Keqi Tang

A NOVEL ION FUNNEL FOR FOCUSING IONS AT ELEVATED PRESSURE USING ELECTROSPRAY IONIZATION MASS SPECTROMETRY

The ability to effectively focus and transmit ions from relatively high pressure ion sources is a key factor that affects sensitivity and dynamic range in mass spectrometry. To improve upon the mass spectrometric sensitivity achievable with electrospray ionization sources a novel ion funnel interface has been developed and implemented with a triple quadrupole mass spectrometer. The ion funnel effectively consists of a series of ring electrodes of progressively smaller internal diamter to which RF and DC electric fields are co-applied. The electric fields create a pseudo-potential causing the collisionally damped ions to be more effectively focused and transmitted as a collimated ion beam. The ion funnel concept we describe is supported by results of SIMION simulations, ion current measurements and implementation with a mass spectrometer. Electrospray ionization mass spectra for an initial ion funnel configuration demonstrated over an order of magnitude increase in signal relative to that of the instrument operated in its standard (capillary inlet-skimmer) configuration under similar conditions.

The Ion Funnel: Theory, Implementations and Applications.

The electrodynamic ion funnel has enabled the manipulation and focusing of ions in a pressure regime (0.1-30 Torr) that has challenged traditional approaches, and provided the basis for much greater mass spectrometer ion transmission efficiencies. The initial ion funnel implementations aimed to efficiently capture ions in the expanding gas jet of an electrospray ionization interface and radially focus them for efficient transfer through a conductance limiting orifice. We review the improvements in fundamental understanding of ion motion in ion funnels, the evolution in its implementations that have brought the ion funnel to its current state of refinement, as well as applications of the ion funnel for purposes such as ion trapping, ion cooling, low pressure electrospray, and ion mobility spectrometry.

Charge competition and the linear dynamic range of detection in electrospray ionization mass spectrometry

An experimental investigation and theoretical analysis are reported on charge competition in electrospray ionization (ESI) and its effects on the linear dynamic range of ESI mass spectrometric (MS) measurements. The experiments confirmed the expected increase of MS sensitivities as the ESI flow rate decreases. However, different compounds show somewhat different mass spectral peak intensities even at the lowest flow rates, at the same concentration and electrospray operating conditions. MS response for each compound solution shows good linearity at lower concentrations and levels off at high concentration, consistent with analyte “saturation” in the ESI process. The extent of charge competition leading to saturation in the ESI process is consistent with the relative magnitude of excess charge in the electrospray compared to the total number of analyte molecules in the solution. This ESI capacity model allows one to predict the sample concentration limits for charge competition and the on-set of ionization suppression effects, as well as the linear dynamic range for ESI-MS. The implications for quantitative MS analysis and possibilities for effectively extending the dynamic range of ESI measurements are discussed.

Antibody-free, targeted mass-spectrometric approach for quantification of proteins at low picogram per milliliter levels in human plasma/serum

Sensitive detection of low-abundance proteins in complex biological samples has typically been achieved by immunoassays that use antibodies specific to target proteins; however, de novo development of antibodies is associated with high costs, long development lead times, and high failure rates. To address these challenges, we developed an antibody-free strategy that involves PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing) for sensitive selected reaction monitoring (SRM)–based targeted protein quantification. The strategy capitalizes on high-resolution reversed-phase liquid chromatographic separations for analyte enrichment, intelligent selection of target fractions via on-line SRM monitoring of internal standards, and fraction multiplexing before nano–liquid chromatography-SRM quantification. Application of this strategy to human plasma/serum demonstrated accurate and reproducible quantification of proteins at concentrations in the 50–100 pg/mL range, which represents a major advance in the sensitivity of targeted protein quantification without the need for specific-affinity reagents. Application to a set of clinical serum samples illustrated an excellent correlation between the results obtained from the PRISM-SRM assay and those from clinical immunoassay for the prostate-specific antigen level.

