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Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging Troy Comi, Seung Woo Ryu, and Richard H. Perry Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03010 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 17, 2015
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Analytical Chemistry
Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging Troy J. Comi, Seung Woo Ryu, and Richard H. Perry* Department of Chemistry, University of Illinois, Urbana, IL 61801 ABSTRACT: Synchronizing the microdroplet spray of a desorption electrospray ionization source (sDESI) with the ion accumulation period (IT) of low duty cycle mass spectrometers has been shown to increase sensitivity and reduce the amount of sample depleted per scan compared with conventional DESI. The observed improvements are thought to result from reduction of deposited solvent volume, which should also minimize analyte spreading and redistribution at the spray impact zone (the “washing effect”). We hypothesize sDESI applied to mass spectrometry imaging (MSI) will demonstrate improved spatial resolution for weakly physisorbed analytes on smooth surfaces, especially for small pixel widths and low duty cycle mass analyzers. Generally, sDESI-MSI coupled to an Orbitrap mass analyzer showed improved sensitivity, decreased sample consumption, and image resolution by factors as high as 4-6 for various analytes and surfaces. The magnitude of improvements depends on spray solvent composition, analyte, and surface type. In a forensics application, low-abundance endogenous fatty acids in latent fingermarks on glass were analyzed by MSI. While DESI failed to generate images with pixel widths of 25 µm, sDESI-MSI produced high resolution chemical maps with a spray solvent composition of 9:1 methanol:water. Simulations of DESI and sDESI-MSI acquisition suggest that improvements in sensitivity and observed image resolution with synchronization is the result of lower desorption rate and surface washing, respectively.
Fingerprint evidence is universally recognized as a reliable method for biometric identification of criminal suspects.1 Latent fingermarks, impressions of fingerprint ridge patterns on surfaces, consist of endo-, semi-exo-, and exogenous compounds related to suspect physiology, diet, and fingertip contact with external chemical compounds including illicit drugs and explosives.2,3 Typically, fingermark visualization at crime scenes is achieved by imaging photoluminescent agents, including ninhydrin or nanoparticles that selectively target endogenous chemical compounds such as glycerides and amino acids.1,2,4,5 Mass spectrometry imaging (MSI) techniques such as secondary ion MS (SIMS),6-9 matrix-assisted laser desorption ionization (MALDI),10-17 and desorption electrospray ionization (DESI),9,18-21 provide higher specificity for mapping the spatial distribution of compounds found within fingermarks. The capabilities of MSI improve the accuracy of suspect identification and provide evidence of recent activities. The main advantage of DESI is that it enables direct fingermark imaging at atmospheric pressure without the need for pretreatment,22-31 thereby preserving evidence integrity. It is often beneficial to perform MSI experiments at high spatial resolutions (< 10 µm) on mass analyzers capable of high m/zresolution (> 60,000) and accurate mass measurements (< 2 ppm), such as the Orbitrap, for high-resolution MS (HRMS).3234 When DESI-MSI is performed on Orbitrap mass spectrometers, the percentage of the desorbed species effectively analyzed per HRMS scan is the ratio of the ion accumulation period (IT) to total HRMS scan time (t = IT + transient acquisition time). For a typical IT = 500 ms and a resolution setting of 100,000 at m/z 400 (1.8 s acquisition), only approximately 22% of sample is utilized.35,36 Thus, ~78% of desorbed mate-
rial is discarded for each MS scan. In addition, the redistribution and spreading of analytes on the surface, known as the “washing effect”,37 negatively affects spatial resolution during the entire scan. These problems are exacerbated for analytes on smooth surfaces such as fingermarks at crime scenes. Cooks and coworkers demonstrated that synchronization of the voltage, nebulizing gas, and IT of rectilinear ion trap miniature mass spectrometers (sDESI-MS) increased sensitivity during static profiling.38,39 In this report, we characterize sDESI-MS as an imaging source for HRMS experiments. The sDESI-MSI source (Figure 1) utilizes two solenoid valves. One valve synchronizes the nebulizing gas flow for desorption/ionization to the IT of a LTQ-Orbitrap XL HRMS. During transient acquisition, the DESI nebulizing gas is turned off and the second valve delivers a perpendicular stream of nitrogen gas (N2) to prevent solvent accumulation at the emitter tip. Thus, solvent is deposited only during IT, minimizing analyte redistribution and the “washing effect”. Our results show that synchronization improves sensitivity, decreases the amount of sample desorbed per HRMS scan, and increases spatial resolution by a factors of ~4-6 for analytes deposited on smooth surfaces. In addition, synchronization in DESI was essential for imaging endogenous fatty acids (FA) in fingermarks on glass with high spatial and m/z resolution simultaneously. Simulations modeling the “washing effect” and surfaceanalyte binding strength in sDESI-MSI were developed and further support these observations. By improving sample utilization and decreasing analyte redistribution, synchronization of desorption/ionization and IT in HRMS extends the range of analytes and surfaces amenable to DESI analysis.
