Electrophoresis of Phosphoproteins on a Tandem Polymerized Gel.

A substrate with n phosphorylated sites may have 2n phosphor-forms for temporal-spatial regulation of biological events. Because phosphates do not significantly change molecular masses but net charges of proteins, those isoforms cannot be separated by regular mass-based sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). A tandem polymerized gel was developed to resolve phosphor-isoforms with different masses, charges, and posttranslational modifications. Without the usage of SDS, the electrophoresis was primarily performed on three adjacent acidic polyacrylamide gels. After being concentrated on a stacking gel, protonated proteins were then separated on the Zr4+ immobilized gel through the coordination of metal ions with phosphates followed by further charge and mass (z/m)-based electrophoretic separation on a TiO2 containing gel. The presence of TiO2 nanoparticles in the third gel is aimed for the initiation of the polymerization of acrylamide in acidic conditions upon ultraviolet irradiation. Distinct isoforms of α-S1-casein, α-S2-casein, ß-casein, and κ casein model proteins located on 11, 8, 8, and 7 different bands of the tandem gel were unambiguously identified, respectively. With the tandem polymerized gel electrophoresis, new phosphorylation events that may occur simultaneously or sequentially were discovered in not only model proteins but also complex biological samples including human saliva, chicken egg, and sprouting maize. This provides a new tool to dissect complex biological processes that are triggered by dynamic phosphorylation events.

Monitoring of adsorption and transfer of organochlorines in soybean seeds and sprouts with mass spectrometric imaging.

Development of analytical techniques that can monitor the adsorption, transfer and in-situ distribution of environmental pollutants in agricultural products is essential to ensure the implementation of stringent food safety standards for consumer protection. A mass spectrometric imaging approach is described herein to investigate the dynamic changes and spatial distributions of 4, 4'-DDT (dichlorodiphenyltrichloroethane) in soybean seeds and sprouts during the growth. Soy beans seeds incubated in DDT containing water were sliced in every 20 µm and directly blotted on the surface of a compressed thin film of (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles. Endogenous molecules and exogenous DDT compounds in soy bean seeds were ionized and dissociated by photoelectrons that are generated on surfaces of semiconductor nanoparticles upon the irradiation of the 3rd harmonic (355 nm) of Nd3+:YAG laser. Structural identification is achieved by the interpretation of fragment ions resulting from electron-initiated specific bond cleavages or hole oxidization. Mass spectrometric images reveal increased quantities of DDT residues in soy bean seeds and sprouts during the growth. It provides an in situ way without extensive sample preparation to monitor the transfer and distribution of exogenous pollutants as well as the possible impacts on plant growth.

Electron Acceptive Mass Tag for Mass Spectrometric Imaging-Guided Synergistic Targeting to Mice Brain Glutamate Receptors.

Dysfunctional glutamate receptors (GluRs) have been implicated in neurological disorders and injuries. Hetero-tetrameric assemblies of different GluR subunits or splicing variants have distinct spatiotemporal expression patterns and pharmacological properties. Mass spectrometric imaging of GluRs-targeted small molecules is important for determining the regional preferences of these compounds. We report herein the development of a mass tag covalently bonded with glutamate or N-methyl-d-aspartate that functions as both an electron acceptor to generate mass spectrometric signals on irradiated (Bi2O3)0.07(CoO)0.03(ZnO)0.9 nanoparticles with the third harmonic (355 nm) of Nd3+:YAG laser and as the core component to target bilobed clamshell-like structures of GluRs. In this approach, different molecules produce the same tag ion. It provides a new avenue for quantitative assessment of spatial densities of different compounds, which cannot be achieved with well-established stable isotope labeling technique due to different ionization efficiency of different compounds. Various coexisting endogenous molecules are also simultaneously detected for investigation of overall physiological changes induced by these compounds. Because semiconductors do not generate background peaks, this method eliminates interferences from organic matrix materials that are used in regular MALDI (matrix assisted laser desorption ionization). The localized ionization provides high spatial resolution that can be down to sub-micrometers.