William F. Danielson

An LC-IMS-MS Platform Providing Increased Dynamic Range for High-Throughput Proteomic Studies

A high-throughput approach and platform using 15 min reversed-phase capillary liquid chromatography (RPLC) separations in conjunction with ion mobility spectrometry-mass spectrometry (IMS-MS) measurements was evaluated for the rapid analysis of complex proteomics samples. To test the separation quality of the short LC gradient, a sample was prepared by spiking 20 reference peptides at varying concentrations from 1 ng/mL to 10 microg/mL into a tryptic digest of mouse blood plasma and analyzed with both a LC-Linear Ion Trap Fourier Transform (FT) MS and LC-IMS-TOF MS. The LC-FT MS detected 13 out of the 20 spiked peptides that had concentrations >or=100 ng/mL. In contrast, the drift time selected mass spectra from the LC-IMS-TOF MS analyses yielded identifications for 19 of the 20 peptides with all spiking levels present. The greater dynamic range of the LC-IMS-TOF MS system could be attributed to two factors. First, the LC-IMS-TOF MS system enabled drift time separation of the low concentration spiked peptides from the high concentration mouse peptide matrix components, reducing signal interference and background, and allowing species to be resolved that would otherwise be obscured by other components. Second, the automatic gain control (AGC) in the linear ion trap of the hybrid FT MS instrument limits the number of ions that are accumulated to reduce space charge effects and achieve high measurement accuracy, but in turn limits the achievable dynamic range compared to the IMS-TOF instrument.

Enhanced ion utilization efficiency using an electrodynamic ion funnel trap as an injection mechanism for ion mobility spectrometry.

Conventional ion mobility spectrometers that sample ion packets from continuous sources have traditionally been constrained by an inherently low duty cycle. As such, ion utilization efficiencies have been limited to <1% in order to maintain instrumental resolving power. Using a modified electrodynamic ion funnel, we demonstrated the ability to accumulate, store, and eject ions in conjunction with ion mobility spectrometry (IMS), which elevated the charge density of the ion packets ejected from the ion funnel trap (IFT) and provided a considerable increase in the overall ion utilization efficiency of the IMS instrument. A 7-fold increase in signal intensity was revealed by comparing continuous ion beam current with the amplitude of the pulsed ion current in IFT-IMS experiments using a Faraday plate. Additionally, we describe the IFT operating characteristics using a time-of-flight mass spectrometer attached to the IMS drift tube.

High-Resolution Differential Ion Mobility Separations Using Helium-Rich Gases

Analyses of complex mixtures and characterization of ions increasingly involve gas-phase separations by ion mobility spectrometry (IMS) and particularly differential or field asymmetric waveform IMS (FAIMS) based on the difference of ion mobility in strong and weak electric fields. The key advantage of FAIMS is substantial orthogonality to mass spectrometry (MS), which makes FAIMS/MS hybrid a powerful analytical platform of broad utility. However, the potential of FAIMS has been constrained by limited resolution. Here, we report that the use of gas mixtures comprising up to 75% He dramatically increases the FAIMS separation capability, with the resolving power for peptides and peak capacity for protein digests reaching and exceeding 100. The resolution gains extend to small molecules, where previously unresolved isomers can now be separated. These performance levels open major new applications of FAIMS in proteomic and other biomolecular analyses.