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Figure 1. (a) Three-dimensional representation of the sDESIMSI source. The timing diagram shows synchronization of the nebulizing gas ON and OFF states with ion accumulation and transient acquisition, respectively. (b) Valve positions of sDESI-MSI during ion accumulation. (c) Valve positions of sDESI-MSI during transient acquisition.
EXPERIMENTAL SECTION Materials. ACS grade chloroform (CHCl3), HPLCgrade water (H2O; Macron Fine Chemicals, Center Valley, PA), HPLC-grade methanol (CH3OH; Fisher Scientific, Pittsburgh, PA), Rhodamine 6G, (R6G; Sigma-Aldrich, St. Louis, MO), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC; Avanti Polar Lipids, Alabaster, AL, USA), 1,2-dipalmitoyl-snglycero-3-phospho-(1'-rac-glycerol) (DPPG; Avanti Polar Lipids), bovine brain total lipid extract (BBE; Avanti Polar Lipids), Ampicillin (AMP; Sigma-Aldrich), Bradykinin (BK; Sigma-Aldrich), and ultra-high purity nitrogen (N2; S.J. Smith Co., Decatur, IL) were used as received. Synchronized Desorption Electrospray Ionization Mass Spectrometry Imaging Source. The sDESI-MSI source
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(Figure 1) involves modifying a commercial Prosolia 2D Omnispray DESI-MSI source (Indianapolis, IN, USA) to include two three-way valves (ASCO, Florham Park, NJ, USA). One of the valves modulates the nebulizing gas (180 psi) and the second regulates a perpendicular N2 gas stream (shutter gas; 20 psi). When the nebulizing gas is off the shutter gas removes solvent delivered to the emitter tip in a direction that is parallel to the sample surface. The three-way valve that regulates the nebulizing gas (V1) is normally closed, while the shutter gas is normally open (V2) (Figure 1c). Gas is emitted from the threeway valve common ports so that N2 backpressure rapidly vents to atmosphere when switching between ion source ON (Figure 1b) and OFF states (Figure 1c). Valve modulation and timing is controlled with a home-built electronic circuit (Figure S1). Direct measurement of gas flow impinging on a microphone shows a valve latency of 16.6 ± 2.9 ms and a 10.5 ± 2.7 ms for switching the shutter gas to the OFF and ON states, respectively, and 494.2 ± 4.6 ms desorption for 500 ms IT (Figure S2). In a typical sDESI-MSI experiment, valve latency is ~3% of IT. Analyte Depletion Rate and Sensitivity. DESI-MS and sDESI-MS extracted ion chromatograms (XIC) were used to estimate the rates of analyte depletion for R6G (100 pg), DPPC (10 ng), DPPG (20 ng), AMP (20 ng), BK (200 µg) or BBE (200 µg) deposited on polytetrafluoroethylene (PTFE) Omni Slides (Prosolia, Inc.). Five XICs were acquired per analyte when the sDESI-MS source was stationary (static mode) and then averaged to generate decay curves. The exponential regression constant reflects the rate of sample depletion from the surface (Figures 3 and S4). Sensitivity was characterized by rastering the ionization source across deposited spots of various analytes in seven evenly distributed rows. The source was continuously moved to investigate sensitivity independent of desorption rates at the spray impact site, maintain good reproducibility without internal standards,40 and simulate imaging conditions. A custom MATLAB (Mathworks, Natick, Massachusetts, USA) script was used to integrate analyte signal intensity across each spot. Differences in sensitivity (i.e. slope of calibration curves) were tested for significance using analysis of covariance (ANCOVA; R software environment). Latent Fingermark Imaging. Sebum-enhanced fingermark impressions on glass microscope slides were produced from a male donor using previously reported methods41 and analyzed immediately (optical images of fingerprints were manually distorted to protect donor privacy). Briefly, after thorough hand-washing, the finger was rubbed against the side of the nose and then pressed against a glass slide. Selected regions of the fingermarks were imaged using either CHCl3:CH3OH (1:1; 2 µL/min) or CH3OH:H2O (9:1; 1 µL/min) with pixel widths of 150 µm, 75 µm, or 25 µm (emitter voltage = -5 kV; capillary temperature = 275 oC; IT = 500 ms; resolution setting = 100,000; m/z range = 100 – 400). At
the smallest pixel width of 25 µm, a 1 mm2 area is analyzed in approximately one hour; sDESI does not affect image acquisition time. Optical images of fingerprints on regular office paper were acquired using a flatbed scanner (Epson Perfection 2400 Photo). The fingerprints were deposited on paper using an inkless fingerprint pad (Lee Products Company, Bloomington, MN).
