Spatial Metabolomics of the Human Kidney Using MALDI Trapped Ion Mobility Imaging Mass Spectrometry Elizabeth K

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Spatial Metabolomics of the Human Kidney using MALDI Trapped Ion Mobility Imaging Mass Spectrometry Elizabeth K. Neumann,1,2 Lukasz G. Migas,3 Jamie L. Allen,2 Richard M. Caprioli,1,2,4-6 Raf Van de Plas,1-3 and Jeffrey M. Spraggins1,2,6*

1Department of Biochemistry, Vanderbilt University, 607 Light Hall, Nashville, TN 37205, USA 2Mass Spectrometry Research Center, Vanderbilt University, 465 21st Ave S #9160, Nashville, TN 37235, USA 3Delft Center for Systems and Control, Delft University of Technology, Mekelweg 2 Building 34, 2628 CD Delft, The Neth- erlands 4Department of Pharmacology, Vanderbilt University, 2220 Pierce Avenue, Nashville, TN 37232, USA 5Department of Medicine, Vanderbilt University, 465 21st Ave S #9160, Nashville, TN 37235, USA 6Department of Chemistry, Vanderbilt University, 7330 Stevenson Center, Station B 351822, Nashville, TN 37235, USA

KEYWORDS. Metabolomics, Human Kidney, Imaging mass spectrometry, Matrix-assisted laser desorption/ionization, Trapped ion mobility spectrometry, Ion mobility mass spectrometry, High spatial resolution imaging

ABSTRACT: Small metabolites are essential for normal and diseased biological function but are difficult to study because of their inherent structural complexity. MALDI imaging mass spectrometry (IMS) of small metabolites is particularly challenging as MALDI matrix clusters are often isobaric with metabolite ions, requiring high resolving power instrumentation or derivatization to circumvent this issue. An alternative to this is to perform ion mobility separation before ion detection, enabling the visualization of metabolites without the interference of matrix ions. Here, we use MALDI timsTOF IMS to image small metabolites at high spatial resolution within the human kidney. Through this, we have found metabolites, such as arginic acid, acetylcarnitine, and choline that localize to the cortex, medulla, and renal pelvis, respectively. We have also demonstrated that trapped ion mobility spectrometry (TIMS) can resolve matrix peaks from metabolite signal and separate both isobaric and isomeric metabolites with different localizations within the kidney. The added ion mobility data dimension dramatically increased the peak capacity for molecular imaging experiments. Future work will involve further exploring the small metabolite profiles of human kidneys as a function of age, gender, and ethnicity.

Small metabolites are essential for normal biological function from small metabolites, lipids and peptides to glycans and pro- as well as in transition to a disease state. The localization of teins, often informing biological processes.22-26 In summary, these metabolites is important for cellular function and may per- frozen tissue is cryosectioned into micron thick sections, coated haps provide insight into the (dis)functional state of human or- with an ultraviolet (UV) light-absorbing chemical matrix, and gans.1 Despite their importance, small metabolites are particu- rastered under a UV laser.24,27 While exceptionally powerful for larly difficult to study within tissue matrices because of their small metabolite analysis,28-31 the application of the organic ma- structural complexity and abundance of isomers.2,3 Traditional trix complicates small molecule analysis by MALDI IMS.32 Pri- means of exploring small metabolites include capillary electro- marily, matrix ions are often isobaric with small metabolites33 phoresis,3-6 electrochemistry,7-9 spectroscopy,10-12 liquid chro- and, secondarily, application of the MALDI matrix can cause matography,13-15 and microscopy.16-18 The discrete metabolites delocalization of easily soluble small molecules, preventing ac- identified from each of these analyses have been integrated into curate visualization of each molecular species at cellular spatial databases, such as Metlin19 and Human Metabolome Data- resolutions. 20 base. Each of these technical approaches have provided im- Several approaches exist for enhancing the detection portant insights into biological functions; however, these tech- of small metabolites within a MALDI IMS experiment, gener- niques are often limited by throughput, specificity, sensitivity, ally by reducing matrix isobaric interferences. The most or cannot be performed in an imaging regime. straightforward is to use high resolving power instruments, such Imaging mass spectrometry (IMS) is a powerful tool as Fourier transform ion cyclotron resonance or Orbitrap mass that enables untargeted, spatial analysis of hundreds of chemi- spectrometers, to mass resolve the metabolites from matrix cals within a biological sample.21 Matrix-assisted laser desorp- ions.34-38 However, these systems cannot resolve isomeric com- tion/ionization (MALDI) IMS is a laser-based imaging system pounds and imaging times tend to be longer at sufficiently long used to study a variety of different chemical classes, ranging transient times. Alternatively, other labs have explored derivat-

