Probe Electrospray Research Articles

Direct Analysis of Reversed-Phase High-Performance Thin Layer Chromatography Separated Tryptic Protein Digests Using a Liquid Microjunction Surface Sampling Probe/Electrospray Ionization Mass Spectrometry System

The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

Detection of biomolecules from solutions with high concentration of salts using probe electrospray and nano-electrospray ionization mass spectrometry

Probe electrospray ionization (PESI) is a recently developed ionization technique which uses a solid needle or wire as sampling probe and ESI emitter instead of capillary. PESI is free from the clogging problem and it has high tolerance to salts and urea. We present herein a comparative study of the probe electrospray ionization (PESI) and nano-electrospray ionization (nano-ESI) for the measurement of biomolecules in the sample solutions with high concentrations of salts and urea. Our results show that PESI could provide equivalent ionization performance with nano-ESI, and in certain cases it could be superior to nano-ESI for the samples with high concentration (>100 mM) of salts and urea. Therefore, PESI can be useful for the direct analysis of cell/tissue extracts and protein digestion without or reduced procedures in sample purification.

Scanning Mass Spectrometry Probe: A Scanning Probe Electrospray Ion Source for Imaging Mass Spectrometry of Submerged Interfaces and Transient Events in Solution

The scanning mass spectrometry (SMS) probe is a new electrospray ion source. Motivated by the need for untargeted chemical imaging of dynamic events in solution, we have exploited an approach to electrospray ionization (ESI) that allows continuous sampling from a highly localized volume (approximately picoliters) in a liquid environment, softly ionizes molecules in the sample to render them amenable for mass spectrometric analysis, and sends the ions to the mass spectrometer. The key underlying concepts for our approach are (1) treating the electrospray capillary inlet as a chemical scanning probe and (2) locating the electrospray point as close as possible to the sampling point, thus providing the shortest response time possible. This approach enables chemical monitoring or imaging of submerged interfaces, providing access to details of spatial heterogeneity and temporal changes within liquid samples. It also permits direct access to liquid/ liquid interfaces for ESI-MS analysis. In this letter we report the first demonstrations of these capabilities of the SMS probe and describe some of the probe's basic characteristics.

Direct Profiling of Phytochemicals in Tulip Tissues and In Vivo Monitoring of the Change of Carbohydrate Content in Tulip Bulbs by Probe Electrospray Ionization Mass Spectrometry

Probe electrospray ionization (PESI) is a recently developed ESI-based ionization technique which generates electrospray from the tip of a solid needle. In this study, we have applied PESI interfaced with a time of flight mass spectrometer (TOF-MS) for direct profiling of phytochemicals in a section of a tulip bulb in different regions, including basal plate, outer and inner rims of scale, flower bud and foliage leaves. Different parts of tulip petals and leaves have also been investigated. Carbohydrates, amino acids and other phytochemicals were detected. A series of in vivo PESI-MS experiments were carried out on the second outermost scales of four living tulip bulbs to monitoring the change of carbohydrate content during the first week of initial growth. The breakdown of carbohydrates was observed which was in accordance with previous reports achieved by other techniques. This study has indicated that PESI-MS can be used for rapid and direct analysis of phytochemicals in living biological systems with advantages of low sample consumption and little sample preparation. Therefore, PESI-MS can be a new choice for direct analysis/profiling of bioactive compounds or monitoring metabolic changes in living biological systems.

