total ion current (TIC), and (C) mass spectra of (1) 1 pmol of sphingomyeline (MW 702 Da), (2) 0.5 pmol of angiotensin I (MW 1295 Da), (3) 1 pmol of bovine insulin (MW 5730 Da), and (4) 2 pmol of ubiquitin (MW 8560 Da). Isotopic distributions are displayed in Figure S12 in the Supporting Information.

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful for support from the WSU Rumble Fellowship Award (to EDI), WSU Schaap Faculty Scholar Award, Eli Lilly Young Investigator Award in Analytical Chemistry, Waters Corporation Center of Innovation Award, DuPont Young Professor Award, the American Society for Mass Spectrometry Research Award, and NSF CAREER Award 0955975 (to ST).

instrument limit. Automation of this process is expected to produce a high-throughput method even for compounds of widely divergent masses. Paper chromatography strips can also be sampled using this method (Figure S13 in the Supporting Information). Small molecules are separated and, instead of staining to observe where analyte is located, the 3-NBN matrix is applied. The surface is then placed against the inlet of the mass spectrometer where ionization commences sequentially as each analyte is

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