Functional magnetic nanoparticle-based label free fluorescence detection of phosphorylated speciesw

Protein phosphorylation is one of the most studied posttranslational modifications (PTMs) that can regulate cellular processes. Thus, developing appropriate quantitative analysis methods for monitoring the levels of phosphorylated species in a biological system is significant. Isotope labeling quantitative phosphoproteomics has been developed for this purpose. However, isotope labeling reagents are expensive. Generally, a sample pretreatment must be performed by either immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC) to concentrate traces of phosphorylated proteins/peptides from complex samples prior to quantitative/qualitative analyses. Phosphorylated species concentrated by these affinity chromatography approaches are generally characterized using biological mass spectrometry. When aiming to monitor the levels of major phosphorylated species in a biological system, quantitative methods possessing the features of simplicity and rapidity are desirable. Label-free fluorescence detection offers a convenient and sensitive means for rapidly sensing target species. Since alumina nanoparticles have been demonstrated to be good affinity probes for phosphorylated species such as phosphopeptides and phosphoproteins, we herein propose a method by immobilizing fluorescence molecules, namely, riboflavin-50monophosphate (RFMP), onto the surface of alumina-coated magnetic nanoparticles (RFMP-Fe3O4@Al2O3 MNPs) via Al-phosphate chelating as sensing probes for phosphorylated species. The magnetic feature of the MNP leads to fast isolation of the MNP-target species from the solution. The fluorescence of RFMP molecules is quenched while the molecules anchor on the surface of the MNPs. However, upon the addition of the RFMP-Fe3O4@Al2O3 MNPs into sample solutions containing phosphorylated species, the phosphorylated species spontaneously exchange with the RFMP molecules from the surfaces of the MNPs, resulting in the appearance of visible fluorescence in the solution. Thus, using RFMPFe3O4@Al2O3 MNPs as sensing probes for phosphorylated species in order to conduct quantitative analysis is possible. Furthermore, the attachment of phosphorylated species on the MNPs can be examined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) for further confirmation. Fig. 1 presents the design of this label free approach for phosphorylated species by using fluorescence spectroscopy (FL) and mass spectrometry (MS) for quantitative and qualitative analysis, respectively. Blood coagulation abnormalities have been found in patients with cancers. Fibrinopeptide A (FPA), one of the products of fibrinogen after digestion by thrombin, has been known to elevate abnormally in the blood of patients with gastric and ovarian cancers. Thus, rapidly determining the FPA level in serum samples can assist medical diagnostics. Furthermore, it has been found that 20–30% of FPA is phosphorylated in human blood. Thus, phosphorylated FPA (PFPA) may be used as a potential risk factor for early detection and screening of high-risk-individuals. We have previously demonstrated that only PFPA-derived ions appear in the MALDI mass spectra after human serum samples were enriched by Fe3O4@Al2O3 MNPs. 4 That is, because PFPA is the major phosphorylated species in serum samples therefore the contribution from traces of other phosphorylated molecules is suppressed and may be neglected. In this paper, we examined the feasibility of employing the RFMP-Fe3O4@Al2O3 MNPs as sensing probes for rapid screening of the presence of PFPA in human serum based on the detection of fluorescence derived from RFMP molecules. We first examined the binding constant of RFMP toward Fe3O4@Al2O3 MNPs according to the Langmuir adsorption equation. The dissociation constant (Kd) is ca. 6.82 10 7 M (see Fig. S1w). The binding process can be accelerated by placing the reaction under microwave-heating. The RFMPFe3O4@Al2O3 MNPs were generated by mixing RFMP with Fe3O4@Al2O3 MNPs under microwave-irradiation (power: 450 W) for 60 s. The maximum binding amount (Qmax) of RFMP molecules on the MNPs was ca. 76 nmol mg , similar to that obtained from vortex-mixing for 2 h at room temperature. Additionally, microwave-assisted exchanges of Fig. 1 Cartoon representation of the quantitative and qualitative detection of phosphorylated peptides/proteins by fluorescence spectroscopy (FL) and mass spectrometry (MS), respectively, using RFMPFe3O4@Al2O3 MNPs as affinity probes and reporters.