Capillary electrophoresis-mass spectrometry methods for tryptic peptide mapping of therapeutic antibodies.

Tryptic peptide mapping is routinely used in the biotech industry to confirm primary sequence, cell line stability, and to analyze posttranslational modifications. Peptide analysis is generally done by reverse phase liquid chromatography with UV or mass spectrometric detection. This method provides excellent resolution and sequence coverage. However, traditional methods are slow, and generally cannot detect small, hydrophilic peptides due to coelution with the column void volume. In this work, complementary CE-MS peptide analysis methods have been developed. The analyses are performed on a traditional CE-MS instrument with a sheath interface, and also on a novel sheathless interface that promises improved resolution and limit of detection. The methods were performed on a tryptic digest of a therapeutic monoclonal antibody for which LC-MS detects 97% sequence coverage. The 3% not covered consists of 11 peptides containing three amino acids or fewer, including two in the critical complementarity binding domain. Without further processing, the same tryptic digest was analyzed by CE-MS. Separation and detection of the 11 small peptides was achieved on CE-MS systems with both interfaces. The sheathless system produced better peak capacity and gave mass spectra with significantly less noise, while the sheath system proved to have better repeatability.

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.

CE-MALDI interface based on inkjet technology.

An ink jet printer valve and a nozzle were used to deliver matrix and sample from an electrophoresis capillary onto a MALDI plate. The system was evaluated by the separation of a set of standard peptides. That separation generated up to 40 000 theoretical plates in less than 3 min. Detection limits were 500 amol for an ABI TOF-TOF instrument and 2 fmol for an ABI Q-TOF instrument. Over 70% coverage was obtained for the tryptic digest of alpha-lactalbumin in less than 2.5 min.

Interface of an array of five capillaries with an array of one-nanoliter wells for high-resolution electrophoretic analysis as an approach to high-throughput chemical cytometry.

We report a system that allows the simultaneous aspiration of one or more cells into each of five capillaries for electrophoresis analysis. A glass wafer was etched to create an array of 1-nL wells. The glass was treated with poly(2-hydroxyethyl methacrylate) to control cell adherence. A suspension of formalin-fixed cells was placed on the surface, and cells were allowed to settle. The concentration of cells and the settling time were chosen so that there was, on average, one cell per well. Next, an array of five capillaries was placed so that the tip of each capillary was in contact with a single well. A pulse of vacuum was applied to the distal end of the capillaries to aspirate the content of each well into a capillary. Next, the tips of the capillaries were placed in running buffer and potential was applied. The cells lysed upon contact with the running buffer, and fluorescent components were detected at the distal end of the capillaries by laser-induced fluorescence. The electrophoretic separation efficiency was outstanding, generating over 750,000 theoretical plates (1,800,000 plates/m). In this example, AtT-20 cells were used that had been treated with TMR-G(M1). The cells were allowed to metabolize this substrate into a series of products before the cells were fixed. The number of cells found in each well was estimated visually under the microscope and was described by a Poisson distribution with mean of 0.98 cell/well. This system provides an approach to high-throughput chemical cytometry.

Synthesis of reference standards to enable single cell metabolomic studies of tetramethylrhodamine-labeled ganglioside GM1.

Ganglioside GM1 and its seven potential catabolic products: asialo-GM1, GM2, asialo-GM2, GM3, Lac-Cer, Glc-Cer and Cer, were labeled with tetramethylrhodamine (TMR) to permit ultra-sensitive analysis using laser-induced fluorescence (LIF) detection. The preparation involved acylation of the homogenous C(18)lyso-forms of GM1, Lac-Cer, Glc-Cer and Cer with the N-hydroxysuccinimide ester of a beta-alanine-tethered 6-TMR derivative, followed by conversion of these labeled products using galactosidase, sialidase, and sialyltransferase enzymes. The TMR-glycolipid analogs produced are detectable on TLC down to the 1 ng level by the naked eye. All eight compounds could be separated within 4 min in capillary electrophoresis where they could be detected at the zeptomole (ca. 1000 molecule) level using LIF.

Six orders of magnitude dynamic range in capillary electrophoresis with ultrasensitive laser-induced fluorescence detection.

