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Biological Sciences

Technology Development

LC-IMS-TOF MS NextGen platform being developed at PNNL.
LC-IMS-TOF MS NextGen platform being developed at PNNL. Enlarged View

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PNNL is a leader in proteomics and mass spectrometry technology development leading to innovative, next-generation, technology platforms. Current areas of development include

  • Ion mobility separation techniques
  • Electrospray interface (ESI) emitters
  • Liquid chromatography (LC)
  • Ion funnel technologies
  • Microfluidics.

NextGen Proteomics Platform. Biomarker discovery efforts will benefit greatly from measurements with improved reproducibility, higher throughput, and increased sensitivity. Mass spectrometry-based platforms currently provide the broadest proteome coverage, but efforts to increase their performance are constrained by issues related to their physical operation (e.g., space charge effects in ion traps) and data content limitations (e.g., peptide ions measured per second).

We are developing a NextGen proteomics platform that combines liquid chromatography (LC), ion mobility spectrometry (IMS), and mass spectrometry (MS). By coupling IMS, which distinguishes ions with differing shapes, to time-of-flight (TOF) MS, samples can be analyzed based on both shape and m/z.

As a result of the IMS shape separation, the ions are spread through a range of IMS drift times so they do not all arrive simultaneously at the detector. This separation allows the NextGen platform to detect low concentration species and use shorter LC gradient times for higher-throughput analyses.

The most important application for the NextGen platform falls in the area of populational proteomics where high throughput is essential but practically unachievable by any conventional LC-MS or LC-MS/MS platforms. For example, a typical human genome-wide association study requires tens of thousands of individuals to be involved; however, conventional platforms would require many years to analyze a sample set of this size. The NextGen platform will be essential for overcoming this throughput barrier and bringing proteomics into the domain of translational research and personalized medicine.

ESI Emitters. We developed encoding translation stages that sequentially position each capillary LC column outlet and electrospray ionization (ESI) emitter at the MS inlet, a critical ability for overlapping the processing of the separation columns. Central to successfully integrating a multi-column system is the reproducibility of the electrosprays between columns.

A 20-emitter array coupled to a 20-capillary inlet ESI interface.
A 20-emitter array coupled to a 20-capillary inlet ESI interface. Enlarged View

Research at PNNL led to a technique for producing ESI emitters based on chemical etching, which has greatly improved reproducibility relative to conventional emitters. The emitters provide better data quality and last ~4 times longer than traditional laser-pulled ESI emitters. The resulting reduction in system downtime increases throughput.

The patented emitter fabrication technique also enables easy creation of emitter arrays. These provide the benefits of nanoESI at the higher flow rates typical of LC separation methods. An ESI emitter array coupled to the outlet of an LC column divides the eluent into several low nL/min flows, each terminating in a nanoESI emitter. This greatly increases ionization efficiency and reduces analyte suppression. The coupling of the arrays to a patented multi-inlet interface ensures that a large fraction of the ions are sampled into the mass spectrometer, increasing instrument sensitivity.

Improvements in commercially available ultra-high pressure valves have made it possible to routinely operate these systems at 10,000 to 20,000 psi, which makes it practical to use smaller packing materials (1.5 μm or 3 μm instead of 5 μm) and smaller inner diameter capillaries (e.g., 50 μm instead of 150 μm) that improve both separation quality and measurement sensitivity.

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LC Column Improvements. We have developed a silica-based monolithic liquid chromatography (LC) column with an inner diameter of 10 μm that provides proteomics measurements with more than 10-fold improved sensitivity relative to those of conventional LC. The sensitivity of proteomics measurements using LC separations interfaced with electrospray ionization-mass spectrometry (ESI-MS) improves as the liquid flow rate decreases, making attractive the use of smaller inner-diameter LC columns.

A low-capacitance ion funnel capable of >30 Torr operation.
A low-capacitance ion funnel capable of >30 Torr operation. Enlarged View

Ion Funnel Technologies. The electrodynamic ion funnel is a device that greatly reduces the ion losses associated with typical skimmer-based ESI interfaces. In contrast to the skimmer interface, the ion funnel can focus essentially all of the ions in a broad m/z range exiting the inlet capillary and transmit them to the next vacuum stage.

Our present ion funnel design consists of a stack of ring electrodes with a front section of constant inner diameter (i.d.) creating a traditional stacked ring ion guide and a back section that linearly decreases in i.d. creating an "ion funnel." A superimposed RF voltage and DC gradient is applied to the rings, which confines and transmits the ions through the device.

The ion funnel has recently been licensed to several mass spectrometer companies, greatly improving the sensitivity of their high-end, commercial instruments.

Recent advances include increasing the operating pressure to >30 Torr by reengineering the device with a lower electrical capacitance. The increased pressure enables effective use of multiple inlets, which can be arranged to better capture the electrospray ion/droplet plume, increasing sampling efficiency and instrument sensitivity.

In addition, increasing the operating pressure of the ion funnel improves the newly developed low pressure ESI source known as Subambient Pressure Ionization with Nanoelectrospray (SPIN). The SPIN source locates the electrospray emitter inside the first vacuum region of the interface at the entrance to an ion funnel. This enables the entire ion/droplet plume to be captured by the ion funnel, eliminating all ion loss associated with typical atmospheric inlets. Greater ion funnel pressures increase the desolvation efficiency of the SPIN source, producing more ions for the mass analyzer.

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Microfluidics. Because of the technology developments described above, we can now separate and detect proteomic samples with exquisite sensitivity. However, because of sample losses associated with conventional sample preparation, a large gap exists between the amount of unprepared sample required (micrograms to milligrams) and prepared sample actually needed for the analysis (picograms to nanograms).

If tools can be developed that reduce losses associated with sample preparation, trace samples, including individual cells, can be analyzed.

Drawing of a microfluidic chip that processes discrete aqueous droplets and transfers the contents to an on-chip ESI emitter providing efficient coupling to a mass spectrometer.
A low-capacitance ion funnel capable of >30 Torr operation. Enlarged View

We are exploring microfluidic platforms capable of isolating, preparing, separating and ionizing small proteomic samples. The integrated microdevices will mitigate the losses associated with bench top processing. To this end, we have developed an effective microfluidic electrospray interface, which operates robustly in the nano-flow regime (as low as 20 nL/min).

We have also coupled droplet-based microfluidics with nanoESI-MS. The droplet format is particularly attractive for small sample analyses, as analytes encapsulated inside aqueous picoliter-sized droplets and surrounded by an immiscible oil can be manipulated and transported without loss. We are currently pursuing single-cell lysis and analysis with MS detection.

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