High-Throughput Characterization of Small and Large Molecules

Mar 10, 2015 - Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals fo...
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High-Throughput Characterization of Small and Large Molecules Using Only a Matrix and the Vacuum of a Mass Spectrometer Daniel W. Woodall,† Beixi Wang,† Ellen D. Inutan,† Srinivas B. Narayan,‡ and Sarah Trimpin*,†,§ †

Department of Chemistry, Wayne State University, 5101 Cass Avenue, Detroit, Michigan 48202, United States Detroit Medical Center: Detroit Hospital (DMC), Detroit, Michigan 48201, United States § Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan 48202, United States ‡

S Supporting Information *

ABSTRACT: Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MSE approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

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possibly because of sensitivity or mass range issues related to singly charged ions.17 Significant efforts have been made toward the development of ionization methods operating at atmospheric pressure utilizing ESI, MALDI, and atmospheric pressure chemical ionization (APCI) to increase analytical utility and throughput.16−20 Previous atmospheric pressure studies using laserspray ionization inlet (LSII)21 and solvent-assisted ionization inlet (SAII)22 for high-throughput analysis relied on the use of significant heat applied to the inlet tube (typically 325 °C) to create analyte ions with ESI-like charge states.23 While these methods are analytically useful, their applicability is currently limited to instruments equipped with a heated transfer capillary (e.g., a Thermo Ion Max source) or instruments that have been retrofitted with a heated inlet tube24,25 or obstruction26,27 to generate analyte ions. The discovery of matrix assisted ionization vacuum (MAIV)28 however, has provided an ionization method that produces ions in charge state and abundance similar to ESI but directly from the solid state, requiring only a MAIV matrix compound, such as 3-nitrobenzonitrile (3-NBN) or coumarin,29 and the vacuum of a mass spectrometer. The vacuum at the inlet aperture to an atmospheric pressure mass spectrometer is sufficient to initiate ionization with little or no heat applied to the inlet, extending applicability to atmospheric pressure mass spectrometers without heated inlet tubes.28,30 Flexibility in instrumentation,

onversion of large nonvolatile molecules such as proteins into gas-phase ions is of immense fundamental and practical importance in the field of mass spectrometry (MS). The 2002 Nobel Prize in Chemistry was awarded for this accomplishment using electrospray ionization (ESI)1 and matrix-assisted laser desorption/ionization (MALDI)2,3 to directly obtain the molecular weights of proteins with high accuracy. MS has been established as a versatile analytical method for use in many fields, such as proteomics,4 metabolomics,5 and environmental6 and food safety.7 As the list of applications continues to grow, so too does the demand for fast, simple, and low-cost analytical methods. Chip-based direct infusion ESI methods allow for automatable analysis,8−10 achieving high sample throughput, on the order of 1 sample per minute, but can require costly additional equipment and consumables. Vacuum MALDI is often the analytical method used for high-throughput analyses because sample preparation and analysis can easily be automated.11,12 However, its use is somewhat limited for the analysis of small molecules, such as drugs and metabolites because of the presence of significant chemical background noise13 in the low mass range that can make data interpretation difficult. MALDI, typically operated on high vacuum time-of-flight mass spectrometers,14,15 also requires the sample to be physically transferred from ambient pressure to vacuum conditions, reducing analytical throughput.16 In addition, the ions generated are primarily singly charged and their utility is limited for MS/MS characterization. Atmospheric pressure MALDI can eliminate some of these issues, but has not yet been widely applied to high throughput, © 2015 American Chemical Society

Received: November 11, 2014 Accepted: March 10, 2015 Published: March 10, 2015 4667

DOI: 10.1021/ac504475x Anal. Chem. 2015, 87, 4667−4674

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Analytical Chemistry

sample to be drawn into the vacuum of the mass spectrometer. Parameters for MSE experiments45,46 were adjusted using the MassLynx 4.1 software on the SYNAPT G2 without performing liquid chromatography (LC) separation prior to analysis.

operating conditions, and sample introduction methods allow MAIV to be compatible with a variety of commercial mass spectrometers with minimal or no source modification as has been shown using intermediate pressure MALDI,28 ESI,29,30 atmospheric solids analysis probe (ASAP),31 and direct analysis in real time (DART)32 sources. MAIV is hypothesized to generate ions by a sublimation driven triboluminescence process,28,29 which may explain earlier studies in which positively charged ice particles were photographed leaving the surface of freezing water droplets.33 Freezing water or methanol solutions have been shown to produce ions from a variety of compounds for analysis by MS.34 The ability of a volatile matrix compound to spontaneously lift nonvolatile molecules into the gas phase as multiply charged ions as the matrix sublimes, without applying voltages, laser power, or significant heat is not easily explained using the current paradigms commonly used to describe ion formation in MS.26,35−42 The observation of positive and negatively charged analyte ions28−30,43 is consistent with the hypothesis that charge separation is a result of the involvement of triboluminescence. Here MAIV is demonstrated as a simple and cost-effective means for rapid analysis and characterization of a variety of compounds ranging from small molecule drugs to proteins. The rate of ion formation is increased by slightly elevating the source temperature (60−80 °C), allowing for shorter acquisition times, higher sample throughput, and automation.

