We report a simple nanospray sheath-flow interface for capillary electrophoresis. This interface relies on electrokinetic flow to drive both the separation and the electrospray; no mechanical pump is used for the sheath flow. This system was interfaced with an LCQ mass spectrometer. The best results were observed with a 2-microm diameter emitter tip and a 1-mm spacing between the separation capillary tip and the emitter tip. Under these conditions, mass detection limits (3sigma) of 100 amol were obtained for insulin receptor fragment 1142-1153. The separation efficiency exceeded 200,000 plates for this compound. The relative standard deviation generated during continual infusion of a 50 microM solution of angiotensin II was 2% for the total ion count and 3% for the extracted ion count over a 40-min period. Finally, the interface was also demonstrated for negative ion mode.

Subattomole analysis of fluorescein isothiocyanate (FITC) derivatives of amino acids is accomplished by combining capillary zone electrophoresis for high-efficiency separation with laser-induced fluorescence for high-sensitivity detection. Concentration detection limits range from 5 x 10(-12) molar for alanine to 9 x 10(-11) molar for lysine, injected in the column; 9 x 10(-21) mole of alanine is contained within the approximately 1-nanoliter injection volume at the detection limit. The alanine detection limit corresponds to fewer than 6000 molecules injected onto the column and represents an improvement of four orders of magnitude in the state of the art for fluorescent detection of amino acids and an improvement of six orders of magnitude in the state of the art for the detection limit for isothiocyanate derivatives of amino acids.

Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics:  a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

We have reported a set of electrokinetically pumped sheath flow nanoelectrospray interfaces to couple capillary zone electrophoresis with mass spectrometry. A separation capillary is threaded through a cross into a glass emitter. A side arm provides fluidic contact with a sheath buffer reservoir that is connected to a power-supply. The potential applied to the sheath buffer drives electro-osmosis in the emitter to pump the sheath fluid at nanoliter/minute rates. Our first generation interface placed a flat-tipped capillary in the emitter. Sensitivity was inversely related to orifice size and to the distance from the capillary tip to the emitter orifice. A second generation interface used a capillary with an etched tip that allowed the capillary exit to approach within a few hundred micrometers of the emitter orifice, resulting in a significant increase in sensitivity. In both the first and second-generation interfaces, the emitter diameter was typically 8-μm; these narrow orifices were susceptible to plugging and tended to have limited lifetime. We now report a third-generation interface that employs a larger diameter emitter orifice with very short distance between the capillary tip and the emitter orifice. This modified interface is much more robust and produces much longer lifetime than our earlier designs with no loss in sensitivity. We evaluated the third-generation interface for a 5,000-min (127 runs, 3.5 days) repetitive analysis of bovine serum albumin digest using an uncoated capillary. We observed a 10% relative standard deviation in peak area, an average of 160,000 theoretical plates, and very low carry-over (much less than 1%). We employed a linear-polyacrylamide (LPA) coated capillary for single-shot, bottom-up proteomic analysis of 300 ng of Xenopus laevis fertilized egg proteome digest, and identified 1,249 protein groups and 4,038 peptides in a 110 min separation using an LTQ-Orbitrap Velos mass spectrometer; peak capacity was ~330. The proteome dataset using this third generation interface based CZE-MS/MS is similar in size to that generated using a commercial ultra-performance liquid chromatographic analysis of the same sample with the same mass spectrometer and similar analysis time.

Femtogram proteomics: We report an ultrasensitive capillary zone electrophoresis-mass spectrometry system based on an improved nanospray interface. This system is used for analysis of picogram to femtogram amounts of E. coli digests. Over 100 proteins were identified based on tandem mass spectra from 16 pg digests; over 60 proteins were identified from 400 fg digests based on accurate mass and time tags in 10 min.

Capillary electrophoresis is coupled with a single molecule detector based on laser-induced fluorescence. Individual molecules migrating from the capillary are detected and counted with 50% efficiency. Injection of 30 000 analyte molecules generates a reproducible peak consisting of at least five components. However, injection of 3000 or fewer molecules leads to a noisy and irreproducible peak. Monte Carlo simulation demonstrates that this irreproducibility results from molecular shot noise or stochastic fluctuations in the number of injected molecules. The model predicts that the relative standard deviations of peak area, peak center, and peak width are inversely proportional to the square root of the number of injected molecules. At least 104 analyte molecules (17 zmol) are required to define peak area and width with 1% relative precision. Fluctuation in the number of molecules taken for chemical analysis is a fundamental and irreducible source of uncertainty.

Capillary electrophoresis is ideally suited to chemical analysis of individual cells. Small mammalian somatic cells (approximately 15 microns in diameter) can be analyzed by injecting the intact cell into a capillary, lysing the cell, separating and detecting the cellular components, and reconditioning the capillary prior to the next injection. In this paper, we report on technical improvements to single-cell analysis. We designed an inexpensive multipurpose single-cell injector that facilitates the following: (i) monitoring of injection, (ii) reproducible pressure- or electrokinetic-driven injection of the cell, (iii) complete cell lysis by SDS within 30 s of injection, and (iv) pressure-driven capillary reconditioning. Furthermore, we report on the analysis of glycosylation and glycolysis in single human carcinoma cells (HT29 cell line). The reliability and quality of the analysis is confirmed by comparing electropherograms from single cells and those from purified cell extracts.

We demonstrate the use of capillary zone electrophoresis with an electrokinetically pumped sheath-flow electrospray interface for the analysis of a tryptic digest of a sample of intermediate protein complexity, the secreted protein fraction of Mycobacterium marinum. For electrophoretic analysis, 11 fractions were generated from the sample using reversed phase liquid chromatography; each fraction was analyzed by CZE-ESI-MS/MS, and 334 peptides corresponding to 140 proteins were identified in 165 min of mass spectrometer time at 95% confidence (FDR<0.15%). In comparison, 388 peptides corresponding to 134 proteins were identified in 180 min of mass spectrometer time by triplicate UPLC-ESI-MS/MS analysis each using 250 ng of the unfractionated peptide mixture at 95% confidence (FDR<0.15%). 62% of peptides identified in CZE-ESI-MS/MS and 67% in UPLC-ESI-MS/MS were unique. CZE-ESI-MS/MS favored basic and hydrophilic peptides with low molecular mass. Combining the two data sets increased the number of unique peptides by 53%. Our approach identified more than twice as many proteins as the previous record for CE proteome analysis. CE-ESI-MS/MS is a useful tool for the analysis of proteome samples of intermediate complexity.