Dynamically Multiplexed Ion Mobility Time-of-Flight Mass Spectrometry

Ion mobility spectrometry-time-of-flight mass spectrometry (IMS-TOFMS) has been increasingly used in analysis of complex biological samples. A major challenge is to transform IMS-TOFMS to a high-sensitivity, high-throughput platform, for example, for proteomics applications. In this work, we have developed and integrated three advanced technologies, including efficient ion accumulation in an ion funnel trap prior to IMS separation, multiplexing (MP) of ion packet introduction into the IMS drift tube, and signal detection with an analog-to-digital converter, into the IMS-TOFMS system for the high-throughput analysis of highly complex proteolytic digests of, for example, blood plasma. To better address variable sample complexity, we have developed and rigorously evaluated a novel dynamic MP approach that ensures correlation of the analyzer performance with an ion source function and provides the improved dynamic range and sensitivity throughout the experiment. The MP IMS-TOFMS instrument has been shown to reliably detect peptides at a concentration of 1 nM in the presence of a highly complex matrix, as well as to provide a 3 orders of magnitude dynamic range and a mass measurement accuracy of better than 5 ppm. When matched against human blood plasma database, the detected IMS-TOF features were found to yield approximately 700 unique peptide identifications at a false discovery rate (FDR) of approximately 7.5%. Accounting for IMS information gave rise to a projected FDR of approximately 4%. Signal reproducibility was found to be greater than 80%, while the variations in the number of unique peptide identifications were <15%. A single sample analysis was completed in 15 min that constitutes almost 1 order of magnitude improvement compared to a more conventional LC-MS approach.

Simultaneous Fragmentation of Multiple Ions Using IMS Drift Time Dependent Collision Energies

Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) was utilized to evaluate an ion collision energy ramping technique that simultaneously fragments a variety of species. To evaluate this technique, the fragmentation patterns of a mixture of ions ranging in mass, charge state, and drift time were analyzed to determine their optimal fragmentation conditions. The precursor ions were pulsed into the IMS-MS instrument and separated in the IMS drift cell based on mobility differences. Two differentially pumped short quadrupoles were used to focus the ions exiting the drift cell, and fragmentation was induced by collision induced dissociation (CID) between the conductance limiting orifice behind the second short quadrupole and before the first octopole in the mass spectrometer. To explore the fragmentation spectrum of each precursor ion, the bias voltages for the short quadrupoles and conductance limiting orifices were increased from 0 to 50 V above nonfragmentation voltage settings. An approximately linear correlation was observed between the optimal fragmentation voltage for each ion and its specific drift time, so a linear voltage gradient was employed to supply less collision energy to high mobility ions (e.g., small conformations or higher charge state ions) and more to low mobility ions. Fragmentation efficiencies were found to be similar for different ions when the fragmentation voltage was linearly ramped with drift time, but varied drastically when only a single voltage was used.

Advancing the High Throughput Identification of Liver Fibrosis Protein Signatures Using Multiplexed Ion Mobility Spectrometry

Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While MS-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry separations with liquid chromatography and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the liquid chromatography - ion mobility spectrometry-MS platform analyzing blood serum samples from 60 postliver transplant patients with recurrent fibrosis progression and 60 nontransplant patients illustrates its potential utility for disease characterization.

Pseudorandom sequence modifications for ion mobility orthogonal time-of-flight mass spectrometry.

Due to the inherently low duty cycle of ion mobility spectrometry (IMS) experiments that sample from continuous ion sources, a range of experimental advances have been developed to maximize ion utilization efficiency. The use of ion trapping and accumulation approaches prior to the ion mobility drift tube has demonstrated significant gains over discrete sampling from continuous sources but have traditionally relied upon a signal averaging (SA) to attain analytically useful signal-to-noise ratios (SNR). Multiplexed (MP) techniques based upon the Hadamard transform offer an alternative experimental approach by which ion utilization efficiency can be elevated from approximately 1 to approximately 50%. Recently, our research group demonstrated a unique multiplexed ion mobility time-of-flight (MP-IMS-TOF) approach that incorporates ion trapping and can extend ion utilization efficiency beyond 50%. However, the spectral reconstruction of the multiplexed signal using this experiment approach requires the use of sample-specific weighting designs. Such general weighting designs have been shown to significantly enhance ion utilization efficiency using this MP technique, but cannot be universally applied. By modifying both the ion trapping and the pseudorandom sequence (PRS) used for the MP experiment, we have eliminated the need for complex weighting matrices. For both simple and complex mixtures, SNR enhancements of up to 13 were routinely observed as compared to the SA-IMS-TOF approach. In addition, this new class of PRS provides a 2-fold enhancement in the number of ion gate pulses per unit time compared to the traditional HT-IMS experiment.

