Measurement of the Spatial Distribution of S1P in Small Quantities of Tissues: Development and Application of a Highly Sensitive LC-MS/MS Method Combined with Laser Microdissection

Sphingosine-1-phosphate (S1P) acts as an extracellular signaling molecule with diverse biological functions. Tissues appear to have an S1P gradient, which is functionally relevant in the biological significance of S1P, although its existence has not been measured directly. Here, we report a highly sensitive method to determine the distribution of S1P, using a column-switching LC-MS/MS system combined with laser microdissection (LMD). Column switching using narrow core Capcell Pak C18 analytical and trap columns with 0.3 mm inner diameter improved the performance of the LC-MS/MS system. The calibration curve of S1P showed good linearity (r>0.999) over the range of 0.05–10 nM (1–200 fmol/injection). The accuracy of the method was confirmed by measuring S1P-spiked laser microdissected mice tissue sections. To evaluate our S1P analytical method, we quantified S1P extracted from micro-dissected mouse brain and spleen. These results show that this method can measure low S1P concentrations and determine S1P distribution in tissue microenvironments.


INTRODUCTION
Sphingosine-1-phosphate (S1P), an important cellsignaling molecule, mediates various pathophysiological processes, such as autoimmunity and vascular development, via ve cell surface receptors (S1P 1-5 ). 1,2) S1P is intracellularly synthesized by the phosphorylation of sphingosine in multiple cells, 3) and then exported to the extracellular environment by speci c transporters. 4) S1P concentration is much higher in blood and lymphatic uid than in interstitial spaces in lymph organs due to the activity of S1P lyase.
is S1P gradient causes lymphocytes to move from lymph organs to the circulation through S1P 1 receptor expressed on lymphocyte. [5][6][7] In addition, mapping S1P in mice by analyzing S1P receptor signaling suggested the presence of a local S1P gradient within tissues. [8][9][10][11] For example, S1P concentration seems to decrease with distance from the marginal zone in the white pulp. 10) However, the existence of an S1P gradient within tissue has not yet been directly measured.
Lipid analysis has been improved greatly in recent years through advances in liquid chromatography (LC) coupled with MS/MS systems. Previously, the limit of detection for sphingolipids had been reduced to the nanomolar level. 12) Although LC-MS/MS has been used widely in lipid research due to its ability to quantify lipids, it is not well-suited for analyzing tissue samples because it requires sample preparation, which destroys spatial information about the analyte. To overcome this problem, imaging mass spectrometry, such as matrix assisted laser desorption ionizationimaging mass spectrometry, has been developed to visualize the spatial distribution of analytes in tissue samples. 13) is technique can clearly visualize sphingolipids including sphingomyelin (SM), which is abundant lipid in eukaryotic cells, in tissue sections. However, imaging S1P with high spatial resolution is challenging because its level is much lower than that of SM. erefore, we sought to develop a more sensitive method to determine the level of S1P with spatial information.
Laser microdissection (LMD) is a contact-and con-tamination-free method for isolating speci c single cells or small areas from a wide variety of samples. LMD is now used in a large number of research elds, such as proteomics, cancer research, neuroscience, and plant research. 14) For instance, the combination of LMD with LC-MS has been successfully applied to explore protein distribution pattern. 15) Previously, we utilized LC-MS/MS combined LMD to quantify sphingolipids in small liver samples. 16) To further improve the performance for S1P, we combined the column-switching LC-MS/MS method with a narrow core column. Column-switching is a two-column liquid chromatography technique that has been used for chromatographic separation, on-line sample cleanup, deproteinization, and trace enrichment. 17) To our knowledge, our method is the highest sensitive one that coupled LMD with LC-MS/MS to investigate lipid tissue distribution.
particle size). Capcell Pak C18 (150 mm×0.3 mm I.D., 3 µm particle size) was applied as analytical column and Capcell Pak C18 (50 mm×0.3 mm I.D., 3 µm particle size) as trap column for column switching system. Columns were maintained at 40°C. e mobile phase was 5 mmol/L ammonium formate (HCOONH 4 )-H 2 O (pH 4) (A) and 5 mmol/L HCOONH 4 -H 2 O/acetonitrile (CH 3 CN) (5 : 95, v/v; pH 4) (B), which was detailed in an earlier study. 18) For the analysis with 150 mm×1.5 mm-I.D. column, the elution gradient with ow rate of 200 µL/min started with 50% B for 0.2 min, increasing to 100% B in 2.7 min, held for 2.9 min and then back to initial conditions. e column was re-equilibrated for 1.1 min until the next injection. For the analysis with 150 mm×0.3 mm-I.D. column, the elution gradient the elution gradient with ow rate of 20 µL/min started with 30% B for 1 min, increasing to 85% B in 5 min and then to 100% in 4 min. A er held in 100% B for 10 min, gradient went back to initial conditions and column was re-equilibrated for 15 min until another injection. e gradient program for column switching system was as follows: the elution with the ow rate of 10 µL/min from rst pump for the separation column was rst 30% B for 8 min, the proportion of B then was linearly increased to 100% B in 7 min, it was held for 15 min and immediately returned to the initial condition and maintained for another 15 min until the end of the run, the elution from the second pump for the trap column was isocratic, with 30% B of 10 µL/min, valve was switched at 10 min to change the direction of elution.

