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Fast, Two-Dimensional, Gas-Phase Separations for Ultrahigh-Throughput Global Analyses

Keqi Tang, Principal Investigator

The Fast, Two-Dimensional, Gas-Phase Separations for Ultrahigh-Throughput Global Analyses project team is developing a new capability at Pacific Northwest National Laboratory (PNNL) for very high-throughput analysis of extremely complex biochemical mixtures. This new capability integrates two technologies: (1) two-dimensional separations of complex analytes fully in the gas phase and (2) subsequent sensitive and accurate mass-spectrometric characterization. The key feature of this innovative approach is extraordinary throughput - as much as 100 times above that of liquid-phase separations of equal quality.

Our approach involves post-ionization separations by low-field ion mobility separation (IMS) in one dimension and high-field field asymmetric waveform ion mobility separation (FAIMS) in another dimension, with their combination interfaced to a time-of-flight (TOF) mass spectrometer using PNNL-developed electrodynamic ion funnels. We are further enhancing the overall separation power, with a moderate reduction of throughput, by addition of a fast liquid chromatography (LC) stage.

FAIMS/IMS separation of proteolytic digests
Two-dimensional FAIMS/IMS separation of proteolytic digests (coupled to TOF MS). Data are for the Kolker standard that has the complexity of a typical protein complex. Click for a larger version.

This new analytical capability will be broadly useful in critical areas of research at PNNL and elsewhere. This technology will enable a throughput of more than 100 samples per day, with overall separation power sufficient to handle the complexity of realistic biological samples. We are also developing a proteomic database search algorithm suitable for FAIMS/IMS/MS methods and increasing the specificity and confidence of the accurate mass and time (AMT) tag approach by incorporation of FAIMS and IMS separation parameters. The high-throughput of the new platform will enable effective implementation of dynamic proteome analyses. The research is expected to yield several patents covering both new designs and methods pertaining to FAIMS, IMS, and their integration.

Mass spectra for protonated insulin
Mass spectra for protonated insulin (+5 m/z = 5734 Da) obtained using electrospray ionization- (ESI-) TOF (left) and ESI/IMS/TOF (right). Signal intensities differ by 2%. Click for a larger version.

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