field is progressively decreased, allowing the trapped ions to elute as a function of their differential mobility (K), which is dependent on the mass, charge, and size of the ion.52,53 TIMS is capable of resolving powers greater than 250, enabling separation of lipid isomeric and isobaric species.54 Other types of ion mobility spectrometry, such as traveling-wave55,56 and drift tube57, can discriminate and differentiate small molecule isomers, but has not been done in an imaging context. Here, we explore the metabolic composition of the human kidney with MALDI trapped ion mobility IMS. Ultimately, we have demonstrated that the use of TIMS enhances the discrimination and peak capacity of small molecule analysis within quadrupole-time of flight (qTOF) MS, enabling high spatial resolution IMS analysis of small molecules (m/z 50-650) without significant interference from MALDI matrix ions. Beyond improving discrimination of metabolites from matrix ions, we also demonstrate that additional metabolite isomers and isobars can be visualized with ion mobility separations compared to qTOF only mode. As such, this developed technology enables imaging-based metabolomics with enhanced sensitivity and specificity at high spatial resolution.

METHODS Materials:

α-Cyano-4-hydroxycinnamic acid (CHCA) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). HPLC- Figure 1: Average mass spectra of metabolites detected within grade acetonitrile, isopropanol, and methanol were purchased the human kidney in qTOF-only mode (A) and with TIMS en- from Fisher Scientific (Pittsburgh, PA, USA). abled (B). Hundreds of metabolites are detected in both modes, but TIMS increases the number of detectable ions with the ad- Sample Preparation: ditional ion mobility component as seen in the mass spectra (B) and gel view image (C). The addition of TIMS separation re- Human kidney tissue was surgically removed during a full ne- duces the detectability of ions below an m/z value of 150. phrectomy and remnant tissue was processed for research pur- poses by the Cooperative Human Tissue Network at Vanderbilt ization of the small metabolites themselves to enhance ioniza- University Medical Center. Remnant biospecimens were col- tion or increase their respective mass-to-charge ratios (m/z), re- lected in compliance with the Cooperative Human Tissue Net- 39-46 ducing isobaric interferences by matrix ions. Derivatization work standard protocols and National Cancer Institute’s Best methods are generally targeted in nature and limit the number Practices for the procurement of remnant surgical research ma- of different metabolite classes that can be simultaneously visu- terial. Participants were consented for remnant tissue collection alized. The derivatization step may also affect localization of in accordance to institutional IRB policies. The excised tissue the small metabolites within tissue, resulting in limited spatial resolutions. Further, others have synthesized matrices that have fewer isobaric interferences.33,47 These matrices either form clusters that have m/z values higher than metabolites of interest or do not effectively cluster, limiting the number of potential isobars. While effective, matrix choice affects the types of detectable molecules, so there is a benefit to developing methods compatible with a wide variety of matrices, particularly common matrices that have been extensively studied. By broaden- ing small metabolite analysis to include a variety of sampling approaches, we can expand and maximize the information obtained from a sample. Ion mobility spectrometry is a gas phase analytical approach that enables the separation of ions based on their appar- ent size, shape, and charge state.48-50 Specifically, trapped ion mobility spectrometry (TIMS) is a fast, high resolution ion mobility technique that we have recently shown is compatible with imaging experiments and time scales, unlike gas or liquid chromatography.51 In brief, ions are initially trapped against oncom- ing gas using an electric field. The magnitude of the electric 2

