Liquid chromatography/mass spectrometry (LC/MS) is increasingly being used for metabolic analysis due in part to its widespread availability and compatibility with biological samples. This invention provides ionizable, isotopic labeling reagents and labeling reaction products, answering the current need for relative quantification using mass spectrometry.
Absolute quantification of an analyte relies upon the addition of an internal standard differing only in its isotopic form. However, it is impractical to add an isotopic standard for every compound when performing more comprehensive metabolic profiling. Relative quantification between samples is more amenable to analyzing broad classes of compounds, and it often provides very useful biological information.
Heavy and light isotopic forms of methylacetimidate have been synthesized and used as labeling reagents for quantification of amine-containing molecules, such as biological samples. Heavy and light isotopic forms of formaldehyde and cholamine have also been synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as these found in biological samples. Advantageously, the labeled end-products are positively charged under normal acidic conditions involving conventional liquid chromatography mass spectrometry (LC/MS) applications.
This invention improves the precision of relative quantification by minimizing or negating errors associated with run-to-run irreproducibility. Such errors can arise from variations in mass spectrometric detection sensitivity, such as those caused by ionization suppression in electrospray, or from retention time differences between runs. The isotopic pair of labeled compounds co-elute within a single run. Therefore, they have identical retention times and are electrosprayed from identical solution conditions.
Embodiments of nanostructured, multilayered metal-dielectric particles suitable for use as Raman spectroscopic probes, as well as methods of designing, making and using such multilayered nanoparticles, and kits including the multilayered nanoparticles. The multilayered nanoparticles include alternating metal and dielectric layers and an outer dielectric shell. One or more of the dielectric layers may include a plurality of reporter molecules. Embodiments of the multilayered nanoparticles are suitable for detecting target analytes in a sample. Some embodiments of the multilayered nanoparticles are suitable for use in multiplexed assays, including assays for multiple target analytes having differing concentrations.