Advancing the sensitivity of selected reaction monitoring-based targeted quantitative proteomics

Selected reaction monitoring (SRM) – also known as multiple reaction monitoring (MRM) – has emerged as a promising high‐throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for, e.g. detecting low‐abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low‐abundance signaling proteins in cells or tissues. Herein, we review recent advances in methods and technologies, including front‐end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications, as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low‐abundance proteins at low‐ to sub‐ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A.2002,99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; ∼3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, ∼900 ORFs detected using LC-TOFMS, with ∼500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.

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.

Improving the Microdialysis Procedure for Electrospray Ionization Mass Spectrometry of Biological Samples

Significant advances in the area of microdialysis which allowed more effective handling of small volumes (microliters) of samples, more efficient desalting and enhanced mass spectrometric detection sensitivity are described. The previously reported on-line coupling of microdialysis with electrospray ionization (ESI) mass spectrometry has been found to be highly effective; however, direct coupling requires relatively high sample flow rates (∽2 μl min-1) to obtain a stable ESI current compared with the flow rates of newer ESI sources (e.g. ‘microspray,’ 10–100 nl min-1). To circumvent this major limitation imposed by the dimensions of currently available materials, the microdialysis procedure was modified to an off-line mode in order to avoid excessive sample consumption. A more than tenfold decrease in sample consumption was achieved using the off-line modevsthe on-line mode, which resulted in a similar quality spectrum. In addition, several other aspects of the microdialysis approach were altered to improve its performance further: (i) an increase in dialysis temperature was found to increase the desalting efficiency greatly and therefore improve the spectrum quality; (ii) the addition of piperidine and imidazole to the dialysis buffer solution resulted in a reduction of charge states and a further increase in detection sensitivity for DNA and (iii) use of low concentrations (0–2.5 mM NH4OAc) of dialysis buffer shifted the DNA negative ions to higher charge states and produced a nearly tenfold increase in detection sensitivity and a slightly decreased desalting efficiency. Protocols for desalting different samples using microdialysis are discussed.

Dilution-Free Analysis from Picoliter Droplets by Nano-Electrospray Ionization Mass Spectrometry†

The expanding role of microfluidics for chemical and biochemical analysis is due to factors including the favorable scaling of separation performance with reduced channel dimensions,[1] flexibility afforded by computer-aided device design, and the ability to integrate multiple sample handling and analysis steps into a single platform.[2] Such devices enable smaller liquid volumes and sample sizes to be handled than can be achieved on the benchtop, where sub-microliter volumes are difficult to work with and where sample losses to the surfaces of multiple reaction vessels become prohibitive. A particularly attractive microfluidic platform for sample-limited analyses employs aqueous droplets or plugs encapsulated by an immiscible oil.[3,4] Each droplet serves as a discrete compartment or reaction chamber enabling, e.g., high throughput screening[5,6] and kinetic studies[7-9] of femto- to nanoliter samples, as well as the encapsulation[10-12] and lysis[10] of individual cells with limited dilution of the cellular contents

A reversed-phase capillary ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method for comprehensive top-down/bottom-up lipid profiling

Lipidomics is a critical part of metabolomics and aims to study all the lipids within a living system. We present here the development and evaluation of a sensitive capillary UPLC-MS method for comprehensive top-down/bottom-up lipid profiling. Three different stationary phases were evaluated in terms of peak capacity, linearity, reproducibility, and limit of quantification (LOQ) using a mixture of lipid standards representative of the lipidome. The relative standard deviations of the retention times and peak abundances of the lipid standards were 0.29% and 7.7%, respectively, when using the optimized method. The linearity was acceptable at >0.99 over 3 orders of magnitude, and the LOQs were sub-fmol. To demonstrate the performance of the method in the analysis of complex samples, we analyzed lipids extracted from a human cell line, rat plasma, and a model human skin tissue, identifying 446, 444, and 370 unique lipids, respectively. Overall, the method provided either higher coverage of the lipidome, greater measurement sensitivity, or both, when compared to other approaches of global, untargeted lipid profiling based on chromatography coupled with MS.