Nanoparticle assisted laser desorption/ionization mass spectrometry (Nano-PALDI MS) is a simple ionization method that eliminates, not only uneven distribution and different sizes of crystals, and the background peaks in the low mass range caused by organic matrices. In previous reports, various analytes such as drugs, lipids, peptides, synthetic polymers and proteins could be ionized and imaged by Nano-PALDI MS.1–5) The nanoparticles used in this study were synthesized by mixing aqueous solutions of 3d transition metal chlorides (MCl2·nH2O) and 3-aminopropyltriethoxysilane in a single step, resulting in particles with a metal oxide core coated with a functionalized silicate sheet and are able to ionize analytes. The nano-PALDI method has problems associated with sensitivity although it is a suitable method for ionizing and detecting low molecular weight compounds.
In the matrix assisted laser desorption/ionization (MALDI) method, chemical matrices are used to enhance the ionization of the high molecular weight analytes. Ion suppression can occur from matrix related ions in the low mass region (Fig. 1). We attempted to improve sensitivity in the low molecular region by LDI method, although we are able to measure the target compound by selecting an ionization method according to molecular weight.
|Fig. 1. Comparison of Nano-PALDI and MALDI MS for detection of small target molecule. Mass spectra of small molecule (carbendazim) by Nano-PALDI (A), by MALDI (B) and only carbendazim (C). Mass spectrum of only nano-particles coated plate (D).|
To enhance sensitivity, stable isotope-labelling reagents were developed to create mass differences for easy comparison of expression levels.6,7) As a new isotopic tagging reagent, we used Py-Tag**, which basically has a pyrylium structure and can specifically react with primary amino groups (e.g., the amino group of a lysine residue). Py-Tag increases the number of identifications due to its high labelling efficiency, wide mass difference, and chemical stability (Fig. 2A). Large mass differences can be created using Py-Tag using only 13C as a stable isotope without the need for other elements such as 15N or 18O.
|Fig. 2. Structure and composition of Py-tag (A) and schematic illustration of reaction mechanism of Py-tag and lysine (B).|
In this research, we attempted to find a better ionization method for increasing the sensitivity of detection of an amino acid compared with MALDI- or Nano-PALDI-coupled with Py-Tag labelling mass spectrometry (Fig. 2B).