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Gordon A Anderson

Publications and source records attributed to Gordon A Anderson.

16 recordsLinked to original sources
PubMed2003

Use of artificial neural networks for the accurate prediction of peptide liquid chromatography elution times in proteome analyses.

The use of artificial neural networks (ANNs) is described for predicting the reversed-phase liquid chromatography retention times of peptides enzymatically digested from proteome-wide proteins. To enable the accurate comparison of the numerous LC/MS data sets, a genetic algorithm was developed to normalize the peptide retention data into a range (from 0 to 1), improving the peptide elution time reproducibility to approximately 1%. The network developed in this study was based on amino acid residue composition and consists of 20 input nodes, 2 hidden nodes, and 1 output node. A data set of approximately 7000 confidently identified peptides from the microorganism Deinococcus radiodurans was used for the training of the ANN. The ANN was then used to predict the elution times for another set of 5200 peptides tentatively identified by MS/MS from a different microorganism (Shewanella oneidensis). The model was found to predict the elution times of peptides with up to 54 amino acid residues (the longest peptide identified after tryptic digestion of S. oneidensis) with an average accuracy of approximately 3%. This predictive capability was then used to distinguish with high confidence isobar peptides otherwise indistinguishable by accurate mass measurements as well as to uncover peptide misidentifications. Thus, integration of ANN peptide elution time prediction in the proteomic research will increase both the number of protein identifications and their confidence.

Amino Acid Sequence↗
PubMed2003

Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results.

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the protein sequence had been appended.

Algorithms↗
PubMed2003

Initial implementation of external accumulation liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance with automated gain control.

Capillary liquid chromatography (LC) separation coupled with external accumulation Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has recently been demonstrated to have significant potential for proteomics research. Accumulation of an excessive space charge external to the FTICR cell ion trap has been shown to result in increased mass measurement error, undesirable ion discrimination and/or fragmentation, potentially causing misrepresentation or incorrect assignments of lower abundance peptides in the acquired mass spectra. In this work we report on the capability of data-dependent adjustment of ion accumulation times in the course of LC separations, further referred to as automated gain control (AGC). Three different AGC approaches were evaluated based on the number of putative peptides from a tryptic digest of four casein proteins detected in the course of LC/FTICR separations. When compared with the conventional technique, AGC was found to increase, up to a factor of 3, the total number of peptides identified.

Automation↗
PubMed2002

High-throughput global peptide proteomic analysis by combining stable isotope amino acid labeling and data-dependent multiplexed-MS/MS.

In this work, we describe the application of a stable isotope amino acid (lysine) labeling in conjunction with data-dependent multiplexed tandem mass spectrometry (MS/MS) to facilitate the characterization and identification of peptides from proteomic (global protein) digests. Lysine auxotrophic yeast was grown in the presence of 13C-labeled or unlabeled lysine and combined after harvesting in equal proportions. Endoproteinase LysC digestion of the cytosolic fraction produced a global proteomic sample, consisting of heavy/light labeled peptide pairs. Then data-dependent multiplexed-MS/MS was applied to simultaneously select and dissociate only labeled peptide ion pairs. The approach allows differentiation between N-terminal (e.g., b-type ions) and C-terminal fragment ions (e.g., y-type ions) in resulting tandem mass spectra, as well as the capability of differentiation between near-isobaric glutamine and lysine residues. We also describe the utility of peptide composition and fragment information to support peptide identifications and examine the potential application of lysine labeling for differential quantitative protein analysis.

Amino Acids↗
PubMed2002

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics.

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.

Chromatography, High Pressure Liquid↗
PubMed2002

Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags.

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Bacteria↗
PubMed2002

Direct mass spectrometric analysis of intact proteins of the yeast large ribosomal subunit using capillary LC/FTICR.

Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry coupled with capillary reverse-phase liquid chromatography was used to characterize intact proteins from the large subunit of the yeast ribosome. High mass measurement accuracy, achieved by "mass locking" with an internal standard from a dual electrospray ionization source, allowed identification of ribosomal proteins. Analyses of the intact proteins revealed information on cotranslational and posttranslational modifications of the ribosomal proteins that included loss of the initiating methionine, acetylation, methylation, and proteolytic maturation. High-resolution separations permitted differentiation of protein isoforms having high structural similarity as well as proteins from their modified forms, facilitating unequivocal assignments. The study identified 42 of the 43 core large ribosomal subunit proteins and 58 (of 64 possible) core large subunit protein isoforms having unique masses in a single analysis. These results demonstrate the basis for the high-throughput analyses of complex mixtures of intact proteins, which we believe will be an important complement to other approaches for defining protein modifications and their changes resulting from physiological processes or environmental perturbations.