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RESULTS AND DISCUSSION The vacuum conditions appear to be important in the MAIV ionization process,28,29 by facilitating sublimation of the matrix:analyte crystals initially from the sample surface holder and subsequently in the gas phase to uncover the bare analyte ions. 47 The temperature of the vacuum inlet where matrix:analyte crystals are introduced has also been shown to influence the intensity of the ionization event.48 Continuing efforts are being made to better understand this process, searching for a relationship between the chemical and physical properties of the matrices, and their ability to impart charge on other molecules as they evaporate36 or sublime.48 The time required for the matrix:analyte crystals to completely sublime, and thus the analysis time of each sample, is directly related to the source temperature (Figure 1) where the matrix:analyte

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EXPERIMENTAL SECTION A brief description of materials used and experimental procedures is provided here. More detailed information regarding materials used, sample preparation and introduction, skimmer cone dimensions, as well as experimental parameters can be found in the Supporting Information. Analyses were performed on a linear ion trap LTQ Velos (Thermo Fisher, San Jose, CA) and a quadrupole ion mobility time-of-flight SYNAPT G2 (Waters Corp., Manchester, U.K.) mass spectrometer, both equipped with ESI sources that were removed and interlocks overridden to grant direct access to the vacuum orifice for sample introduction as previously described.30,44 A commercial ESI skimmer cone, and a modified skimmer cone with a wider opening to the vacuum orifice were used on the SYNAPT G2 as previously described.29 All analytes were prepared in 50% aqueous methanol and diluted to concentrations ranging from 1 to 5 pmol·μL−1 unless otherwise specified. 3-NBN matrix was dissolved in acetonitrile to a concentration of 100 mg·mL−1 (∼650 mM) and mixed with an equal volume of analyte solution to yield final analyte concentrations of 500 fmol·μL−1 to 5 pmol·μL−1 (∼500 000:1 molar ratio of matrix:analyte). Using a multichannel pipet (8 or 64 tip), 1 μL of the matrix:analyte mixture is drawn into each of the pipet tips and allowed to dry on the end as a crystalline droplet or spotted onto the surface of the respective sample holder. After the matrix:analyte samples had crystallized, each respective sample holder was aligned successively to make contact with the vacuum orifice for 1−5 s per sample to allow the matrix:analyte crystals to be drawn into the inlet of the mass spectrometer. This is accomplished by aligning the matrix:analyte samples to the vacuum orifice by hand, or with an automated stage. Automation of the sample introduction process was accomplished by mounting the sample holder to a programmable XYZ stage. The stage was programmed to briefly align each sample with the vacuum orifice in order to allow the

Figure 1. Effect of the source block temperature on sublimation time, and ion abundance for the [M + 2H]2+ charge state of the peptide bradykinin with the matrix 3-NBN. Mean values of triplicate measurements of sublimation time and ion abundance are plotted over the range of standard operating temperatures of the Z-Spray source of the Waters SYNAPT G2 (30−150 °C). The melting point (mp) of the matrix (117 °C)49 is denoted above the x-axis. All samples were introduced manually using the pipet tip method.

crystals are introduced. Using the pipet tip introduction method (Scheme 1.I.A) at the lowest temperature setting of 30 °C on the Z-Spray source block of the SYNAPT G2, the sublimation time of 3-NBN is ∼1 min and decreases rapidly with increasing temperature down to ∼4.0 s at the maximum temperature setting of 150 °C. As the source temperature is increased from 30−150 °C, 3-NBN shows a trend of decreased ion abundance proportional to the decrease in sublimation time shown in Figure 1. This trend is apparent until the source block temperature reaches or exceeds the melting point of 3-NBN (mp 117 °C),49 where the performance of the matrix begins to drop notably from ∼100 000 ion abundance at 100 °C to 5000 at 130 °C. While lower source block temperatures on the SYNAPT G2 favor higher ion abundances which are useful for structural characterization by tandem MS (MS/MS), higher temperature offers high-throughput capabilities by decreasing the sublimation time. Thus, the temperature dependence of sublimation represents a fundamental aspect of MAIV, in addition to vacuum.48 4668

