We thank Ayumi Iizuka for operating the PESI-MS and Kazuko Sawa-nobori for her secretarial assistance. We thank Bronwen Gardner, Ph.D., from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript.

The institutional committee for animal care at the University of Yamanashi approved all the protocols and the use of experimental animals stated herein. Human sample usage was approved by the institutional ethics board at the University of Yamanashi.
1. Solid tissue preparation
NOTE: Samples must be kept on ice after their removal from the animal or human body to preserve the tissue freshness. If measurements do not immediately follow dissection, it is recommended to ...

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The corresponding author was funded by Shimadzu, a manufacturer and supplier of PESI-MS instrument.

Although PESI is a derivative of ESI for mass spectrometry4, it is most advantageous for monitoring real-time metabolomics, as well as for analyzing biochemical reactions without performing any complex or time-consuming pretreatments5,14,15,17. It is an easy and instantaneous mass spectrometry technique that can be applied to the integrated state of living organisms. Sin...

Mass spectrometry (MS) is a technological realization of reductionism; it reduces the object of analysis to a unit that can be interpreted on the basis of molecular species or cascades. Therefore, it is a representative method of analytical chemistry. It is made up of four processes: ionization, analysis, detection, and spectral acquisition. Because ionization of the molecule is the first process in mass spectrometry, it generally restricts the form of the analytes to be processed. Most ionization procedures require the destruction of the structure, morphology, and real-time biological processes of organic samples. For example, electrospray ionization (ESI) MS requires that the samples be in a liquid state for efficient ionization1. Samples, therefore, must go through a complex biochemical preparation, which alters the composition of molecules. Alternatively, while matrix-assisted laser desorption ionization (MALDI) MS can reconstruct molecular maps of thin sectioned tissue2,3, its ionization efficiency is too low to detect all molecules in the samples, and it is particularly poor at analyzing fatty acids. Considering these limitations, probe electrospray ionization (PESI)4 can be used to observe the real-time changes in biological systems in situ without destroying the structural integrity5, while the biological organism being observed is technically in a living state. A very fine needle is used in this case that serves simultaneously as a sample picker and an ion emitter. This means that the complex sample pretreatment sequences can be bypassed to obtain mass spectra that reflect the molecular components of the living system in situ.
There are several other ionization methods that rival PESI-MS. One is rapid evaporative ionization mass spectrometry (REIMS)6. This technique works well during surgery because it is assembled with an electrical knife and collects the ion plume generated during incision. While REIMS is very useful for the surgery, it is essentially a destructive method that requires the electrical ablation of the tissue. Therefore, it is not useful for the detailed analysis of cells and tissues in a preparative sample or in laboratory analyses. Moreover, because it collects a large amount of plume containing tissue debris, it requires lengthy maintenance of the devices after each use, thus limiting the use of this machine to special surgical procedures. A similar method, called laser desorption ionization mass spectrometry (LDI-MS)7, is another technique that is noninvasive and useful for the surface analysis. Because this technique is good at scanning the surface of a specimen, it achieves comprehensive two-dimensional analysis like MALDI imaging mass spectrometry8,9. However, because LDI-MS is only applicable to the surface analysis, PESI-MS is advantageous for analyzing the samples e.g., within the tissue. Another technique, the MasSpec Pen10, was reported to achieve high specificity and sensitivity in diagnosing thyroid cancer, but the diameter of the probe is in the order of mm and it is specific for the surface analysis, meaning that it cannot detect small nodules of cancer or deeply localized lesions. Moreover, as this method uses a microcapillary flow canal embedded in the probe pen, cross-contamination must be taken into consideration, similar to LDI-MS. Other techniques exist that have been applied to clinical settings, such as the flow probe and ionization form swab11, but they are not widespread.
PESI is extreme miniaturization of ESI, wherein the capillary of the nano-electrospray converges on a solid needle with a tip curvature radius of several hundred nm. Ionization takes place in the extremely restricted area of the needle tip by forming a Taylor cone, on which samples remain until ionization of all the fluid on the tip is completed12. If the analyte stays on the tip of the metal needle, excess charge is continuously generated at the interface between the metal needle and the analytes. Therefore, sequential ionization of molecules occurs depending on their surface activity. This property makes the needle tip a kind of chromatogram, separating the analytes depending on their surface activity. More technically, molecules with the stronger surface activity come to the surface of the Taylor cone and are ionized earlier than those with weaker surface activity, which adhere to the surface of the needle until the end of the ionization process. Thus, complete ionization of all molecules picked up by the needle is achieved13. Moreover, because this technique does not involve the addition of superfluous solvent to the sample, several hundreds of femtoliters are sufficient to get mass spectra strong enough for further analysis14. These properties are advantageous for the analysis of intact biological samples. However, a major disadvantage of PESI-MS lies in the discontinuity in ionization because of the reciprocating movement of the needle along the vertical axis, similar to a sawing machine. Ionization only takes place when the tip of the probe reaches the highest point when the height of the ion orifice is aligned on the horizontal axis. Ionization ceases while the needle picks up samples, and so the stability of ionization is not equal to that in conventional ESI. Therefore, PESI-MS is not an ideal method for proteomics.
