Bottom-up proteome analysis of E. coli using capillary zone electrophoresis-tandem mass spectrometry with an electrokinetic sheath-flow electrospray interface.

The Escherichia coli proteome was digested with trypsin and fractionated using SPE on a C18 SPE column. Seven fractions were collected and analyzed by CZE-ESI-MS/MS. The separation was performed in a 60-cm-long linear polyacrylamide-coated capillary with a 0.1% v/v formic acid separation buffer. An electrokinetic sheath-flow electrospray interface was used to couple the separation capillary with an Orbitrap-Velos operating in higher-energy collisional dissociation mode. Each CZE-ESI-MS/MS run lasted 50 min and total MS time was 350 min. A total of 23 706 peptide spectra matches, 4902 peptide IDs, and 871 protein group IDs were generated using MASCOT with false discovery rate less than 1% on the peptide level. The total mass spectrometer analysis time was less than 6 h, the sample identification rate (145 proteins/h) was more than two times higher than previous studies of the E. coli proteome, and the amount of sample consumed (<1 µg) was roughly fourfold less than previous studies. These results demonstrate that CZE is a useful tool for the bottom-up analysis of prokaryote proteomes.

Capillary electrophoresis with three-color fluorescence detection for the analysis of glycosphingolipid metabolism.

A capillary electrophoresis system with an ultrasensitive three-color laser-induced fluorescence detector was constructed for the simultaneous measurement of glycosphingolipids conjugated with a family of BODIPY fluorophores. The compounds were separated by capillary electrophoresis and detected by laser-induced fluorescence excited within a sheath-flow cuvette. Diode-pumped solid-state lasers operating at 473 nm and 532 nm, and a diode laser operating at 633 nm were used to excite glycosphingolipids tagged with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 fluorophores. Detection limits were 34 ± 1 molecules, 67 ± 7 molecules, and 36 ± 13 molecules of BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled glycosphingolipids. Separation efficiencies were typically one million theoretical plates. To test the ability of the system to analyze cellular contents in an in vitro biological model, differentiated PC12 cells were co-incubated with BODIPY-FL, BODIPY-TMR, and BODIPY-650/665 labeled lactosylceramide substrates. Cells were homogenized. The metabolic products originating from the glycosphingolipid substrates were simultaneously analyzed using the system.

Metabolic cytometry: capillary electrophoresis with two-color fluorescence detection for the simultaneous study of two glycosphingolipid metabolic pathways in single primary neurons.

Metabolic cytometry is a form of chemical cytometry wherein metabolic cascades are monitored in single cells. We report the first example of metabolic cytometry where two different metabolic pathways are simultaneously monitored. Glycolipid catabolism in primary rat cerebella neurons was probed by incubation with tetramethylrhodamine-labeled GM1 (GM1-TMR). Simultaneously, both catabolism and anabolism were probed by coincubation with BODIPY-FL labeled LacCer (LacCer-BODIPY-FL). In a metabolic cytometry experiment, single cells were incubated with substrate, washed, aspirated into a capillary, and lysed. The components were separated by capillary electrophoresis equipped with a two-spectral channel laser-induced fluorescence detector. One channel monitored fluorescence generated by the metabolic products produced from GM1-TMR and the other monitored the metabolic products produced from LacCer-BODIPY-FL. The metabolic products were identified by comparison with the mobility of a set of standards. The detection system produced at least 6 orders of magnitude dynamic range in each spectral channel with negligible spectral crosstalk. Detection limits were 1 zmol for BODIPY-FL and 500 ymol for tetramethylrhodamine standard solutions.

Single cell ganglioside catabolism in primary cerebellar neurons and glia.

Cell-to-cell heterogeneity in ganglioside catabolism was determined by profiling fluorescent tetramethylrhodamine-labeled GM1 (TMR-GM1) breakdown in individual primary neurons and glia from the rat cerebellum. Cells isolated from 5 to 6 day old rat cerebella were cultured for 7 days, and then incubated for 14 h with TMR-GM1. Intact cells were recovered from cultures by mild proteolysis, paraformaldehyde fixed, and subjected to single cell analysis. Individual cells were captured in a capillary, lysed, and the released single-cell contents analyzed by capillary electrophoresis with quantitative laser-induced fluorescent detection of metabolites. Non-neuronal cells on average took up much more exogenous TMR-GM1 than neuronal cells, and catabolized it more extensively. After 14 h of incubation, non-neuronal cells retained only 14% of the TMR products as GM1 and GM2, compared to >50% for neurons. On average, non-neuronal cells contained 74% of TMR-labeled product as TMR-ceramide, compared to only 42% for neurons. Non-neuronal cells retained seven times as much TMR-GM3 (7%) compared to neuronal cells (1%). To confirm the observed single cell metabolomics, we lysed and compared TMR-GM1 catabolic profiles from mixed neuron/glial cell cultures and from cultures depleted of non-neuronal cells by treatment with the antimitotic agent cytosine arabinoside. The lysed culture catabolic profiles were consistent with the average profiles of single neurons and glia. We conclude that the ultrasensitive analytic methods described accurately reflect single cell ganglioside catabolism in different cell populations from the brain.

Nine orders of magnitude dynamic range: picomolar to millimolar concentration measurement in capillary electrophoresis with laser induced fluorescence detection employing cascaded avalanche photodiode photon counters.

The dynamic range of capillary electrophoresis analysis is ultimately limited by molecular shot noise at low concentrations and by concentration-induced band broadening at high concentrations. We report a system that approaches these fundamental limits. A laser-induced fluorescence detector is reported that employs a cascade of four fiber-optic beam splitters connected in series to generate a primary signal and four attenuated signals, each monitored by a single-photon counting avalanche photodiode. Appropriate scaling of the signals from the five photodiodes produces a linear optical calibration curve for 5-carboxyl-tetramethylrhodamine from the concentration detection limit of 1 pM to the upper limit of 1 mM. Mass detection limits are 120 yoctomoles (70 molecules) injected into the instrument. The very-wide dynamic range instrument was used to study the metabolic products of the fluorescently labeled glycosphingolipid tetramethylrhodamine labeled GM1 (GM1-TMR) produced by single cells isolated from the rat cerebellum.

Monitoring the uptake of glycosphingolipids in Plasmodium falciparum-infected erythrocytes using both fluorescence microscopy and capillary electrophoresis with laser-induced fluorescence detection.

The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal 20 standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique.

Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells.

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

Preparation and electrophoretic separation of Bodipy-Fl-labeled glycosphingolipids.

Several glycosphingolipids were labeled with the fluorphore Bodipy-Fl and analyzed using capillary electrophoresis with laser-induced fluorescence detection. GM1-, LacCer-, and Cer-Bodipy-Fl were prepared through acylation using the N-hydroxysuccinimide ester of Bodipy-Fl. Several other glycosphingolipids including GT1a-, GD1a-, GM2-, GM3-, GD3-, and GlcCer-Bodipy-Fl were enzymatically synthesized. Micellar electrokinetic capillary chromatography with a TRIS/CHES/SDS/α-cyclodextrin buffer produced better separation than an established borate/deoxycholate/methyl-ß-cyclodextrin buffer. The nine Bodipy-Fl-labeled glycosphingolipid standards were separated in under 5 min, theoretical plate counts were between 640,000 and 740,000, and the limit of detection was approximately 3 pM or 240 ymol analyte injected onto the capillary.