Biomedical subjects

Keqi Tang

Publications and source records attributed to Keqi Tang.

25 records · Page 2Linked to original sources

PubMed2004

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.

Caffeine↗

PubMed2004

Modeling the resolution and sensitivity of FAIMS analyses.

Field asymmetric waveform ion mobility spectrometry (FAIMS) is rapidly gaining acceptance as a robust, versatile tool for post-ionization separations prior to mass-spectrometric analyses. The separation is based on differences between ion mobilities at high and low electric fields, and proceeds at atmospheric pressure. Two major advantages of FAIMS over condensed-phase separations are its high speed and an ion focusing effect that often improves sensitivity. While selected aspects of FAIMS performance are understood empirically, no physical model rationalizing the resolving power and sensitivity of the method and revealing their dependence on instrumental variables has existed. Here we present a first-principles computational treatment capable of simulating the FAIMS analyzer for virtually any geometry (including the known cylindrical and planar designs) and arbitrary operational parameters. The approach involves propagating an ensemble of ion trajectories through the device in real time under the influence of applied asymmetric potential, diffusional motion incorporating the high-field and anisotropic phenomena, and mutual Coulomb repulsion of ionic charges. Calculations for both resolution and sensitivity are validated by excellent agreement with measurements in different FAIMS modes for ions representing diverse types and analyte classes.

Amino Acids↗

PubMed2004

Ultrasensitive and quantitative analyses from combined separations-mass spectrometry for the characterization of proteomes.

This article describes developments in fundamental and applied aspects of separations, electrospray ionization phenomena, and mass spectrometric instrumentation that are interrelated and important for making more effective and quantitative measurements, particularly for proteomics applications. The basis for better quantitation and ultrahigh sensitivity is highlighted for high-resolution capillary liquid chromatography separations that provide low nanoliter per minute flow rates to an electrospray ionization interface. The increased dynamic range of measurements and low zeptomole regime detection limits obtainable open new avenues for biological research.

Chromatography, Liquid↗

PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗

PubMed2003

Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance.

When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a approximately 10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods.

Journal Article↗

PubMed2003

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; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 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.

Amino Acid Sequence↗

PubMed2002

Independent control of ion transmission in a jet disrupter dual-channel ion funnel electrospray ionization MS interface.

A new atmospheric pressure ionization mass spectrometer (API-MS) interface has been developed to allow the control of ion transmission through the first vacuum stage of the mass spectrometer. The described interface uses a dual-heated capillary and a dual-inlet ion funnel design. Two electrosprays, aligned with the dual-capillary inlet, are used to introduce ions from different solutions independently into the MS. The initial design was specifically aimed at developing a method for the controlled introduction of calibrant ions in highly accurate mass measurements using Fourier transform ion cyclotron resonance mass spectrometer (FTICR). The dual-channel ion funnel has different inlet diameters that are aligned with the dual capillaries. The large diameter main channel of the ion funnel is used for analyte introduction to provide optimum ion transmission. The second, smaller diameter channel inlet includes a jet disrupter in the ion funnel to modulate the ion transmission through the channel. The two inlet channels converge into a single-channel ion funnel where ions from both channels are mixed, focused, and transmitted to the mass analyzer. Both theoretical simulations and experimental results show that the transmission of different m/z species in the small diameter channel of the ion funnel can be effectively modulated by varying the bias voltage on the jet disrupter. Both static and dynamic modulations of ion transmission are demonstrated experimentally by applying either a constant DC or a square waveform voltage to the jet disrupter. High ion transmission efficiency, similar to the standard single-channel ion funnel, is maintained in the main analyte channel inlet of the ion funnel over a broad m/z range with negligible "cross talk" between the two ion funnel inlet channels. Several possible applications of the new interface (e.g., for high-accuracy MS analysis of complex biological samples) are described.

Calibration↗