Chromatography↗
PubMed2002

An accurate mass tag strategy for quantitative and high-throughput proteome measurements.

We describe and demonstrate a global strategy that extends the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements based upon the use of peptide "accurate mass tags" (AMTs) produced by global protein enzymatic digestion. The two-stage strategy exploits Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry to validate peptide AMTs for a specific organism, tissue or cell type from "potential mass tags" identified using conventional tandem mass spectrometry (MS/MS) methods, providing greater confidence in identifications as well as the basis for subsequent measurements without the need for MS/MS, and thus with greater sensitivity and increased throughput. A single high resolution capillary liquid chromatography separation combined with high sensitivity, high resolution and accurate FT-ICR measurements has been shown capable of characterizing peptide mixtures of significantly more than 10(5) components with mass accuracies of < 1 ppm, sufficient for broad protein identification using AMTs. Other attractions of the approach include the broad and relatively unbiased proteome coverage, the capability for exploiting stable isotope labeling methods to realize high precision for relative protein abundance measurements, and the projected potential for study of mammalian proteomes when combined with additional sample fractionation. Using this strategy, in our first application we have been able to identify AMTs for >60% of the potentially expressed proteins in the organism Deinococcus radiodurans.

Amino Acid Sequence↗
PubMed2002

Selective incorporation of isotopically labeled amino acids for identification of intact proteins on a proteome-wide level.

The post-genomic era and increased demands for broad proteome measurements have greatly increased the needs for protein identification. We describe a strategy that uses accurate mass measurements and partial amino acid content information to unambiguously identify intact proteins, and show its initial application to the proteomes of Escherichia coli and Saccharomyces cerevisiae. Proteins were extracted from the organisms grown in minimal medium or minimal medium to which isotopically labeled leucine (Leu-D(10)) had been added. The two protein extracts were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled residue has no effect on the CIEF separation of proteins, and both isotopically labeled and unlabeled versions of specific proteins are observed within the same mass spectrum. The difference in the mass of the unlabeled and labeled proteins is used to determine the number of Leu residues present in a particular protein. Proteins can then often be unambiguously identified based on their accurately determined molecular mass and the additional constraint provided by number of Leu residues. The identities of proteins were further confirmed by repeating CIEF/FTICR measurements with samples that contain other isotopically labeled amino acid residues (e.g. His, Arg, Ile, Phe, Lys). A theoretical study of the amino acid composition (for a difference in the amino acid sequence) showed the constraints needed in order to identify the protein unambiguously. Additionally, the mass differences between the predicted and the experimental accurate mass measurement provide insights into the nature of simple post-translational modifications.

Amino Acids↗
PubMed2002

Gene expression profiling using advanced mass spectrometric approaches.

In the era of systems biology, computational and high-throughput experimental biological approaches are increasingly being combined to provide global snapshots of entire genomes and proteomes under tissue- and disease-specific conditions. The aim is to identify proteins changing in concentration and/or post-translational state and/or location, and develop a better molecular level understanding of the operation of biological systems. Here we describe an approach for comparative proteomics that builds upon the combination of high-performance nano-scale separations with the high-mass measurement accuracy, mass-resolving power and sensitivity of Fourier transform ion cyclotron resonance mass spectrometry to provide broad dynamic range, comprehensive and quantitative proteome measurements.

Amino Acid Sequence↗
PubMed2002

Mass measurement errors caused by 'local" frequency perturbations in FTICR mass spectrometry.

