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Phosphoinositide (PPI) lipids are a crucial class of low abundance signaling molecules that regulate many processes within cells. Methods that enable simultaneous detection of all PPI lipid species provide a wholistic snapshot of the PPI profile of cells, which is critical for probing PPI biology. Here we describe a method for the simultaneous measurement of cellular PPI levels by metabolically labeling yeast or mammalian cells with myo-(3)H-inositol, extracting radiolabeled glycerophosphoinositides, and separating lipid species on an anion exchange column via HPLC.

An HPLC system has been optimized for the rapid resolution and quantitation of IgG in mouse ascites fluids, making use of newly developed HPLC columns (MA7P). Chromatography using MA7P columns is characterized by remarkably narrow band widths and very short retention times. The HPLC system is capable of analyzing the IgG content of a typical ascites fluid with a cycle-to-cycle time of under 5 minutes, regardless of IgG subtype. Separation times under 1 minute are possible. Recoveries of 100 μg injections of IgG and other proteins are quantitative. Using a UV monitor, the system was optimized to generate a linear plot of peak area vs. amount of IgG injected from 100 ng to 3 μg per injection. These properties make the MA7P column very useful for both analytical and micropreparative applications. Ascites fluids from eleven different IgG1-producing hybridomas were analyzed. The IgG concentrations in those eleven ascites varied considerably. Retention times of the IgG varied slightly but significantly from hybridoma to hybridoma. Some monoclonals produce heterogeneous IgG. This heterogeneity can be detected by chromatography using MA7P columns.