Figure 2: Selected ion images of small metabolites at 20 µm pixel size in comparison to autofluorescence (AF) microscopy (A), showing the spatial and size diversity of the detectable analytes (B-H). Each metabolite localizes to different regions within the kidney, such as the cortex, medulla, and renal pelvis. The selected ion images demonstrate our molecular coverage from smaller m/z values (A) to larger values (H) with many ions detected between those values (C-G). Scale bars are 1.5 mm. was flash frozen over an isopentane, dry ice slurry, embedded time was set to 200 ms. Instrumental parameter metadata is pro- in carboxymethylcellulose, and stored at -80 °C until use. Kid- vided in the supplementary materials and special tuning of the ney tissues were cryosectioned to a 10 µm thickness, thaw collision funnel RF values, TIMS ramp time, and TOF transfer mounted onto indium tin-oxide (ITO) coated glass slides (Delta time were required for metabolite imaging. All qTOF mode im- Technologies, Loveland, CO, USA), and returned to 20 °C aging data were visualized using SCiLS Lab Version 2019 within a vacuum desiccator. Autofluorescence microscopy im- (Bruker Daltonics, Bremen, Germany) and TIMS imaging data ages were acquired using EGFP, DAPI and DsRed filters on a were analyzed using custom in-house developed software. Me- Zeiss AxioScan Z1 slide scanner (Carl Zeiss Microscopy tabolites were identified using a combination of, high mass ac- GmbH, Oberkochen, Germany) prior to matrix application. curacy and METLIN /Human Metabolome Database searching. Samples were coated with a 5 mg/mL solution of All putatively identified metabolites had mass accuracies of ≤ 4 CHCA dissolved in a 70% methanol solution using an HTX TM ppm. Select ions were fragmented using tandem MS. Sprayer (HTX Technologies, LLC, Chapel Hill, NC, USA) yielding a 2.67 mg coating (1200 mm2, 0.12 mL/hr, 8 passes, 4 Data processing: sec drying time, 80 °C spray nozzle). Tissue samples were im- aged immediately after matrix deposition and stored within a The kidney data was exported into a custom binary file format vacuum desiccator for subsequent tandem MS analysis. optimized for storage and speed of analysis of ion mobility-IMS data. Each individual frame contains between 10,000-100,000 MALDI timsTOF IMS: centroid peaks that span the acquisition range of m/z 50-1500 -2 and 1/K0 of 0.6-0.95 Vscm with 400,577 and 589 bins in the MALDI TIMS IMS was performed on a prototype Bruker tim- MS and ion mobility-dimensions, respectively. The processing 51 sTOF fleX MS system (Bruker Daltonics, Bremen, Germany) pipeline requires common m/z and 1/K0 axes, hence individual in both qTOF and TIMS analysis modes. Unless otherwise centroid peaks were inserted at their correct bin position along specified, qTOF ion images were collected in positive ion mode the MS and IM-dimensions; missing values were set to zero. at 20 µm pixel size with the beam scan set to 18 µm2 using 600 Following the conversion process, a mean mass spectrum of the laser shots per pixel and 29% laser power (30% global attenua- entire dataset was retrieved and peak-picked. A total of ~500 tor and 99% local laser power) at 10 kHz. Data were collected most intense ions were then selected and examined to identify from m/z 50 – 1500 for small metabolite analysis. Imaging data multi-conformational species, and they were visualised to ex- collected with TIMS activated were acquired at 20 µm pixel amine conformation-specific ion localization in the spatial do- size with the beam scan set to 18 µm2, 600 laser shots per pixel main. and 29% laser power (30% global attenuator and 99% local laser power) at 10 kHz. The TIMS electric field gradient scan RESULTS AND DISCUSSION 3

Figure 3: Ion mobility analysis can separate MALDI matrix ions from metabolite signals, increasing specificity of metabolite imaging. Composite image (A inset) encompasses the distribution of an isobaric matrix and metabolite ion pair that can be separated in ion mobility space (A). Separate ion images of glycerophosphocholine (GPC) (m/z 258.1067, 0.677 ppm error, B) and the matrix (C) ions with localization outside and inside the kidney, respectively. Scale bars are 1.5 mm.

