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Vacuum Matrix-Assisted Ionization Source offering Simplicity, Sensitivity, and Exceptional Robustness in Mass Spectrometry Sarah Trimpin, Milan Pophristic, Adetoun Adeniji-Adele, John W. Tomsho, and Charles N McEwen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03378 • Publication Date (Web): 21 Aug 2018 Downloaded from http://pubs.acs.org on August 23, 2018
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Analytical Chemistry
Vacuum Matrix-Assisted Ionization Source offering Simplicity, Sensitivity, and Exceptional Robustness in Mass Spectrometry Sarah Trimpin,1-3* Milan Pophristic,3* Adetoun Adeniji-Adele,4 John W. Tomsho,4 Charles N. McEwen3,4* 1
Department of Chemistry, Wayne State University, 5101 Cass Ave, Detroit, MI, 48202, USA
2
Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, MI 48202, USA 3
4
MSTM LLC, Newark, DE 19711, USA Department of Chemistry & Biochemistry, University of the Sciences, Philadelphia, PA 19104, USA
[email protected],
[email protected],
[email protected] Abstract Vacuum matrix-assisted ionization (vMAI) uses select matrix compounds which when exposed to the vacuum of a mass spectrometer produce gas-phase ions from associated volatile or nonvolatile analyte without external energy input. Here, a vacuum MAI source was constructed to replace the commercial inlet of a Thermo Orbitrap mass spectrometer. This allowed for rapid introduction of the matrix:analyte sample by a probe, contrary to vacuum matrix-assisted laser desorption/ionization (MALDI) sources.
The matrix:analyte sample is
inserted into a region of the ‘S-lens’ entrance, where the spontaneously formed ions can be effectively transferred to the mass analyzer. This specifically designed ion source requires no laser, high voltage, heat, or nebulizing gases. A low voltage is used to transmit the ions through the commercial ‘S-lens’ assembly. Airflow can be used to modulate the ionization event. A few picograms of the drug erythromycin, assisted by the 3-nitrobenzonitrile vMAI matrix, is sufficient to produce mass spectra for over 1 min with the MH+ ion as the base peak in each mass spectrum. There is minimal carryover when loading high concentration samples and complex mixtures, contrary to direct infusion electrospray ionization, providing the probe is thoroughly cleaned between each new sample acquisition. Analyses of biological fluids, bacterial extracts, tissue, and high concentration samples have so far shown no indication of inlet or instrument contamination with these samples. The typical ultra-high resolution and mass accuracy of the mass spectrometer are achieved, and a path forward to potential high throughput acquisitions demonstrated. It is expected that robustness can be introduced to any mass spectrometer through implementation of such a simple source.
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Introduction An exciting advantage of matrix-assisted ionization (MAI) is its simplicity.1 An ion source with high voltage, laser, nebulizing gas, or capillary tubing, that can be clogged, is unnecessary, thus avoiding common complications associated with mass spectrometry (MS).2-5
MAI has
been most commonly used with atmospheric pressure inlet mass spectrometers with the matrix:analyte sample introduced from close proximity, or directly into, the inlet aperture.6,7 Over 40 MAI matrices have been discovered, some of which show specificity for certain analytes.8,9 This method has been commercialized and automated, and the approach used with a portable10 as well as high-end mass spectrometers11,12 obtaining results directly from e.g. diluted urine without cleanup procedures.
Notably, inlet ionization processes including MAI,6,10,13-26 give
competitive sensitivity using inlets designed and optimized for nearly 30 years for electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI).