Ambient imaging mass spectrometry by electrospray ionization using solid needle as sampling probe

Although being an atmospheric pressure ion source, electrospray ionization (ESI) has rarely been used directly for ambient imaging mass spectrometry because the sample has to be introduced as liquid solution through the capillary. Instead of capillary, probe electrospray ionization (PESI), which has been developed recently, uses a solid needle as the sampling probe, as well as the electrospray emitter, and has been applied not only for liquid solutions but also for the direct sampling on wet samples. Biological tissues are composed of cells that contain 70-90% water, and when the surface is probed by the needle tip, the biological fluid adhering to the needle can be electrosprayed directly or assisted by additional solvent added onto the needle surface. Here, we demonstrate ambient imaging mass spectrometry of mouse brain section using PESI, incorporated with an auxiliary heated capillary sprayer. The solvent vapor generated from the sprayer condensed on the needle tip, re-dissolving the adhered sample, and at the same time, providing an indirect means for needle cleaning. The histological sections were prepared by fixation using paraformaldehyde, and the spatial analysis was automated by maintaining an equal sampling depth into the sample in addition to raster scan. Phospholipids and galactosylceramides were readily detected from the mouse brain section in the positive ion mode, and were mapped with 60 microm lateral resolution to form mass spectrometric images.

Physical properties of the probe electrospray ionization (PESI) needle applied to the biological samples

Probe electrospray ionization (PESI) is a recently developed technique that uses a fine solid needle as a probe for sampling biological materials. In this study, we quantified the volume of liquid sample picked up by the solid needle with the tip diameter of approximately 700 nm and the apex angle of approximately 60 degrees. The amounts of low-viscosity samples (rat urine) loaded on the tip of the needle by a single stroke were 0.35 +/- 0.09 pl. Interestingly, the amount of liquid adhered to the tip did not significantly depend on the protein concentration, but viscosity and surface tension of the sample. Under these conditions, we successfully obtained mass spectra for each biological sample.

Direct and Real-Time Surface Analysis and Imaging of Biological Samples by Probe Electrospray

Recently, we have developed the probe electrospray ionization (PESI) that uses the solid needle. In this system, the probe needle moves up and down along the vertical axis by a motor-driven system. At the highest position of the probe needle, electrospray is generated by applying the high voltage. In this study, we used PESI directly to the biological samples such as urine, mouse brain, mouse liver, salmon egg, artificial salmon egg, and fruits (orange, banana, etc.). Strong ion signals for almost all the samples were obtained. The amount of liquid sample picked up by the needle is as small as pL or less, making the PESI the most promising non-invasive technique for detecting biomolecules in a living systems such as cells. Therefore, PESI may be useful as versatile and ready-to-use semi-on line analyses in the fields of surface chemistry, medicine, pharmaceutical, agriculture, food science, etc.

Application of Probe Electrospray Ionization for Biological Sample Measurements

Electrospray has been used as one of the most powerful techniques for the analysis of biological samples. Recently, we have developed the probe electrospray (PESI) (modified version of the conventional electrospray) that uses the fine solid needle. In this method, the probe needle moves up and down along the vertical axis by a motor-driven system. At the highest position of the probe needle, electrospray is generated by applying a high voltage. In this study, we used PESI directly to the biological samples such as human milk, bovine milk, beverages, mouse brains, flower petals, plant leaves, bananas, tulip bulbs, etc. Strong ion signals for almost all samples were obtained. The amount of liquid sample picked up by the needle is as small as a few pL or less, making the PESI one of the most promising less-invasive analysis and imaging for biological samples. PESI may be useful as versatile and ready-to-use semi-on line analyses in the fields of agriculture, food science, medicine, pharmaceutical, etc. An example of imaging for mouse brain is demonstrated.

Characteristics of Probe Electrospray Generated from a Solid Needle

Probe electrospray ionization (PESI) has recently been developed, in which the electrospray was generated from a solid needle instead of by using a capillary. In this paper, the characteristics of probe electrospray ionization were studied based on the measurement of spray current, optical microscopy, and PESI mass spectrometry. In the experiment, the solid needle was moved up and down a vertical axis, and a small amount of sample was repeatedly loaded to the needle when the tip of the needle touched the surface of the liquid sample at the lowest position. After the application of high voltage, a liquid droplet was formed on the tip of the solid needle probe, with its size was determined by the size of the needle tip. The liquid flow rate to the tip, as indicated by the spray current, depends on the voltage applied to the needle as well as the loaded liquid amount. Stable electrospray can be maintained until the total consumption of liquid sample. The kilohertz current pulsation takes place in the case of overloading the sample to the needle. The influences of the applied voltage and the liquid flow rate on the PESI mass spectra were also examined.