Small Metabolite Analysis using MALDI timsTOF MS enabled. The increase in the number of detectable ions by acti- vating TIMS likely results from the accumulation of ions within We have developed a method for visualizing small molecules the TIMS funnel prior to analysis. Moreover, the detected ions (m/z 86-650) with MALDI ion mobility MS. In general, the de- were further separated into >900 additional features after incor- tectability of small molecules using MALDI MS is mostly de- porating the ion mobility dimension (Figure 1). The improved pendent upon careful sample preparation to maintain metabolite number of detected molecules after ion mobility separation localization, maximize signal intensity, and instrument tuning demonstrates the effectivity and power of MALDI TIMS for to transmit smaller m/z values. Sample preservation was not ex- small molecule analysis and subsequent detection. In total, we tensively optimized within this manuscript as it has been stud- have detected different classes of metabolites, ranging from nu- ied elsewhere,58 but we preserved metabolites by minimizing trients and preservatives to drugs and lipid precursors, demon- the time the tissue was not stabilized and stored, since many of strating the wide range of detectable ion classes with MALDI these molecules are easily oxidized or are otherwise degraded ion mobility IMS. Interestingly, there is a shift in detectable in atmospheric conditions. For these experiments, kidney resec- ions when TIMS is enabled, decreasing the number of ions de- tions were embedded immediately after they were obtained, fro- tected below an m/z value of 150. Because the only instrumental zen at -80 °C until cryosectioning. The tissue was coated with parameters that were changed between qTOF mode and TIMS CHCA and analyzed within one to three days of sectioning to were those concerning ion mobility separation, this may indi- reduce degradation within the freezer. Moreover, several matri- cate that lighter ions are lost during TIMS separations. Despite ces and application parameters were examined to maximize an- tuning each optic separately, the sensitivity of ions below an m/z alyte extraction without causing significant analyte delocaliza- value of 150 could not be readily reestablished, with some no- tion (SI Table 1). As expected, matrix application with too high table exceptions such as choline. Choline and choline-related of an aqueous content results in efficient metabolite extraction ions are likely detected because of their naturally high abun- but causes significant delocalization of these molecules pre- dance within biological systems. venting high spatial resolution images (20 µm pixel size) from being obtained. High spatial resolution was achieved by apply- In both qTOF and TIMS TOF modalities, the detecta- ing matrix at a lower flow rate with additional passes from what ble features are difficult to identify due to their structural com- was recorded in literature59, providing a drier matrix applica- plexity and nondescript fragmentation profiles, which is a com- tion. Finally, instrumental parameters must be optimized to monly stated limitation of metabolite analysis. In these initial transmit m/z values between 50 and 650 in addition to achieving experiments, mass accuracy and a combination of Metlin and sufficient ion mobility resolving power without significantly re- Human Metabolome Databases were used to putatively identify ducing instrument sensitivity. While careful tuning of each op- 46 of the detected ions (Table 1). Future work will involve pur- tic improves ion transmission, we found that the accumulation chasing standards to confirm the detected features in the dataset funnel RF values, TIMS ramp time, and TOF transfer time af- and determining which isomers are present within the ion mo- fected transmission of m/z values between 50 and 650 the most bility dimension as this is beyond the scope of the current man- (SI Figure 1). In general, lowering the magnitude of the voltage uscript. The large number of detectable metabolites, particu- applied to each optic increases the transmittance of small mass larly compared to what can be putatively identified, demon- ions. strates the necessity for continually developing and improving untargeted metabolomic approaches, such as this one. Further, Using this approach, we have successfully detected TIMS can detect metabolites from a variety of classes showing >200 distinct species within the human kidney using qTOF- the power and utility of untargeted, imaging metabolomics da- only mode and >350 discrete m/z values with TIMS separation tasets. 4

Figure 4: Ion mobility separations enable visualization of metabolite isobars/isomers increasing the peak capacity of MALDI IMS. Composite image (A inset, m/z 267.956) of four components separated within the ion mobility dimension (A). Each component can be separately visualized with the first component localizing to the renal pelvis (B) and the other three detected through the entire kidney section (C-E). Scale bars are 1.5 mm.