Hydrodynamic injection with pneumatic valving for microchip electrophoresis with total analyte utilization

A novel hydrodynamic injector that is directly controlled by a pneumatic valve has been developed for reproducible microchip CE separations. The PDMS devices used for the evaluation comprise a separation channel, a side channel for sample introduction, and a pneumatic valve aligned at the intersection of the channels. A low pressure (≤3 psi) applied to the sample reservoir is sufficient to drive sample into the separation channel. The rapidly actuated pneumatic valve enables injection of discrete sample plugs as small as ∼100 pL for CE separation. The injection volume can be easily controlled by adjusting the intersection geometry, the solution back pressure, and the valve actuation time. Sample injection could be reliably operated at different frequencies (<0.1 Hz to >2 Hz) with good reproducibility (peak height relative standard deviation ≤3.6%) and no sampling biases associated with the conventional electrokinetic injections. The separation channel was dynamically coated with a cationic polymer, and FITC‐labeled amino acids were employed to evaluate the CE separation. Highly efficient (≥7.0×103 theoretical plates for the ∼2.4‐cm‐long channel) and reproducible CE separations were obtained. The demonstrated method has numerous advantages compared with the conventional techniques, including repeatable and unbiased injections, little sample waste, high duty cycle, controllable injected sample volume, and fewer electrodes with no need for voltage switching. The prospects of implementing this injection method for coupling multidimensional separations for multiplexing CE separations and for sample‐limited bioanalyses are discussed.

An Efficient Data Format for Mass Spectrometry-Based Proteomics

The diverse range of mass spectrometry (MS) instrumentation along with corresponding proprietary and nonproprietary data formats has generated a proteomics community driven call for a standardized format to facilitate management, processing, storing, visualization, and exchange of both experimental and processed data. To date, significant efforts have been extended towards standardizing XML-based formats for mass spectrometry data representation, despite the recognized inefficiencies associated with storing large numeric datasets in XML. The proteomics community has periodically entertained alternate strategies for data exchange, e.g., using a common application programming interface or a database-derived format. However, these efforts have yet to gain significant attention, mostly because they have not demonstrated significant performance benefits over existing standards, but also due to issues such as extensibility to multidimensional separation systems, robustness of operation, and incomplete or mismatched vocabulary. Here, we describe a format based on standard database principles that offers multiple benefits over existing formats in terms of storage size, ease of processing, data retrieval times, and extensibility to accommodate multidimensional separation systems.

Pulsed multiple reaction monitoring approach to enhancing sensitivity of a tandem quadrupole mass spectrometer.

Liquid chromatography (LC)-triple quadrupole mass spectrometers operating in a multiple reaction monitoring (MRM) mode are increasingly used for quantitative analysis of low-abundance analytes in highly complex biochemical matrixes. After development and selection of optimum MRM transitions, sensitivity and data quality limitations are largely related to mass spectral peak interferences from sample or matrix constituents and statistical limitations at low number of ions reaching the detector. Herein, we report on a new approach to enhancing MRM sensitivity by converting the continuous stream of ions from the ion source into a pulsed ion beam through the use of an ion funnel trap (IFT). Evaluation of the pulsed MRM approach was performed with a tryptic digest of Shewanella oneidensis strain MR-1 spiked with several model peptides. The sensitivity improvement observed with the IFT coupled in to the triple quadrupole instrument is based on several unique features. First, ion accumulation radio frequency (rf) ion trap facilitates improved droplet desolvation, which is manifested in the reduced background ion noise at the detector. Second, signal amplitude for a given transition is enhanced because of an order-of-magnitude increase in the ion charge density compared to a continuous mode of operation. Third, signal detection at the full duty cycle is obtained, as the trap use eliminates dead times between transitions, which are inevitable with continuous ion streams. In comparison with the conventional approach, the pulsed MRM signals showed 5-fold enhanced peak amplitude and 2-3-fold reduced chemical background, resulting in an improvement in the limit of detection (LOD) by a factor of ∼4-8.