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We designed a software package (C# programming language) to process and visualize MSI data. Thermo Fisher Scientific RAW data files are converted to the mzXML file format using ProteoWizard.42 The software loads the mzXML files into memory without data reduction or binning of m/z values, and then generates chemical images using false colors to represent signal intensities (Figure S3). The software package also provides the capability to average spectra within userdrawn regions of interest (ROI), subtract spectra between ROIs, export spectra as comma-separated value text, and perform hyperspectral visualization43 with m/z binning as described by Xiong et. al.44 Spatial Resolution. The spatial resolution of DESI-MSI and sDESI-MSI were compared using patterns of R6G dots on photographic paper (Epson, Long Beach, CA). The patterns were generated from a red Sharpie marker (Sanford Corp., Oak Brook, IL) and an unpolished stainless steel mesh template (Small Parts Inc., Miramar, FL). The R6G patterned surfaces were fabricated by pressing the SS mesh on top of the photographic paper immediately after it was drawn on with Sharpie, which produces an array of R6G dots (~100 µm diameter) spaced by ~500 µm. Then, a second array was superimposed on the first, generating a R6G dots with variable spacing. Optical images of the R6G dots were acquired using an EVOS fl Inverted Fluorescence Microscope (Advanced Microscopy Group, Life Technologies, Thermo Fisher Scientific). Pattern dimensions were estimated from the fluorescence images using ImageJ (http://imagej.nih.gov/ij/). DESI MS Simulation. Simulations estimating the relative impact of the desorption/ionization and washing (W) efficiencies on sensitivity, decay rate, and spatial resolution, were performed using the finite difference method45 (implemented in MATLAB). For simplicity, the model estimates a circular spray profile at the surface (Figure 2), instantaneous analyte dissolution and immediate thin film formation upon spray impact. The rate of desorption is modeled as a twodimensional Gaussian distribution that accounts for concentric regions of high (H; 100 µm diameter) and low (L; 500 µm diameter) ionization efficiency within the spray profile at the surface.46 The model for desorption is combined with a cosine-distributed “washing effect” parameter to describe analyte movement on the surface relative to the center of the spray profile (; Figure 2). The magnitudes of the washing and desorption/ionization efficiencies were modified by adjusting their corresponding rate constants (washing: Rw; desorption/ionization: RL + RH). At each time step Δ (10 ms), the amount of analyte () at a given input pixel () changes due to low efficiency desorption from the outer spray plume, high efficiency desorption/ionization from the inner spray plume, washing from to neighboring pixels (Wout), and washing from neighboring pixels to (Win). I for a given pixel is updated for the next time step by the following relationship: + Δ = − + + Δ + where Wout, is distributed to neighboring pixels at position i ) based on the magnitude of their projections on the radial ( vector = − . The fraction of analyte redistributed to pixel i is:
= =
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Figure 2. Scheme showing the variables used in simulating analyte removal and redistribution during DESI-MSI. 〉 denotes an inner product. After each ∆t, the where, 〈, center of the spray profile at the surface was moved a distance equal to spray profile velocity × ∆t = 1.3 µm. At the end of each row of the output image, the spray profile is moved to the beginning of the next row, simulating the fly back motion of a xy-translation stage used for DESI-MSI. Simulations of sDESI-MSI were performed identically but the input analyte distribution was only updated during IT.