Mass spectrometric conditions
e MS system was a TSQ Quantiva ( ermo Fisher Scienti c, San Jose, CA) triple quadrupole mass spectrometer equipped with a heated-electrospray ionization-II (HESI-II) source. e nebulizing gas and collision gas were nitrogen and argon, respectively. Standard solutions dissolved in MeOH (1.0 µmol/L) were infused into MS continuously by a syringe pump at the rate of 5 µL/min. HESI was performed in positive mode for S1P. Samples were analyzed in SRM mode, using the transitions of the [M+H] + precursor ions to their product ions. e MS/MS transitions were determined in full scan mode (m/z 50-450). Optimized parameters are described in Table 1.

Validation parameters
A 10 µmol/L stock solution of S1P was prepared in methanol. It was evaporated and resuspended in 0.1% BSA-PBS and diluted to the concentration of 0.125, 0.25, 1.25, 2.5, 12.5, 25 µmol/L as standard solution. C17-dhS1P (100 nmol/L, nal concentration) was selected as the internal standard (IS) in the present method. We con rmed that ion intensity and peak sharpness of C17-dhS1P were almost equal to those of C17-S1P, which is another commercially available IS. Standard calibration solutions were prepared with LMD-dissected tissue fragments (liver, spleen, kidney, brain) obtained from male C57BL/6 mice. 0.2 µL of each standard solution were spiked on the surface of tissue cryosections and dried in room temperature. LMD-dissected sections were collected with 500 µL IS solution added in each tube. e obtained mixture was homogenized for 10 min in an ultrasonic bath. A er centrifugation at 15,000×g for 10 min at 4°C, the supernatant was collected, ltered and centrifuged at 6,500×g for 5 min at 4°C. An aliquot (20 µL) of ltered solution was analyzed by columnswitching LC-MS/MS. For validation of the method,  S1P standard spiked on each LMD-dissected liver, spleen, kidney and brain tissue sections were extracted and applied to column-switching LC-MS/MS system. samples prepared at concentrations of 1, 2, 10, 20, 100 and 200 fmol/injection were injected four times on the same day and analyzed. is procedure was repeated for 3 times in independent experiments. e accuracy was calculated as [(found concentration−endogenous concentration)/spiked concentration−1]×100 (%), and the precision was evaluated using the coe cient of variation. Peak areas and calibration curves were obtained using Xcalibur so ware ( ermo Fisher Scienti c).

Preparation of biological samples
Mice tissues (liver, spleen, kidney, brain) were obtained from male C57BL/6 mice. Mice were rst anesthetized with ethyl carbamate and then rapidly perfused transcardially with PBS through the le ventricle and sacri ced. Tissue was quickly removed, embedded in OCT compound and snap frozen by liquid nitrogen. Cryosections of 15 µm thickness were made with a Leica CM1950 cryostat. Tissue sections attached to PEN membrane glass slides were laser microdissected (Leica LMD7000). To dissect spleen white pulp and red pulp, more than 20 spleen slices stained by 0.5% toluidine blue were microdissected and the tissue dissectates of the same sub-region were pooled. We conrmed that the level of S1P in spleen section was unchanged by staining. Laser parameters for dissection were laser power: 45, aperture: 20 and speed: 10. Five hundred µL IS (100 nmol/L) was added to LMD-dissected sections. S1P was extracted from the dissected samples as described in the 'Method validation' section.