An ultrasensitive laser-induced fluorescence detector was used with capillary electrophoresis for the study of 5-carboxy-tetramethylrhodamine. The raw signal from the detector provided roughly three orders of magnitude dynamic range. The signal saturated at high analyte concentrations due to the dead time associated with the single-photon counting avalanche photodiode employed in the detector. The signal can be corrected for the detector dead time, providing an additional order of magnitude dynamic range. To further increase dynamic range, two fiber-optic beam-splitters were cascaded to generate a primary signal and two attenuated signals, each monitored by a single-photon counting avalanche photodiode. The combined signals from the three photodiodes are reasonably linear from the concentration detection limit of 3 pM to 10 microM, the maximum concentration investigated, a range of 3,000,000. Mass detection limits were 150 yoctomoles injected onto the capillary.

Chemical cytometry: fluorescence-based single-cell analysis.

Cytometry deals with the analysis of the composition of single cells. Flow and image cytometry employ antibody-based stains to characterize a handful of components in single cells. Chemical cytometry, in contrast, employs a suite of powerful analytical tools to characterize a large number of components. Tools have been developed to characterize nucleic acids, proteins, and metabolites in single cells. Whereas nucleic acid analysis employs powerful polymerase chain reaction-based amplification techniques, protein and metabolite analysis tends to employ capillary electrophoresis separation and ultrasensitive laser-induced fluorescence detection. It is now possible to detect yoctomole amounts of many analytes in single cells.

Metabolic cytometry. Glycosphingolipid metabolism in single cells.

Glycosphingolipids are found on all vertebrate cells and constitute major cell surface determinants on all nerve cells, where they contribute to cellular diversity and function. We report a method for the analysis of glycosphingolipid metabolism in single cells. The ganglioside GM1 was tagged with the fluorescent dye tetramethylrhodamine. This labeled compound was taken up and metabolized by a culture of pituitary tumor (AtT-20) cells. After 50 h, the cells were formalin fixed. Cells were aspirated into a fused-silica capillary and lysed, and components were separated by capillary electrophoresis with a laser-induced fluorescence detector. All metabolic products that retained the fluorescent dye could be detected at the low-zeptomole level. A total of 54 AtT-20 cells were individually analyzed using this procedure. The electrophoretic profiles were remarkably reproducible, which facilitated identification of components based on the migration time of fluorescently labeled standards. Eleven components were detected, and the average peak height of these components spanned more than 2 orders of magnitude, so that trace metabolites can be detected in the presence of abundant components. The most highly abundant components generated 10% relative standard deviation in normalized abundance. The average cell took up roughly 2 amol (10(6) copies) of the labeled substrate. This method allows determination of cell-to-cell diversity and regulation of glycosphingolipid metabolism.

Yoctomole analysis of ganglioside metabolism in PC12 cellular homogenates.

We report an ultrasensitive method for the analysis of glycosphingolipid catabolism. The substrate G(M1) and the set of seven metabolites into which it can be degraded (G(A1), G(M2), G(A2), G(M3), LacCer, GlcCer, and Cer) were labeled with the highly fluorescent dye tetramethylrhodamine. CE with LIF detection was used to assay these compounds with 150 +/- 80 yoctomole mass (1 ymol = 10(-24) mol = 0.6 copies) detection limits and 5 +/- 3 pM concentration detection limits. An alignment algorithm based on migration of two components was employed to correct for drift in the separation. The within-day and between-day precision in peak height was 20%, in peak width 15%, and in adjusted migration time 0.03%. After normalization to total sample injected, the RSD in peak height reduced to 2-6%, which approaches the limit set by molecular shot noise in the number of molecules taken for analysis. PC12 cells were incubated with the labeled G(M1). Fluorescent microscopy demonstrated uptake by the cells. CE was used to separate a cellular homogenate prepared from these cells. A set of peaks was observed, which were tentatively identified based on comigration with the standards. Roughly 120 pL of homogenate was injected, which contained a total of 150 zmol of labeled substrate and products. Metabolite that preserves the fluorescent label can be detected at the yoctomole level, which should allow characterization of this metabolic pathway in single cells.

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection.

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.