RESULTS AND DISCUSSION The impact of synchronization on sensitivity was evaluated by measuring R6G deposited onto Omni Slides with emitter velocities of 100 µm/s, 50 µm/s, 25 µm/s, and 0 µm/s, to simulate a typical imaging experiment. Sensitivity was measured as the slope of a calibration curve acquired at each scan rate and compared between DESI and sDESI by ANCOVA. The sensitivity with sDESI improved by factors of 1.77 ± 0.13, 2.02 ± 0.15, 2.96 ± 0.43, and 3.51 ± 0.55, respectively. Further investigations demonstrated that the magnitude of sensitivity improvements depended on the composition of the microdroplet spray, the sample surface, and the nature of the analyte (Figure S4 and Table S1), which are in agreement with previous reports.38,39 The sensitivity improvement with slower raster speeds is particularly important for MSI as it suggests that synchronization will largely benefit images acquired at high spatial resolution. The depletion rate was next estimated by recording the intensity of specific lipid signals from BBE samples as function of time and then calculating the decay constant from exponential regression curves (Figure 3). With synchronization, signals for [36:1 PS – H]- (m/z 788.536), [40:6 PS – H]- (m/z 834.520), [38:4 PI – H]- (m/z 885.541), and [42:2 sulfatide (ST) – H]- (m/z 888.614) decayed slower by an average factor of 5.90 ± 0.71 (Figure 3b – 3e), which is in agreement with a desorption period of ~0.2t for sDESI. The decay curves also show that the DESI and sDESI variances are relatively similar (Figure S4), indicating that synchronization does not significantly degrade reproducibility between technical replicates. When performing MSI of R6G spots at 40 µm pixel widths, optical images following MSI show a higher amount of residual R6G after sDESI-MSI (Figures 4a and 4b), as ex-
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Relative Abundance
pected since desorption only occurs during the IT and in agreement with the improved decay rates. MS images and XICs of R6G (a) 788.536 100 888.614 DESI 885.541 sDESI
is less than 10%, these results demonstrate that synchronization improves spatial resolution by a factor of ~4. The dimensions of the DESI and sDESI microdroplet spray profiles at the (a)
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Figure 3. (a) Mass spectra of BBE for DESI (blue) and sDESI (red). (b) Signal decay of species at m/z 788.537 (b), m/z 834.520 (c), m/z 885.541 (d), and m/z 888.614 (e) for static profiling. Shaded areas represent ±1 S.D. of five measurements and dotted lines show the time at which signal decreases to 30% of the highest intensity. The time point at which signal decayed to 30% for [38:4 PI –H]- was determined directly from the graph, owing to a poor exponential fit.
spots on photographic paper show baseline-resolution for sDESI (Figure 4a), while the DESI XICs contain valleys between spots with intensities as high as ~20-25% (Figure 4b). By considering two spots as resolved when the valley intensity
Figure 4. (a) sDESI-MS and (b) DESI-MS analysis of R6G dots deposited on photographic paper. Images of the R6G dots were acquired using fluorescence microscopy, MSI (1:1 CHCl3:CH3OH spray solvent and 40 µm pixel width), and a flatbed scanner in the listed order. XICs for one row (red arrows) are also shown for each MS image. Note that while the R6G dots are centered on the XIC for 4b, several dots are partially sampled in 4a leading to varied relative intensity. (c) Microdroplet spray profiles at the surface of water-sensitive paper with emitter velocities of 100 µm/s, 50 µm/s, 25 µm/s,
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which the rates of washing (Rw) and desorption (RH, RL) impact MSI performance (Figure 6). Generally, the desorption parameter is proportional to the sensitivity while washing is inversely proportional. The ratio sDESI/DESI intensity is always greater than unity, reflecting the more efficient sample
and 10 µm/s. The paper changes color from yellow to blue upon interaction with water in the solvent. surface of water-sensitive paper are relatively similar (Figure 4c). However, the sDESI source deposits less liquid on the [24:0 FA – H]m/z 367.390
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Figure 5. Optical scan of a fingerprint and MS images showing the ridge patterns of a latent fingermark on glass analyzed with CHCl3:CH3OH (1:1) (a) and CH3OH:H2O (9:1) spray solvent (b). MS images were generated by plotting the spatial distribution of the fatty acids (FA) lignoceric acid (24:0 FA) and cerotic acid (26:0 FA). MS images grouped by braces have the same intensity scale. surface, which reduces the redistribution and spreading of analytes during desorption. For example, when the emitter travels at 10 µm/s, the spray impact region was mostly blue with DESI, indicating solvent pooling at the surface. Weakly bound analytes, such as the R6G spots, rapidly dissolve and diffuse within the liquid pool, which degrades spatial resolution via the “washing effect”. One potential application of sDESI-MSI is the highresolution imaging of low abundance chemical compounds in latent fingermarks found on smooth surfaces such as glass. Using a sampling period of 150 µm and microdroplet spray composition of CHCl3:CH3OH (1:1), endogenous FAs with ion abundance less than 10% relative intensity, such as [24:0 FA – H]- (m/z 367.390) and [26:0 FA – H]- (m/z 395.424) produce distinct ridge patterns with and without synchronization (Figures 5 and S6; see Table S2 for the identification of other endogenous FAs). Chemical images of these FAs in fingermarks were observed at sampling periods of 75 µm (CHCl3:CH3OH (1:1)) and 25 µm (CH3OH:H2O (9:1)) with sDESI but not DESI (Figure 5). We hypothesize that the lower sensitivity in DESI under these conditions is due to a faster rate of the spreading of analytes along the surface compared to desorption/ionization. Synchronization should significantly reduce the “washing effect”, thereby expanding the scope of DESI-MSI. To test our hypothesis of sDESI improving resolution through reducing the “washing effect”, we simulated analyte movement along a surface during DESI MSI. Using one of the fluorescent images of the R6G spots as input for simulations, we obtained preliminary insights about the mechanism by