The MSTM manual
platform interfaced with an Orbitrap Q-Exactive achieved sensitivities equivalent to nanoESI with less background using the MAI method.19 Reproducibility and quantification of drugs with internal standards were shown to provide equivalent results to ESI, but faster.16 However, while MAI sample introduction from atmospheric pressure into the inlet of the mass spectrometer is simple and sensitive, similar to ESI, the method is prone to carryover for concentrated samples as well as inlet contamination over time from direct analyses of biological samples.3 However, the MAI matrix:analyte sample can be introduced on a substrate (plate or probe) directly into vacuum, greatly reducing these issues.6,27
This approach for sample introduction is here
referred to as vacuum (v) MAI. Initial studies with vMAI were performed on a Waters SYNAPT G2 intermediate pressure MALDI source without the use of a laser, and primarily using 3-nitrobenzonitrile (3-NBN) as matrix, although other matrices have also been studied. For example, 66 kDa bovine serum albumin (BSA) protein produces gas-phase molecular ions with up to 37 charges (protons) when dried with 3-NBN, and exposed to the vacuum MALDI source without initiating the laser.6 Using this vMAI method, urine, blood and tissue samples were analyzed for drugs and their metabolites directly, or after simple dilution with water, and proteins are ionized by protonation from buffered and detergent conditions as demonstrated with e.g. bacteriorhodopsin, a membrane protein.11,28,29 Metal cation adducts or other undesired attachments (e.g., matrix molecules) produced using MALDI or ESI are generally not observed.8,9 Negative ions are spontaneously produced6,8,9,11,28 allowing, for example, detection of ganglioside and other lipids (e.g. cardiolipins directly from a simple mitochondria extract or directly from tissue) and carbohydrate conjugates as singly- and multiply-deprotonated ions with minimal fragmentation, 2 ACS Paragon Plus Environment
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Analytical Chemistry
which is often not possible using ESI or MALDI. In several applications, MALDI, ESI, or both, failed or produced poor results (e.g., high chemical background) relative to MAI.30,31 Acquisition times similar to MALDI were demonstrated using the commercial MALDI source of a Waters SYNAPT G2S. Without the use of a laser, 24 samples were acquired in 4 minutes (ca. 10 seconds per sample) without cross-talk between the samples.32 The typical use of MALDI is in conjunction with TOF mass spectrometers with limited mass resolution and mass accuracy,33 which
is
a
spectrometers,
distinct
2,12,15,19,20,23,29
disadvantage
relative
to
ultra-high
resolution
mass
33
especially with increasingly complex samples.
In order to simplify the vMAI source the ion extraction lens, ion guide and laser were removed from the Waters MALDI source and a flange with ball valve replaced the MALDI sample plate introduction module so that a probe with the matrix:analyte sample could be inserted near the entrance aperture to the mass analyzer.27
Fundamental research
demonstrated that nonvolatile materials which are not ionized remain on the probe after complete sublimation of the matrix indicating high resistance to instrument contamination relative to other ionization methods used in MS. Further, carryover between samples, which can occur with ESI, SAI, and MAI, especially with increasing concentration, degree of complexity, and ‘dirtiness’ of a sample is eliminated with the vMAI method so long as the probe tip is replaced between acquisitions. Here, we report the construction of a vMAI source for Thermo mass spectrometers, including Orbitraps, as well as studies demonstrating the effectiveness of this source with an Orbitrap Focus. This vMAI source provides exceptional results with high sensitivity, simplicity, speed, and robustness. The typical ESI-like charge states are observed and the ultrahigh mass resolution and mass measurement accuracy of the employed mass spectrometer are achieved. Experimental details have been added to the Supplementary Information. Results A vacuum ionization source based on solid probe introduction was constructed and implemented on a Thermo Orbitrap Focus mass spectrometer (Figure 1 and S1 of the Supplemental).
The temperature and pressure are deciding factors in the analyte ion
abundance and duration with MAI. However, with vMAI, excellent ion abundance is obtained at room temperature. Too low of a pressure slows the ionization process, thus some gas is favorable for ionization as can be seen in Figure S2. The higher pressure in the ionization region relative to the mass analyzer may help in ion transfer to the mass analyzer, but a potential difference of a few volts placed between the probe tip and the entrance to the mass 3 ACS Paragon Plus Environment
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analyzer can be used instead of, or to aid, gas flow for ion transfer. Clearly, too high a pressure is detrimental to ion abundance, which is likely related to ion loss in the instrument.
Gas,
through collisions with the matrix surface, also counteracts evaporative cooling, helping to maintain the rate of sublimation of the matrix, which tracks gas phase ion formation. Most likely the ions observed originate from charged gas-phase matrix:analyte particles released during sublimation of the matrix.34 In this study, different samples, ranging from attomoles to micromoles of analyte, mixed with the 3-NBN matrix solution and dried on the probe tip, were introduced near the entrance to the ‘S-lens’. In one example, mass spectra were acquired for ca. 1 min applying only 20 pg of erythromycin, along with the 3-NBN matrix, to the probe. The base peak in all spectra was the protonated molecular ion (Figure 2). A single 0.5 sec acquisition at a mass resolution of 70,000 (m/∆m 200) shows good ion abundance (Figure 2B) and represents < 250 attomole of the total analyte consumed acquired at mass resolution m/∆m 40,000.
Increasing the amount of
erythromycin by 10,000 times to 200 ng substantially increased ion abundance, and after thorough cleaning of the probe tip, carryover from erythromycin was reduced to 0.05% of the original signal. A comparison of single acquisition mass spectra from 20 pg, 200 ng, and a blank after thorough cleaning of the probe tip is shown in Figure S3. The transfer of the analyte ions into the mass spectrometer occurs when the sample reaches the ‘S-lens’ region (Figure S4). The molar matrix:analyte ratios and sample sizes (
