Gary A. Valaskovic

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 protein characterization by capillary electrophoresis-mass spectrometry

Electrospray ionization with an ultralow flow rate (≤4 nanoliters per minute) was used to directly couple capillary electrophoresis with tandem mass spectrometry for the analysis and identification of biomolecules in mixtures. A Fourier transform mass spectrometer provided full spectra (>30 kilodaltons) at a resolving power of ≈60,000 for injections of 0.7 × 10−18 to 3 × 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 × 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.

Size-Sorting Combined with Improved Nanocapillary Liquid Chromatography-Mass Spectrometry for Identification of Intact Proteins up to 80 kDa

Despite the availability of ultra-high-resolution mass spectrometers, methods for separation and detection of intact proteins for proteome-scale analyses are still in a developmental phase. Here we report robust protocols for online LC-MS to drive high-throughput top-down proteomics in a fashion similar to that of bottom-up proteomics. Comparative work on protein standards showed that a polymeric stationary phase led to superior sensitivity over a silica-based medium in reversed-phase nanocapillary LC, with detection of proteins >50 kDa routinely accomplished in the linear ion trap of a hybrid Fourier transform mass spectrometer. Protein identification was enabled by nozzle-skimmer dissociation and detection of fragment ions with <10 ppm mass accuracy for highly specific database searching using tailored software. This overall approach led to identification of proteins up to 80 kDa, with 10-60 proteins identified in single LC-MS runs of samples from yeast and human cell lines prefractionated by their molecular mass using a gel-based sieving system.

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 SiOx 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.

Analysis of Samples Stored as Individual Plugs in a Capillary by Electrospray Ionization Mass Spectrometry

Droplets or plugs within multiphase microfluidic systems have rapidly gained interest as a way to manipulate samples and chemical reactions on the femtoliter to microliter scale. Chemical analysis of the plugs remains a challenge. We have discovered that nanoliter plugs of sample separated by air or oil can be analyzed by electrospray ionization mass spectrometry when pumped directly into a fused silica nanospray emitter tip. Using leucine-enkephalin in methanol and 1% acetic acid in water (50:50 v:v) as a model sample, we found carry-over between plugs was <0.1% and relative standard deviation of signal for a series of plugs was 3%. Detection limits were 1 nM. Sample analysis rates of 0.8 Hz were achieved by pumping 13 nL samples separated by 3 mm long air gaps in a 75 microm inner diameter tube. Analysis rates were limited by the scan time of the ion trap mass spectrometer. The system provides a robust, rapid, and information-rich method for chemical analysis of sample in segmented flow systems.

Effects of Column and Gradient Lengths on Peak Capacity and Peptide Identification in Nanoflow LC-MS/MS of Complex Proteomic Samples

AbstractReversed-phase liquid chromatography is the most commonly used separation method for shotgun proteomics. Nanoflow chromatography has emerged as the preferred chromatography method for its increased sensitivity and separation. Despite its common use, there are a wide range of parameters and conditions used across research groups. These parameters have an effect on the quality of the chromatographic separation, which is critical to maximizing the number of peptide identifications and minimizing ion suppression. Here we examined the relationship between column lengths, gradient lengths, peptide identifications, and peptide peak capacity. We found that while longer column and gradient lengths generally increase peptide identifications, the degree of improvement is dependent on both parameters and is diminished at longer column and gradients. Peak capacity, in comparison, showed a more linear increase with column and gradient lengths. We discuss the discrepancy between these two results and some of the considerations that should be taken into account when deciding on the chromatographic conditions for a proteomics experiment. FigureThe effects of column and gradient lengths on the performance of nanoflow LC-MS/MS is examined in complex proteomic samples.

Image contrast of dielectric specimens in transmission mode near-field scanning optical microscopy: imaging properties and tip artefacts

Near‐field scanning optical microscopy (NSOM) is a scanned probe technique utilizing a subwavelength‐sized light source for high‐resolution imaging of surfaces. Although NSOM has the potential to exploit and extend the experimental utility of the modern light microscope, the interpretation of image contrast is not straightforward. In near‐field microscopy the illumination intensity of the source (probe) is not a constant value, rather it is a function of the probe–sample electronic environment. A number of dielectric specimens have been studied by NSOM to elucidate the contrast role of specimen type, topography and crystallinity; a summary of metallic specimen observations is presented for comparative purposes. Near‐field image contrast is found to be a result of lateral changes in optical density and edge scattering for specimens with little sample topography. For surfaces with considerable topography the contributions of topographic (Z) axis contrast to lateral (X,Y) changes in optical density have been characterized. Selected near‐field probes have also been shown to exhibit a variety of unusual contrast artefacts. Thorough study of polarization contrast, optical edge (scattering) contrast, as well as molecular orientation in crystalline specimens, can be used to distinguish lateral contrast from topographic components. In a few cases Fourier filtering can be successfully applied to separate the topographic and lateral contrast components.

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.

Online nanoelectrospray/high-field asymmetric waveform ion mobility spectrometry as a potential tool for discovery pharmaceutical bioanalysis.

Nanoelectrospray ionization (nESI) coupled online with high-field asymmetric waveform ion mobility spectrometry (FAIMS) for small molecule analysis in a discovery pharmaceutical setting was examined. A conventional capillary pump, autosampler and nESI source were used to introduce samples directly into the FAIMS device. The FAIMS device was used to separate gas-phase ions on a timescale that was compatible with the mass spectrometer. The capability of the nESI-FAIMS combination to efficiently remove metabolite interferences from the parent drug, and reduce ion suppression effects, was demonstrated. On average, 85% of the signal intensity obtained from a neat sample was preserved in the extracted plasma samples. Standard curves were prepared for several compounds. Linearity was obtained over approximately 3 to 4 orders of magnitude. Comparison of results from nESI-FAIMS with those from conventional LC/MS for a mouse pharmacokinetic study yielded concentration values differing by no more than 30%.

Biological near-field scanning optical microscopy: instrumentation and sample issues for shear-force feedback

Abstract The current technology of near-field microscopy is limited in two important aspects: photon throughput and scan rate. In this paper we will consider the second limitation only. Furthermore, we will consider it as a function of probe-sample feedback response time and not one of low light level imaging; i.e. scan times are not limited by any need to integrate the optical signal. Our considerations will focus on shear-force feedback response times and how it places a limit on the type of biological investigations that can be accomplished.