High Spatial Resolution TIMS Imaging Beyond detection, one of the most difficult challenges facing small metabolite analysis is unequivocal identification of The kidney is composed of a variety of structures (e.g. glomer- each ion. As seen by the number of ion mobility-resolved peaks, uli and tubules) and each of these functional units exist within isobaric and isomeric compounds significantly complicate in- defined kidney regions. The cortex, medulla, and renal pelvis terpretation of fragmentation spectra in addition to database are functional regions of the human kidney and can be discrim- searching. These difficulties and challenges multiply when inated with autofluorescence microscopy (Figure 2A). For vis- identifying low intensity species or compounds closely resolved ualization of small metabolites within the different regions of in the ion mobility dimension. While not exhaustive, we have the kidney, IMS was performed at 20 µm pixel size without sig- performed fragmentation and subsequent identification of sev- nificant delocalization artifacts and high signal-to-noise ratios eral of the detectable small metabolites within the kidney (SI (S/N, Figure 2B-H). Many of the detectable metabolites are lo- figure 2). We obtained diagnostic fragments for different me- calized throughout the entire kidney, such as inosine (Figure 2 tabolites at high S/N by summing together spectra from the iso- E&G), although several show localizations within the different lated ion and different collision energies. Because spectra are segments of the kidney. For instance, choline and sapropterin obtained from a range of collision energies, each metabolite is both localize to the medulla and renal pelvis (Figure 2B&F), more thoroughly characterized than by just using a single colli- while acetylcarnitine is most abundant within the medulla (Fig- sion energy. Many fragments, however, are related to water loss ure 2D) and arginic acid is mostly detected within the cortex rather than diagnostic fragments. As such, absolute identifica- (Figure 2 C). Finally, heme shows a more sporadic localization tion of all detectable metabolites is beyond the scope of this ar- within the kidney tissue but is most abundant within blood ves- ticle, but the successful identification of a fraction of the small sels, likely due to the natural abundance of heme within blood metabolites demonstrates the power and utility of the MALDI (Figure 2 H). While only a subset of detected molecules is dis- TIMS MS for small ion fragmentation and, further, metabolom- played here, further examples of metabolites that localize to ics experiments. each portion of the kidney can be found within the dataset. The function of many of these metabolites is well understood, so High-Performance Ion Mobility Separations of Small Mole- their localization within different regions of the kidney lends cules potential functional insight, such as lipid metabolism (choline)60 and fatty acid transport (acetylcarnitine)61 or disordered The most cited limitation of MALDI analysis of small metabo- urea metabolism (arginic acid).62 Further, the spatial correlation lites in both a profiling and imaging context is the isobaric in- of small metabolites can be used to develop functional profiles terference of matrix ions with key biological features.63-66 While of cell types and organ functional units within different systems high resolving power instrumentation can resolve some of these with implications to cellular heterogeneity, efficiency, or dis- isobaric interferences, these experiments often require longer ease progression. transient scan times, drastically increasing the time an imaging experiment takes or limiting its spatial resolution. Moreover, 5