Application of probe electrospray to direct ambient analysis of biological samples

Recently, we have developed probe electrospray ionization (PESI) that uses a solid needle. In this system, the probe needle moves up and down along the vertical axis by a motor-driven system. At the highest position of the probe needle, electrospray is generated by applying a high voltage. In this study, we applied PESI directly to biological samples such as urine, mouse brain, mouse liver, salmon egg, and fruits (orange, banana, etc.). Strong ion signals for almost all the samples were obtained. The amount of liquid sample picked up by the needle is as small as pL or less, making PESI a promising non-invasive technique for detecting biomolecules in living systems such as cells. Therefore, PESI may be useful as a versatile and ready-to-use semi-online analytical tool in the fields of medicine, pharmaceuticals, agriculture, food science, etc.

Liquid microjunction surface sampling probe electrospray mass spectrometry for detection of drugs and metabolites in thin tissue sections

A self-aspirating, liquid microjunction surface sampling probe/electrospray emitter mass spectrometry system was demonstrated for use in the direct analysis of spotted and dosed drugs and their metabolites in thin tissue sections. Proof-of-principle sampling and analysis directly from tissue without the need for sample preparation was demonstrated first by raster scanning a region on a section of rat liver onto which reserpine was spotted. The mass spectral signal from selected reaction monitoring was used to develop a chemical image of the spotted drug on the tissue. The probe was also used to selectively spot sample areas of sagittal whole-body tissue from a mouse that had been dosed orally (90 mg/kg) with R,S-sulforaphane 3 h prior to sacrifice. Sulforaphane and its glutathione and N-acetyl cysteine conjugates were monitored with selected reaction monitoring and detected in the stomach and various other tissues from the dosed mouse. No signal for these species was observed in the tissue from a control mouse. The same dosed-tissue section was used to illustrate the possibility of obtaining a lane scan across the whole-body section. In total, these results illustrate the potential for rapid screening of the distribution of drugs and metabolites in thin tissue sections with the liquid micro-junction surface sampling probe/electrospray mass spectrometry approach. Published in 2007 by John Wiley & Sons, Ltd.

Evaluation of a surface‐sampling probe electrospray mass spectrometry system for the analysis of surface‐deposited and affinity‐captured proteins

A combined self-aspirating electrospray emitter/surfacing-sampling probe coupled with an ion trap mass spectrometer was used to sample and mass analyze proteins from surfaces. The sampling probe mass spectrometer system was used to sample and detect lysozyme that had been deposited onto a glass slide using a piezoelectric spotter or murine gamma-interferon affinity captured on a glass slide using surface-immobilized anti-gamma-interferon antibody. The detection level for surface-deposited lysozyme (spot size < or =200 microm) was approximately 1.0 fmol (approximately 100 fmol/mm2) as determined from the ability to measure accurately the protein molecular mass from the mass spectrum acquired by sampling the deposit. These detection limits may be sufficient for certain applications in which protein fractions from a separation method are collected onto a surface. Radiolabeled proteins were used to quantify the surface density of immobilized antibody and the efficiency of capture of the gamma-interferon on glass and higher surface area ceramic supports. The capture density of gamma-interferon at surface saturation ranged from about 23 to 50 fmol/mm2 depending on the capture surface. Nonetheless, mass spectrometric detection of affinity capture protein was successful in some cases, but the results were not reproducible. Thus, improvement of the sampling system, ionization efficiency and/or capture density will be necessary for practical sampling of affinity-captured proteins. The means to accomplish improved sampling system detection limits and to increase the absolute amounts of protein captured per unit area are discussed.