Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Summary

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.

Abstract

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.

Introduction

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 require....

Protocol

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 store tissue at -80 °C. It is not advisable to place the tissue in any kind of buffer or saline, because they may extract certain contents from t....

Results

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 .......

Discussion

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 pretreatments^5,14,15,17. It is an easy and instantaneous mass spectrometry technique that can be applied to the integrated state of living organisms. Sin.......

Materials

Name	Company	Catalog Number	Comments
5-Fluoro-2'-deoxyuridine (5-FdU)	Sigma-Aldrich	F8791-25MG	25mg
disposable biposy punch (Trepan)	kai Europa GmbH	BP-30F	bore size 3mm
ethanol	nacalai tesque	14710-25	extra pure reagent
LabSolutions	Shimadzu		ver. 5.96, Data analyzer
micropestle	United Scientific Supplies	S13091
microtube	Treff	982855	0.5 mL clear
PESI-MS (Direct Probe Ionization-MS)	Shimadzu	DPiMS-2020	Mass spectrometer equipped with PESI
PPGT solition	Shimadzu	ND	Attached to DPiMS-2020