Attomole protein characterization by capillary electrophoresis-mass spectrometry.

Electrospray ionization with an ultralow flow rate (30 kilodaltons) at a resolving power of approximately 60,000 for injections of 0.7 x 10(-18) to 3 x 10(-18) mole of 8- to 29-kilodalton proteins with errors of <1 dalton in molecular mass. Using a crude isolate from human blood, a value of 28,780.6 daltons (calculated, 28,780.4 daltons) was measured for carbonic anhydrase, representing 1 percent by weight of the protein in a single red blood cell. Dissociation of molecular ions from 9 x 10(-18) mole of carbonic anhydrase gave nine sequence-specific fragment ions, more data than required for unique retrieval of this enzyme from the protein database.

Long-lived metallized tips for nanoliter electrospray mass spectrometry.

Sheathless electrospray at nL/min flow rates combined with Fourier-transform mass spectrometry has made possible high resolving power (>50,000) mass spectra of subattomole samples of >8 kDa proteins separated by capillary electrophoresis (Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science, 1996, 273, 1199-1202). However, for this new method the mechanical stability of the thin (35 to 100 nm) gold film electrodes has limited tip lifetime to 15 to 30 min. A technique for SiO x coating of the gold is described that provides a steady ion current (±10 pA) for 1 to 2 h, even with arcs or interruptions of the electrospray voltage.

Quantitative imaging ion microscopy: a short review.

A short review of recent efforts being made in the quantification of images in ion microscopy is given. Special aspects of instrumentation, detection and acquisition, which are unique to direct imaging secondary ion mass spectrometry, are discussed in relation to the successful application of traditional empirical quantification schemes. Application of such quantification schemes requires proper sample preparation, standardization, analysis, and quite often, special techniques in image processing and the correlation of ion microscopy with other microscopies. Quantification within this technique is a difficult goal which can only be realized if the analyst pays strict attention to every step of the analytical process.

Sampling error in small-bore sheathless capillary electrophoresis/electrospray-ionization mass spectrometry.

Two previously unreported sources of systematic error in electrokinetic injection caused by induced (hydrodynamic) flow in sheathless capillary electrophoresis/electrospray have been characterized for an interface constructed from 5 microns i.d. capillary column(s) with a 2-5 microns i.d. electrospray tip(s). The tip of a sheathless interface is usually exposed to the atmosphere, resulting in evaporation of buffer solvent and inducing flow inside the column; such flow can cause a significant underestimation of injection size for quantitative electrokinetic sampling by as much as 50%. This bias can be eliminated during the injection process by temporarily immersing the tip in buffer solvent. The second source of hydrodynamic flow results from the electrostatic pull exerted by the electrospray process on the buffer, reducing migration times. Reduction in migration times causes an overestimation of sampling volume if there are no electrospray processes during the sampling event. The magnitude of this effect is a complex function of electrophoretic and electrospray parameters and has been measured to be as high as 15% for 5 microns i.d. capillaries. The possible deterioration of electrophoretic resolution caused by these processes is not yet clarified.

Parameter control, characterization, and optimization in the fabrication of optical fiber near-field probes.

Tip diameter and transmission efficiency of a visible-wavelength near-field optic probe determine both the lateral spatial resolution and experimental utility of the near-field scanning optical microscope. The commonly used tip fabrication technique, laser-heated pulling of fused-silica optical fiber followed by aperture formation through aluminization, is a complex process governed by a large number of parameters. An extensive study of the pulling parameter space has revealed a time-dependent functionality between the various pulling parameters dominated by a photon-based heating mechanism. The photon-based heat source results in a temperature and viscosity dependence that is a complex function of time and fiber diameter. Changing the taper of the optical probe can affect transmission efficiency by an order of magnitude or more.

Attomole-sensitivity electrospray source for large-molecule mass spectrometry.

Full mass spectra of high resolving power are obtained from 0.2 nL sample volumes of large (> 10 kDa) nucleotides and proteins using a new electrospray ionization (ESI) system combined with Fourier transform mass spectrometry. The ESI needles are fabricated by laser-heated pulling of fused-silica tubing (5-20 microns i.d.), followed by chemical etching and surface metalization. Total analyte loaded at the instrument of 8.6 fmol and 216 amol produces signal-to-noise ratios of 400:1 and 60:1, respectively, and resolving power of > 10(5) for full mass spectra, while the total amount of material consumed is approximately 150 and 10 amol, respectively.