To date, PESI-MS has been applied chiefly to the analysis of biological systems, covering a broad range of fields from basic research to clinical settings. For example, the direct analysis of human tissue prepared during the surgery was able to reveal the accumulation of triacylglycerol in both renal cell carcinoma15 and pharyngeal squamous carcinoma16. This method can also measure liquid samples, such as blood, to focus on the lipid profile. For example, some molecules have been delineated during dietary changes in rabbits; it was reported that some of these molecules decreased at very early stages of the experiments, indicating the high sensitivity and usefulness of this system for clinical diagnosis17. Furthermore, direct application to a living animal allowed the detection of biochemical changes of the liver after just one night of fasting5. Zaitsu et al.18 revisited this experiment5 and analyzed the metabolic profiles of the liver in almost the same way, with results that reinforced the stability and reproducibility of our original method. Furthermore, we were able to discriminate the cancer tissue from surrounding non-cancerous liver in mice using this technique19. Therefore, this is a versatile mass spectrometry technique that is useful in various settings, both in vivo and in vitro. From another standpoint, the PESI module can be made to fit various mass spectrometers by adjusting the mounting attachment. In this short article, we introduce the basics and examples of applications (Figure 1), including applications with living animals5.
According to the regulations and laws in each country, parts of this protocol will need to be revised to meet the criteria of each institution. Application to the living organism is the most interesting and challenging because it can provide biochemical or metabolic changes in tissues or organs in living animals in situ. While this application was approved by the institutional committee for animal care at the University of Yamanashi, in 20135, another round of approval will now be necessary because of recent changes in regulations for the animal experiments. Several modifications in the experimental scheme are, therefore, advisable. Regarding the mass spectra obtained in experiments, taking the fluctuations of mass spectra between each measurement into account, there is no spectral information sharing system that is common to the nucleotide sequencing community. Care must be taken when the operator handles the needle to avoid needle-stick accidents, especially when removing the needle from the needle holder. A special device for detaching the needle is very useful for this purpose. Since the compartment of the PESI module is an airtight, closed chamber, leakage of the ion plume does not occur if the mass spectrometer is operated according to the instructions.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-Fluoro-2&#39;-deoxyuridine (5-FdU)</td>
			<td>Sigma-Aldrich</td>
			<td>F8791-25MG</td>
			<td>25mg</td>
		</tr>
		<tr>
			<td>disposable biposy punch (Trepan)</td>
			<td>kai Europa GmbH</td>
			<td>BP-30F</td>
			<td>bore size 3mm</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>nacalai tesque</td>
			<td>14710-25</td>
			<td>extra pure reagent</td>
		</tr>
		<tr>
			<td>LabSolutions</td>
			<td>Shimadzu</td>
			<td></td>
			<td>ver. 5.96, Data analyzer</td>
		</tr>
		<tr>
			<td>micropestle</td>
			<td>United Scientific Supplies</td>
			<td>S13091</td>
			<td></td>
		</tr>
		<tr>
			<td>microtube</td>
			<td>Treff</td>
			<td>982855</td>
			<td>0.5 mL clear</td>
		</tr>
		<tr>
			<td>PESI-MS (Direct Probe Ionization-MS)</td>
			<td>Shimadzu</td>
			<td>DPiMS-2020</td>
			<td>Mass spectrometer equipped with PESI</td>
		</tr>
		<tr>
			<td>PPGT solition</td>
			<td>Shimadzu</td>
			<td>ND</td>
			<td>Attached to DPiMS-2020</td>
		</tr>
	</tbody>
</table>

sample preparation for probe electrospray ionization mass spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

As depicted in Figure 3, the data obtained by PESI-MS technique are the mass spectra, whose m/z range from 10 to 1,200 in this system. While one can detect molecules up to m/z 2,000, there were few peaks obtained using this technique over the mass range of m/z 1,200. Therefore, we analyzed peaks from m/z 10 to 1,200. There were conspicuous groups of peaks around m/z 800 and 900; the former represents cell membrane ...

Watch this Scientific Journal Video about Sample Preparation for Probe Electrospray Ionization Mass Spectrometry at JoVE.com

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

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9.2K visualizações.  University of Yamanashi School of Medicine. Então, esta é uma espectrometria de massa ambiente única, particularmente adequada para a análise de amostras biológicas, tanto in vitro quanto in vivo. Como isso não requer regularmente um pré-tratamento especial de amostras, como classificação celular, fracionamento e concentração, pode analisar uma amostra com perda mínima de componentes biológicos, especialmente aqueles que existem como quantidade muito pequena. Esta técnica permite analisar as matérias-primas diretamente e...