utilization of sDESI (Figure 6a). Simulating DESI-MS images at various washing and desorption parameters yielded extracted line scans (Figure 6b), which show broadening and distortion of peaks at higher magnitudes of Rw and RL. In contrast, the peak shapes are unaffected with synchronization. These observations are reflected in the lower spatial resolution of simulated DESI-MS images at high magnitude of Rw (Figure 6c). Interestingly, no DESI-MS signal is observed when the magnitudes of Rw and RH, RL are large, indicating that the analytes are washed away by the outer region of the spray plume before contacting the central region of highest ionization efficiency (Figure 2). Conversely, spatial resolution is similar for analytes strongly bound to the surface (i.e., the magnitude of Rw is low) such as lipids embedded in the extracellular matrix of rat brain tissue sections (Figure S8). Our phenomenological model of desorption/ionization and washing processes that occur during DESI and sDESI MSI replicates experimental MS images. These simulations show that the loss of sensitivity and spatial resolution is proportional to Rw, which is substantially alleviated by synchronizing desorption/ionization with IT. While momentum transfer and droplet dynamics have been previously modeled for the DESI spray impact, this represents the first report of phenomenological simulations of analyte removal and redistribution during DESI MSI. With a simple model, the critical aspects affecting DESI imaging performance are recapitulated successfully.
CONCLUSIONS
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Analytical Chemistry Synchronization of DESI with the ion accumulation period of an ion trap mass spectrometer (sDESI) improves sensitivity and reduces the “washing effect”. Herein we demonstrate that these benefits transfer to operation of sDESI in imaging mode by mapping the spatial distribution of weakly bound samples such as latent fingermarks on smooth surfaces. Our results
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ASSOCIATED CONTENT Supporting Information Figures S1 – S8 and Tables S1 – S2. This material is available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION Corresponding Author * E-mail:
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 400
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Notes The authors declare no competing financial interest
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Figure 6. Simulated DESI and sDESI analysis. (a) Comparison of sensitivity and decay rate for static profiling. (b) Line scans from an image corresponding to the XIC in Figure 4b. (c) Simulated images for DESI and sDESI. These values represent contributions from numerous physical properties and were chosen to coincide with experimental chemical images. show that sDESI-MSI achieves higher sensitivity, spatial resolution, and lower rates of sample consumption per MS scan compared to DESI-MSI (factors of up to ~4-6 times improvement). One of the most exciting observations is that sDESIMSI enables acquisition of images using small pixel widths (≤ 75 µm) at which analyte redistribution negates DESI-MSI.
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ACKNOWLEDGMENTS
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The Perry Research Laboratory acknowledges financial support from the University of Illinois at Urbana-Champaign (UIUC), NIH Training Program at Chemistry-Interface with Biology (NIH T32 GM070421), National Science Foundation Graduate Research Fellowship Program, and the UIUC Springborn Fellowship. We also thank Dr. Edward T. Chainani for help in designing initial circuitry, assisting with valve latency measurements, and many useful conversations.
REFERENCES
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(1) Green, F. M.; Salter, T. L.; Stokes, P.; Gilmore, I. S.; O'Connor, G. Surface and Interface Analysis 2010, 42, 347. (2) Francese, S.; Bradshaw, R.; Ferguson, L. S.; Wolstenholme, R.; Clench, M. R.; Bleay, S. Analyst 2013, 138, 4215. (3) Weyermann, C.; Roux, C.; Champod, C. Journal of Forensic Sciences 2011, 56, 102. (4) Jelly, R.; Patton, E. L. T.; Lennard, C.; Lewis, S. W.; Lim, K. F. Analytica Chimica Acta 2009, 652, 128. (5) Choi, M. J.; McDonagh, A. M.; Maynard, P.; Roux, C. Forensic Science International, 179, 87.
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Analytical Chemistry
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GRAPHICAL ABSTRACT N2 Shutter Gas
Ion Trap Closed Spray Off Gate Lens
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Transient Acquisition Time
ACS Paragon Plus Environment
DESI Emitter
MS Inlet
Ion Trap Open Spray On Ion Accumulation
