Enrichment of phospholipids using magnetic Fe3O4/TiO2 nanoparticles for quantitative detection at single cell levels by electrospray ionization mass spectrometry
Graphical abstract
Introduction
Serving as essential components of cell membranes, phospholipids (PLs) participate various crucial life activities in biological systems, such as signal transduction, energy storage, cell proliferation, apoptosis [1]. The abnormal fluctuation of PLs metabolism may lead to a variety of pathophysiological diseases (e.g., diabetes, Alzheimerʼs disease and cancer) [2]. Because of the intercellular heterogeneities, the molecular information of individual cells is of significant importance in many research fields (e.g., early diagnosis and treatment of disease, as well as screening of effective drugs) [3,4]. Thus, it is highly desirable to measure PLs and metabolites [5] at the single cell level.
In the past few decades, the analysis of PLs in single cell has attracted increasing interest [[6], [7], [8], [9]]. However, the substances in a single cell are scarce yet of great varieties, making PLs analysis of single cell difficult. Numerous analytical methods including nuclear magnetic resonance (NMR) spectroscopy [10], fluorometry [11], Raman scattering [12], microelectrode [13], capillary electrophoresis (CE) [14], high performance liquid chromatography mass spectrometry (HPLC-MS) [15], and mass spectrometry (MS) [[6], [7], [8], [9]] have been tested for characterization of PLs in cell matrixes. Among these techniques, MS stands out as a powerful tool for the analysis of trace cell contents due to its high sensitivity, excellent selectivity and information-richness [[6], [7], [8], [9]]. Matrix-assisted laser desorption/ionization (MALDI-MS) [7,16] and secondary ion mass spectrometry (SIMS) [17] are the conventional MS ionization techniques for the analysis of chemicals in single cells, which usually require high vacuum conditions to generate the analyte ions.
Ambient MS is increasing implemented for analysis of complex biological samples [18,19], featuring rapid sampling and ionizing biological samples under ambient conditions. So far, a series of ambient MS methods have been developed for PLs analysis of various biological cells. For instance, desorption electrospray ionization (DESI)-MS was used for phospholipid profiling of mouse individual oocytes [20]. Probe electrospray ionization (PESI)-MS was applied for analysis of PLs from renal cell carcinoma [21]. Easy ambient sonic-spray ionization (EASI)-MS was employed for investigation of the changes of acidic PLs in unicellular cyanobacteria [22]. Subcellular metabolite and phospholipid analysis of single sea urchin eggs were demonstrated by laser ablation electrospray ionization (LAESI)-MS [23]. Differences in the phospholipid profiles between normal and apoptotic HEK cells were observed by laser desorption/ionization droplet delivery (LDIDD)-MS [24]. Although these techniques have their own features for rapid and direct characterization of PLs at the single-cell level, they are focused on the analysis of nontarget metabolites without the extraction and enrichment of PLs and thus their sensitivity and selectivity are limited. In addition, the existing studies are mainly focused on the qualitative detection of phospholipid rather than the quantitative analysis.
With the development of functional materials, the sensitivity and selectivity of MS for single cell analysis are obviously improved. Various functional materials, such as surface modified wooden-tip [25], coated blade [26], biocompatible surface-coated probe [9], metal oxides [27,28], superparamagnetic nanoparticle [29], have been used for the selective chemical extraction from complex biological matrixes prior to MS detection. One of the powerful and promising approaches that have appeared in recent years is metal oxide affinity chromatography (MOAC) which takes advantage of the particular affinity of metal oxides to phosphate groups. The mechanism is based on Lewis acid-base interactions between the phosphate group and metal atoms [30]. PLs are a unique type of lipids containing the phosphate group, constituting the major part of biological membranes in the form of lipid bilayers. Thus, Fe3O4/TiO2 nanoparticles theoretically can be applied for any types of PLs with the phosphate group. Noted that the study of selectivity variation in different type of PLs will be carried out in the future. In addition to TiO2, there are other metal oxides [28], such as zirconium dioxide(ZrO2), aluminum oxide(Al2O3), gallium oxide(Ga2O3), iron oxide(Fe3O4), and hafnium oxide(HfO2), which are often used to selectively enrich phosphorylated compounds. Although different metal oxides have been applied for enrichment of phosphorylated compounds, their specificity and application are limited. For instance, large-scale studies revealed more substantial binding of non-phosphorylated peptides to the ZrO2 material when compared to TiO2. A small number of phosphopeptides enriched by Ga2O3-coated magnetic particles were identified. Al2O3 has weak acid-base tolerance. HfO2 has not so far been applied to more complex samples. TiO2 continues to the metal oxide most widely applied due to the superior specificity in combination with optimized binding and washing conditions. Thus, the functional material based on TiO2 combined with ambient ionization techniques should be a potential solution for the rapid determination of PLs or phosphopeptides in single-cell samples.
In this article, magnetic TiO2-coated Fe3O4 nanoparticles featured to easy separation and selective enrichment were synthesized for the selective extraction of PLs in single cell samples without derivatization or labeling procedure. Then, the analytes-loaded Fe3O4/TiO2 nanoparticles were eluted by 1.5% ammonia/methanol (w/w). Finally, the eluent with PLs was injected to electrospray ionization mass spectrometry (ESI-MS) for sensitive determination. The analytical performance and potential applications of the method were characterized for several types of cells, showing that the method is potentially useful for future applications in quantification of PLs and cell subpopulation identification for related disease diagnosis.
Section snippets
Chemicals and materials
Methanol (HPLC grade) and isopropanol (HPLC grade) were purchased from Merck KGaA (Darmstadt, Germany), and ammonium hydroxide solution (w/w, 20%) was obtained from CNW Technologies GmbH (Düsseldorf, Germany). Trifluoroacetic acid (TFA) (HPLC grade) was bought from Fisher Scientific (Waltham, MA, USA). Tetrabutyl titanate, ethylene glycol (EG), ethanol, ethylene diamine (ED), ferric trichloride hexahydrate (FeCl3·6H2O), sodium acetate (NaAc) and sodium hydroxide (NaOH) were got from Sinopharm
Fe3O4/TiO2-ESI-MS
The synthesized Fe3O4–TiO2 nanoparticles were characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive spectrometer (EDS) and X-ray diffraction (XRD) (Fig. S2). As shown in Fig. S2a, SEM image indicates that Fe3O4/TiO2 nanoparticles was spherical with size about 450 nm. Thus, adsorption efficiency was further improved. In Fig. S2b, the core-shell Fe3O4–TiO2 nanoparticle was apparently observed from the TEM image. The outer layer of
Conclusions
In summary, the method of Fe3O4/TiO2-ESI-MS was developed for selectively enriching trace PLs and quantitative analysis at single cell levels. The capability of the proposed method was demonstrated by qualitative determination of eighteen PLs quantitative detection of PC(34:1) and PC(36:2) in six real single-cell samples. In addition, differentiations of six kinds of cell subpopulations were also conducted by PLS-DA. The proposed method is featured by the high sensitivity, good selectivity,
Acknowledgement
The work was supported by National Natural Science Foundation of China (Nos. 21765001, 21565004 and 21427802) and Natural Science Foundation of Jiangxi Province (No. 20165BCB19013).
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