One of the key qualities of mass spectrometric measurements for biomolecules is the mass measurement accuracy (MMA) obtained. FTICR presently provides the highest MMA over a broad m/z range. However, due to space charge effects, the achievable MMA crucially depends on the number of ions trapped in the ICR cell for a measurement. Thus, beyond some point, as the effective sensitivity and dynamic range of a measurement increase, MMA tends to decrease. While analyzing deviations from the commonly used calibration law in FTICR we have found systematic errors which are not accounted for by a "global" space charge correction approach. The analysis of these errors and their dependence on charge population and post-excite radius have led us to conclude that each ion cloud experiences a different interaction with other ion clouds. We propose a novel calibration function which is shown to provide an improvement in MMA for all the spectra studied.

Chemistry, Physical↗
PubMed2002

ESI-FTICR mass spectrometry employing data-dependent external ion selection and accumulation.

Data-dependent external m/z selection and accumulation of ions is demonstrated in use with ESI-FTICR instrumentation, with two different methods for ion selection being explored. One method uses RF/DC quadrupole filtering and is described in use with an 11.5 tesla (T) FTICR instrument, while the second method employs RF-only resonance dipolar excitation selection and is described in use with a 3.5 T FTICR instrument. In both methods ions are data-dependently selected on the fly in a linear quadrupole ion guide, then accumulated in a second linear RF-only quadrupole trap that immediately follows. A major benefit of ion preselection prior to external accumulation is the enhancement of ion populations for low-level species. This development is expected to expand the dynamic range and sensitivity of FTICR for applications including analysis of complex polypeptide mixtures (e.g., proteomics).

Journal Article↗
PubMed2002

Frequency shifts due to the interference of resolved peaks in magnitude-mode Fourier-transform ion cyclotron resonance mass spectra.

We have obtained relationships for frequency shifts resulting from the interference of spectral components for the magnitude mode Fourier transform. The approximation of a weak perturbation of well resolved peaks has been used. Both the low- and high-pressure limits for Fourier-transform ion cyclotron resonance (FTICR) operation have been considered. We have found that the shifts can be either negative or positive, depending on the initial phase and/or the choice of the time-domain interval. The magnitude of shifts generally does not exceed the peak width. In the approximation of small perturbations the shifts produced by multiple peaks are additive. We have compared theoretical results with experimental shifts for isotopic clusters of multiply charged insulin. Up to 1 ppm frequency variations were experimentally observed for the insulin 5+ charge state, consistent with theoretical estimates. The peak interference is of particular significance in the case of bio-molecular mass spectra having a large number of peaks and covering a considerable dynamic range (i.e., relative abundance). We conclude that the common mass measurement procedure based on the location of the magnitude mode maxima of well resolved peaks can result in systematic mass measurement errors. The relationships obtained provide corrections for the frequency shifts and thus improve the mass measurement accuracy.

Algorithms↗
PubMed2002

Increased proteome coverage for quantitative peptide abundance measurements based upon high performance separations and DREAMS FTICR mass spectrometry.

A primary challenge in proteome measurements is to be able to detect, identify, and quantify the extremely complex mixtures of proteins. The relative abundances of interest span at least six orders of magnitude for mammalian proteomes, and this constitutes an intractable challenge for high throughput proteome studies. We have recently described a new approach, Dynamic Range Enhancement Applied to Mass Spectrometry (DREAMS), which is based upon the selective ejection of the most abundant species to expand the dynamic range of Fourier transform ion cyclotron resonanace (FTICR) measurements. The basis of our approach is on-the-fly data-dependent selective ejection of highly abundant species, followed by prolonged accumulation of remaining low-abundance species in a quadrupole external to the FTICR ion trap. Here we report the initial implementation of this approach with high efficiency capillary reverse phase LC separations and high magnetic field electrospray ionization FTICR mass spectrometry for obtaining enhanced coverage in quantitative measurements for mammalian proteomes. We describe the analysis of a sample derived from a tryptic digest of proteins from mouse B16 cells cultured in both natural isotopic abundance and 15N-labeled media. The FTICR mass spectrometric analysis allows the assignment of peptide pairs (corresponding to the two distinctive versions of each peptide), and thus provides the basis for quantiative measurements when one of the two proteomes in the mixture is perturbed or altered in some fashion. We show that implementation of the DREAMS approach allows assignment of approximately 80% more peptide pairs, thus providing quantitative information for approximately 18,000 peptide pairs in a single analysis.

Animals↗
PubMed2002

The use of accurate mass tags for high-throughput microbial proteomics.

We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.

Amino Acid Sequence↗