Jo-Il Kim, Jong-Min Park, Joo-Yoon Noh, Jae-Chul Pyun
Two kinds of solid nanostructure matrices, TiO2 nanowires and functional nanowebs, were synthesized to detect small molecules from human serum and dairy milk samples by MALDI-TOF MS. TiO2 nanowires were synthesized by top-down hydrothermal process, and functional nanowebs were synthesized by electrospinning on the metal plate. The feasibility of applying solid nanostructure matrices to MALDI-TOF MS was demonstrated by the detection of short peptides and amino acids. For the real sample analysis, amino acids and antibiotics were spiked into human sera and milk, respectively. The spiked analytes in serum and milk were detected qualitatively and quantitatively using solid nanostructure matrices.
MALDI-TOF mass spectrometry has been widely applied for the analysis of biomolecules with high molecular weights, such as proteins and peptides, because of its advantages on soft ionization, easy sample preparation, sensitive detection, and so on. Recently, the application of MALDI-TOF MS has been widely studied not only proteomics but also clinically relevant fields such as bacterial identification, newborn screening and drug monitoring. Amongst the new applications, more and more clinical fields require easy and rapid detection and quantitation of small molecules for MALDI-TOF MS. However, the larger amount of conventional organic matrices are ionized and flown to detector to produce their own peaks at low mass-to-charge (m/z) ratio range (<500) while the sample analytes are ionized by pulsed laser. Moreover, the organic matrices formed non-uniform co-crystals with samples while drying on the surface of target plates. Resultingly, these characteristics of MALDI made identification and quantitation difficult. More specifically, the peaks from small molecule analytes are overlapped with matrix peaks and spot-to-spot deviations are too high to apply MALDI-TOF mass spectrometry to the quantitative analysis for the clinical purpose. From such reasons, the application of MALDI-TOF has difficulties in applying to the clinical diagnosis by small molecular weight molecules detection so the other kinds of matrices are required. In this work, two kinds of solid matrices, TiO2 nanowire arrays and functional nanoweb matrix, were synthesized and applied to MALDI-TOF mass spectrometry. Furthermore, clinically important small molecules were detected from human serum and milk samples qualitatively and quantitatively using solid nanostructure matrices.
In order to utilize TiO2 nanowires for MALDI-TOF mass spectrometry as solid matrices, TiO2 nanowires were synthesized by top-down hydrothermal process directly on the surface of thin titanium plates using alkali solution, subsequently treated with water and heat. For the actual, the surface of TiO2 nanowire target plates was patterned with parylene thin film to have 81 nanowire array zones on the surface. The structures and electrical properties of synthesized TiO2 nanowire arrays were then characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Raman spectrometry and photo luminescence spectrometry. In case of functional nanoweb matrix, nanoweb matrices were synthesized by a simultaneous process of electrospinning of nanoweb and electrospraying of TiO2 nanoparticles on the metal target plate. The synthesis process was monitored by a quartz crystal microbalance sensor. The characteristic of functional nanoweb matrices were determined by SEM and a dynamic light scattering method.
The feasibility of using these solid nanostructure matrices to MALDI-TOF mass spectrometry to small molecule analysis was demonstrated by the detection of short peptides (leu-enkephalin) and amino acids. Leu-enkephalin is a short endogenous opioid peptide neurotransmitter, which is consisted with 5 amino acids. The molecular weight of leu-enkephalin is 555.62 g/mol, so solid nanostructure matrices should be capable of detecting this neurotransmitter as well as an organic matrix for further applications. Other model analytes amino acids are generally categorized into 4 groups; polar, non-polar, acidic and basic. Here, 2 amino acids from each group were selected as model small biomolecules. With TiO2 nanowire arrays and functional nanoweb matrix, the selected amino acids were clearly detected regardless of their categories without matrix-induced noise which hindered identification of amino acids in conventional MALDI-TOF mass spectrometry. Even quantitation were achieved using the solid nanostructure matrices, and the limit-of-detection (LOD) was established about 10 pM for amino acids using TiO2 nanowire arrays and 10 pM for leu-enkephalin using functional nanoweb matrix. The inter-spot relative standard deviation values were then calculated from the peak intensities of three different sample spots. From the aspect of generated ion species, [M+K]+ and [M+H]+ ions were obtained from TiO2 nanowire arrays and functional nanoweb matrices respectively.
In order to use solid nanostructure matrices to the clinical applications, the small analytes molecules have to be detected from the complex biological samples like blood. As well known, human sera include various kinds of biomolecules, such as albumins, globulins, antibodies and amino acids, even sugar. The presence of such numerous large proteins in a sample was also known to have an influence on desorption/ionization of analytes with low molecular weights. To reduce the influence of proteins, large proteins were precipitated out from arginine-spiked serum by a methanol precipitation method and a supernatant was used to analysis in this study. As a result, arginine could be clearly detected from methanol treated arginine-spiked sera using functional nanoweb matrices, whereas non-treated arginine-spiked sera could not. These results implied that solid nanostructure matrices can be applied for the clinical purpose.
Analysis of antibiotics in milk has become a big issue of public health. Commercially available dairy milk also contains lot of components including fats and proteins, and showed the same phenomenon as sera. However, the presence of such large molecules challenges the detection of the trace amount of antibiotic molecules. In this study, benzylpenicillin-spiked milk samples were analyzed by MALDI-TOF mass spectrometry using TiO2 nanowires after simple centrifugation as a preparation step. As results, benzylpenicillin in milk was clearly detected as sodium-adduct ions and the quantification of benzylpenicillin was achieved with established LOD of 0.4 ng/mL in dairy milk samples. As the cut-off concentration of antibiotics in milk was known to be 4 ng/mL by EU directives, these results show applying MALDI-TOF MS with solid nanostructure matrices to the clinical applications is feasible.