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allowing only 4 s between samples. Increasing the temperature to 80 °C proved effective for eliminating sample carry-over by decreasing the sublimation time. As a result, the total ion current (TIC) signal from the previous sample rapidly decays back to the baseline before the next sample is introduced. Previous studies toward high-throughput analysis using heated inlet tube ionization methods required higher inlet temperatures (up to 450 °C)21 and the use of a cleaning solvent between subsequent sample introductions to avoid cross contamination.22 Another advantage in using MAIV matrices is that they readily sublime at ambient temperature in vacuum so that they will be pumped from the instrument. Several methods for manual and automated introduction of matrix:analyte samples to the mass spectrometer were studied (Scheme 1) to determine maximum throughput and the most adaptable methods to fit experimental needs and differences in instrumentation such as physical dimensions and instrument capabilities. Disposable pipet tips (Scheme 1.I.A) and filter paper (Supporting Information Scheme I.A) were determined to be the most effective and reliable methods for both manual (Scheme 1.I) and automated (Scheme 1.II) sample introductions. Various other surfaces were tested including nylon meshes (Supporting Information Scheme I.B), plastic, or wooden toothpicks (Supporting Information Figure S-2). Aluminum foil, glass microscope slides, and metal wires and pins were less suitable for high-throughput. One significant limitation is undesired chemical background and metal cation adductions. Porous materials that allow too much air to flow through (e.g., wide pore meshes and fast filtration paper) did not make good sample holders as they may cause the mass spectrometer to vent if operated improperly. The commercial ESI skimmer cone assembly of the SYNAPT G2 (Supporting Information Scheme II.A) provides adequate airflow for all sample introduction methods; however, the filter paper and nylon mesh introduction methods perform best with larger inlet apertures (Supporting Information Scheme II.B−C). Manual Sample Introduction. Pipet Tips. Disposable pipet tips serve as an effective and inexpensive sample introduction method suitable for high-throughput analyses, while also streamlining the workflow by allowing multiplexed sample preparation and sample introduction. This is advantageous in that it does not require the sample to be deposited on any kind of surface. A 64-channel pipet was used to mix matrix and analyte solutions in a microtiter plate and then draw 1 μL of the matrix:analyte solution into each of the disposable pipet tips. After being allowed to crystallize on the tips briefly, each of the tips were manually aligned with the vacuum orifice for analysis (Scheme 1.I.A). Analysis of 64 samples consisting of eight repeated cycles of eight analytes (clozapine, leucine enkephalin, angiotensin I, [Glu1]-fibrinopeptide B, galanin, bovine insulin, ubiquitin, and lysozyme) was performed in less than 6 min (Figure 2) with the source block temperature of the Waters Z-Spray source set to 70 °C. One representative cycle of the eight analytes is shown in the inset of Figure 2.I. Mass spectra for a single cycle of analytes consisting of clozapine (MW 326 Da) (Figure 2.II.A), leucine enkephalin (MW 555 Da) (Figure 2.II.B), angiotensin I (MW 1296 Da) (Figure 2.II.C), [Glu1]-fibrinopeptide B (MW 1570 Da) (Figure 2.II.D), galanin (MW 3157 Da) (Figure 2.II.E), insulin (MW 5.7 kDa) (Figure 2.II.F), lysozyme (MW 14.3 kDa) (Figure 2.II.G), and ubiquitin (MW 8.5 kDa) (Figure 2.II.H) were obtained by summing the indicated region of the TIC inset. The detected ions are ESI-like in charge states and ion

Scheme 1. Photographs of MAIV-MS Sample Introduction Options to Bring the Matrix:Analyte Sample in Close Proximity Briefly with the Vacuum Orifice to Allow the Crystals to Be Drawn into the Respective Mass Spectrometer Orificea

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(I) Manual sample introduction using (A) a 64-channel multi-pipet with 1 μL of matrix:analyte mixture crystallized on the individual pipet tips. (II) Automated sample introduction using a programmable XYZ stage to align and move samples to the vacuum orifice from (A) an 8channel multi-pipet with 1 μL of matrix:analyte mixture crystallized on the individual pipet tips and (B) a strip of filter paper spotted with 6 matrix:analyte samples.