Measured Assignment Mass Error Measured Assignment Mass Error m/z Value (ppm) m/z Value (ppm) 86.097 Choline-H2O -1.131 229.152 N-Decanoylglycine -3.650 104.107 Choline -0.394 232.151 Isobutyryl-carnitine 1.393 116.050 Mandelonitrile -0.021 250.086 Gly-Ala-Cys -0.198 3-Acetylpyridine/2- M-Chlorohippuric acid 144.044 Acetylpyridine -1.190 251.982 -0.028 146.981 Phosphonoacetaldehyde 0.589 258.107 Glycerophosphocholine 0.677 156.043 N-phenyl-Glycine -0.779 275.146 Gln-Lys 3.936 4-Amino-2-Hydroxylamino- Sapropterin 166.061 6-Nitrotoluene -0.179 280.081 -2.652 Dihydrocaffeic Acid 3- 172.038 5,6-Dihydroxyindole -0.466 285.004 Sulfate -0.265 184.072 PC Head Group 0.935 307.044 Inosine 1.516 189.040 Methylisocitric acid -0.336 311.074 Phlorin -0.277 190.047 Pyridoxal (Vitamin B6) -0.019 313.091 N2-Succinoylarginine 0.153 190.062 N-Methyltyramine 0.301 317.115 α-CEHC -0.137 196.073 Pyroquilon -0.008 346.041 5'-CMP -0.384 197.105 N-Methyltryptamine -0.041 355.101 Gly-Pro-Gly-Ser 0.181 198.088 Arginic Acid -1.49 357.081 Asp-Gly-Ala-Gly 0.170 N-Nitrosothiazolidine-4-Car- His-Gly-OH 200.972 boxylic Acid 0.433 373.054 0.212 Met-Cys-Gly-Thr/Met- 204.121 Acetylcarnitine 1.518 393.126 Cys-Ala-Ser -0.105 5-Hydroxy-3,3',4',7,8-Pen- 206.054 Phosphocholine 0.773 427.079 tamethoxyflavone 0.111 7,11,12-Triacetoxycou- 208.073 α-Naphthylacetamide 0.137 449.027 mestan 0.387 214.063 Baclofen 0.202 504.342 PC(O-16:0) 0.108 3-(4-Isopropylphenyl)Pro- Leu-Tyr-Lys-Glu 215.083 panal 0.359 534.292 -0.182 216.063 Betamipron -0.086 616.197 Heme 3.305 Table 1: Table of putatively identified (<5 ppm mass error) metabolites detected within the human kidney. While only one metabolite is shown here, we are likely detecting multiple isomers at each of these different m/z values. high resolving power alone cannot discriminate isomeric spe- the utility of ion mobility for separating isobaric matrix clusters cies because they have the same m/z value. The MALDI TIMS from small mass metabolites. In this case, GPC is one of the system has been used recently to discriminate isomeric species, molecules used for lipid storage in the body and protects renal such as lipids, within an LC experiment54 and isobaric lipid spe- cells from urea damage during normal kidney function, demon- cies in an imaging context51 both of which lead us to our current strating key biological insights that can be gleaned from metab- study. olites that are isobaric with matrix ions. While only one example is featured here, ion mobility could separate background We have expanded this concept to include small mol- ions from biological metabolic information, specificity, and ecule analysis and separation of isobaric MALDI matrix ions sensitivity in over 30 cases, drastically increasing the spatial in- and small metabolites (m/z 256.8, Figure 3). The composite im- formation we obtain from matrix isobaric metabolite ions. age of matrix ion and metabolite mainly shows the localization of the matrix ion with the metabolite (putatively identified as Further, ion mobility was used to separate metabolic glycerophosphocholine or GPC) initially appearing as a low in- features that are isobaric with one another and show differential tensity signal within the tissue (Figure 3 inset). The extracted localization within the kidney (Figure 4). In this example, the mobilogram of this m/z value shows two components, with the m/z value 267.956 separates into four different structures by ion lower drift time associated with GPC (Figure 3B) and higher mobility (Figure 4A), where the species with the lowest 1/K0 drift time associated with the matrix ion (Figure 3C). The local- value localizes almost exclusively in the renal pelvis (Figure ization of the metabolite within the tissue is easier to discrimi- 4B) and the other three species localize through all three layers nate within the ion mobility extracted image when compared to of the kidney (Figure 4C-E). The localization of the fourth peak the composite image. Interestingly, the abundance of GPC of is lost almost entirely in the composite image (Figure 4A inset), higher within the mobilogram than the matrix ion, despite hav- demonstrating the information that can be gained from employ- ing a lower intensity within an image. We believe this is due to ing ion mobility separations to the small metabolite mass range the signal of GPC being spread across more pixels than the ma- to bring low-abundant signals into a detectable range. The first trix ion. The increased specificity of the ion image demonstrates 6 three ion mobility-resolved peaks display very similar localiza- and Kidney Diseases (U54DK120058 awarded to J.M.S. and tion within the kidney with only slight differences in ion abun- R.M.C.), NIH National Institute of Allergy and Infectious Dis- dances, perhaps indicating these are isomers or conformers, as ease (R01 AI138581 awarded to J.M.S.), and the National Sci- opposed to isobars. Even if they are different conformers, it is ence Foundation Major Research Instrument Program (CBET – still useful to probe how many conformers are present within a 1828299 awarded to J.M.S. and R.M.C.). E.K.N. is supported single m/z values as it could result from gas phase transitions or by a National Institute of Environmental Health Sciences train- biologically-relevant differences. Future work will involve ing grant (T32ES007028). The Cooperative Human Tissue Net- more complete identification of the ion mobility resolved peaks work is supported by the NIH National Cancer Institute (5 UM1 observed in this kidney tissue with the use of standards for a CA183727-08). biological study. REFERENCES In these examples, the addition of TIMS separations (1) Weiss, R. H.; Kim, K. Nature Reviews Nephrology 2012, 8, 22. increases the amount of information that can be garnered from (2) Dettmer, K.; Aronov, P. A.; Hammock, B. D. Mass Spectrometry each pixel within the metabolite IMS experiment, both in terms Reviews 2007, 26, 51. of specificity and sensitivity. Practically, the image acquisition (3) Monton, M. R. N.; Soga, T. 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