6 research outputs found
Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry
With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis
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Electrospray ionization mass spectrometry as a tool to study transferrin interactions with metals of biological interest
Transferrins constitute a class of metalloproteins that sequester and transport iron in vertebrates. Understanding the metal-binding properties of transferrin is essential for studies of iron metabolism and metabolic disorders. However, dynamics of these systems is poorly understood, since they are not amendable to analysis by NMR due to the high spin of the metal ion and the large molecular weight of the protein (35–80 kDa). In this work we successfully developed and utilized an ESI MS based approach to study metal-release properties of the human serum transferrin N-lobe (hTF/2N), as well as the intact transferrin from human (hTF). Both composition and conformation of a protein complex was monitored under a variety of conditions that are designed to mimic extracellular and endosomal environments. It was determined that iron dissociation occurs in vitro at typical endosomal pH 5.5 when physiological chelators (e.g. citrate) are present at endogenous levels. In the absence of such chelating agents, detectable iron release occurs at much lower pH (\u3c4.5) and is accompanied by unfolding of the protein. ESI MS data also indicated the transient nature of the anion in the holo-protein. Furthermore, studies of hTF revealed that the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. Another surprising discovery was that the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. These results provide strong support for the earlier suggestions that hTF interacts with its receptor (TfR) primarily through the C-lobe both at the cell surface and inside the endosome. We also successfully extended the above ESI MS approach to characterize interaction of intact transferrin (80kDa) with metals of medicinal relevance, such as In3+ and Bi3+. The results of our studies have revealed that indium-hTF complex exhibits the same pattern of conformational and metal release properties as iron-hTF. Because of conformational similarities between indium-hTF and iron-hTF, it is possible that indium-hTF is recognized by transferrin receptor protein (TfR) at the cell surface and is transported into the cells by the same mechanism of receptor mediated endocytosis as iron. On the other hand the results of our bismuth studies suggest that hTF adopts a semi-open conformation upon bismuth uptake. This conformation is different from that of iron-hTF, therefore it is likely that bismuth transport does not occur via the similar mechanism