A source block temperature of 80 °C was determined to be a good compromise for high-throughput analyses, requiring only ∼4.0 s per analysis, and yet sensitive enough to detect the drug clozapine (molecular weight [MW] 326) at a concentration of 5 fmol·μL−1, and the peptide hormone bradykinin (MW 1060 Da) at 15 fmol·μL−1(Supporting Information Figure S-1) using a matrix:analyte sample size of 1 μL. When the source temperature was set to 70 °C, there were some instances where carry-over was observed from the previous sample when 4669

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Figure 2. Analysis of 64 samples in 6.04 min using a 64-channel pipet for sample introduction to the Z-Spray source on a Waters SYNAPT G2 with source block temperature set to 70 °C. (I) TIC of the eight analytes analyzed in eight cycles, with an inset showing a single cycle of eight samples. Mass spectra extracted from the fifth cycle of (A) clozapine, (B) leucine enkephalin, (C) angiotensin I, (D) [Glu1]-fibrinopeptide, (E) galanin, (F) insulin, (G) lysozyme, and (H) ubiquitin. Fragment ion is denoted with a blue asterisk in panel A and assigned to the singly protonated [M − C3H8N + H]+ fragment of clozapine.50 Ion abundance is displayed in the top right corner of each spectra in red.

abundances, as reported previously.28 It is interesting to note the presence of only a limited amount chemical background which is typically associated with the use of a matrix in MALDI.13 The amount of chemical background observed may vary depending on instrumentation, as has been observed with the inlet tube design of the LTQ Velos compared to the skimmer cone design of the SYNAPT G2. When preparing and analyzing samples manually, reproducibility presents an issue. Five measurements taken from the same sample of insulin using manual pipet tip introduction within 1 min at a temperature of 80 °C (Supporting Information Figure S-3) resulted in a standard deviation in relative ion abundance of 29%. It is expected that reproducibility will be improved by method development and automation as reported with MALDI.12,51,52 Fragment ions are sometimes observed when analyzing small molecules on both mass spectrometers. In-source fragmentation for small molecules has been studied by Fenner and McEwen using ESI, SAII, and MAIV.53 Porous Materials. Eleven analytes were analyzed from a single piece of filter paper (Supporting Information Scheme I.A) without carry-over in less than 1 min with the inlet capillary of the LTQ Velos set to 70 °C (Supporting Information Figure S-4). Mass spectra for each analyte are extracted from the TIC by summing the region indicated for each matrix:analyte introduction (Supporting Information Figure S-4.I.). Two representative mass spectra are shown of a small molecule drug clozapine (Supporting Information Figure S-4.II.A) and a larger protein, myoglobin (MW 16.9 kDa) (Supporting Information Figure S-4.II.B). Mass spectral data for the remaining sample introductions consisting of angiotensin I, myelin basic protein fragment peptide, insulin, and ubiquitin, are displayed in Supporting Information Figure S-5. Nine samples of the three nitrogen containing pesticides,

atrazine (MW 215 Da), imazamox (MW 305 Da), and tralkoxidym (MW 329 Da), were also analyzed in this manner in 1.80 min using a Waters SYNAPT G2 mass spectrometer with a modified wide-bore skimmer cone (Supporting Information Figure S-6). The matrix 3-NBN ionizes molecules with basic moieties especially well.48 Nylon mesh with matrix:analyte solution spotted onto it was also used to introduce samples to the vacuum of the mass spectrometer (Supporting Information Scheme I.B). The mesh was pulled taught across two glass rods, and each spot is sequentially brought in contact with the vacuum opening of a modified medium-bore skimmer cone (1.0 mm) on a Waters SYNAPT G2. Using this method, 6 alternating samples of the drug clozapine and the peptide angiotensin I were analyzed without carryover in 0.60 min (Supporting Information Figure S-7). Automated Sample Introduction. To increase total analytical throughput and utility, a simple automation method was developed for both the pipet tip and filter paper sample introduction methods. A programmable XYZ stage and motion controller were used to automate first the sample introduction using pipet tips by mounting an 8-channel pipet to the stage (Scheme 1.II.A) for introduction to the vacuum inlet of a Thermo LTQ Velos. Each tip was loaded with 1 μL of matrix:analyte mixtures in a microtiter plate. Each well contained a different analyte (clozapine, codeine-6-β-Dglucuronide (MW 475 Da), bradykinin, angiotensin I, insulin, ubiquitin, lysozyme, and α-chymotrypsinogen A (MW 25.6 kDa). The stage was programmed to move the row of pipet tips horizontally at a rate of 0.50 cm s−1 and aligned so that each tip was brought within close proximity (