LC-MS/MS method development
In order to improve LC-MS/MS detection of S1P, a highly sensitive mass spectrometer, TSQ Quantiva ( ermo Scienti c) was used. e optimized parameters are shown in Table 1. SRM transitions are m/z 368.3 270.2 for C17-dhS1P and 380.3 264.2 for C18-S1P. e quanti cation limit of S1P (signal to noise ratio of ≥10) was 100 pM (2 fmol/injection) (Fig. 1a, d). To improve peak sharpness, Fig. 3. S1P distribution in brain tissue sections. we used a narrow (0.3 mm I.D.) C18 column with a low elution ow rate. We obtained a symmetrical peak shape for S1P using the narrow column. Although the narrow column and low ow rate did not improve the detection limit, the precision, as expressed by the coe cient of variation (CV), was improved (Fig. 1b, e). We then added column switching, which is a technique for concentrating an analyte. In these systems, the analyte is rst trapped in a trap column and then the direction of elution is switched to back-ush the enriched analyte to an analytical column. With this system, the detection limit for S1P fell to 50 pM (1 fmol/injection) (Fig. 1c, f), a 20-fold improvement compared with a previously reported method. 9)

Method validation
To evaluate the method, we examined linearity, recovery, precision and the study of matrix e ects. e relative standard deviation ranged from 0.429 to 3.06% for the 0.125 µmol/L spiked samples and the recovery varied from 83.1 (-16.9) to 96.94 (−3.06)% (Table 2). Calibration curves for S1P were linear from 1 to 200 fmol/injection, tted to a linear equation of the slope and intercept (y=ax+b). Correlation coe cients were better than 0.99 (Table 3). As shown in Fig. 2, the calculated S1P concentration that subtracted naturally presented S1P in naïve samples, against the spiked S1P concentration was plotted. Linear recoveries were yielded for each tissue samples. ese data suggest that this method is accurate and reproducible for analyzing S1P in biological samples and demonstrate its reliability.

Application of LMD-combined column-switching LC-MS/MS for investigating distribution of S1P in brain and spleen tissue sections
To test the utility of this technique, we examined the levels of S1P in di erent sub-regions of the brain and spleen. Brain is an S1P-rich tissue. 12) Furthermore, S1P-related molecules (sphingosine kinases, transporters and receptors) have di erent functions in di erent parts of the central nervous system. [19][20][21] To see these di erences more clearly, we prepared eight transverse sections of an adult mouse brain and then microdissected parts of these sections corresponding to di erent brain parts (corpus callosum, cerebral cortex, etc.) (Fig. 3a). In this gure, the microdissected parts are indicated by dashed red lines. We then measured the level of S1P in each part. S1P was detected in each part, with the highest levels in the corpus callosum ( Fig. 3b A), pons and medullar (Fig. 3b F). In the cerebellum, the level of S1P was signi cantly higher in the white matter (Fig. 3b G) than in the grey matter (Fig. 3b H). e cerebral cortex, which is the outer covering of the gray matter, also had a low level of S1P (Fig. 3b B). Unexpectedly, these di erences in S1P levels were not consistent with the sphingosine kinase (SPHK) activity. 22) at is, high SPHK activity was found in cortex, which showed the lowest levels of S1P in the present study (Fig. 3b B). is discrepancy may be due to di erences in the level of sphingosine that act as a substrate of SPHK, and in the S1P degradation by S1P lyase. 23,24) We also examined the distribution of S1P in the spleen. e spleen mainly contains two di erent tissues, red pulp and white pulp. Red pulp is connective tissue that receives circulating blood containing abundant S1P and removes abnormal red blood cells. Red pulp is composed of splenic cords and venous sinuses. Splenic cords are consisted from reticular bers, myo broblasts and macrophages, while venous sinuses are lined by endothelial cells. In contrast, white pulp is lymphatic tissue consisting of lymphocytes, macrophages, dendritic cells, plasma cells, arterioles, and capillaries in a reticular framework similar to that found in the red pulp. 25) In a spleen section, we microdissected a white pulp area and the adjacent red pulp area (Fig.  4a), and determined S1P level.
e S1P concentrations in the spleen (<1 pmol/mg) were much lower than those in the brain (<35 pmol/mg). Interestingly, S1P concentration was higher in red pulp than in white pulp (Fig. 4b). However, it was previously reported that mouse red pulp had little signaling-available S1P, 10) the distribution of S1P remains controversial.
column-switching technique to a TSQ Quantiva ™ mass spectrometer, increased the sensitivity to S1P dramatically, reaching a detection limit of 50 pM (1 fmol/injection). e system also had good linearity, recoveries and precision. Our analyses of mouse brain and spleen samples demonstrate that combining column-switching LC-MS/MS with laser microdissection is feasible for studying the distribution of S1P in tissue sections. e technique developed in this study will allow further analysis for other lipids in the future.