Biomedical subjects

Peter B O'Connor

Publications and source records attributed to Peter B O'Connor.

At least 19 recordsLinked to original sources

PubMed2006

Quantitating the relative abundance of isoaspartyl residues in deamidated proteins by electron capture dissociation.

Relative quantitation of aspartyl and isoaspartyl residue mixtures from asparagine deamidation is demonstrated using electron capture dissociation without prior HPLC separation. The method utilizes the linear relationship found between the relative abundance of the isoaspartyl diagnostic ion, z(n)-57, and % isoaspartyl content based on the ECD spectra of known isoaspartyl/aspartyl mixtures of synthetic peptides. The observed linearity appears to be sequence independent because the relationship exists despite sequence variations and changes in backbone fragment abundances when isoaspartyl and aspartyl residues are interchanged. Furthermore, a new method to calculate the relative abundances of isomer from protein deamidation without synthetic peptides is proposed and tested using a linear peptide released by protein digestion that contains the deamidation site. The proteolytic peptide can be rapidly aged to the expected 3:1 (isoaspartyl:aspartyl) mixture to generate a two-point calibration standard for ECD analysis. The procedure can then be used to determine the relative abundance of deamidation products from in vivo or in vitro protein aging experiments.

Amino Acid Sequence↗

PubMed2006

Use of a double resonance electron capture dissociation experiment to probe fragment intermediate lifetimes.

The relative abundances of fragment ions in electron capture dissociation (ECD) are often greatly affected by the secondary and tertiary structures of the precursor ion, and have been used to derive the gas-phase conformations of the protein ions. In this study, it is found that resonance ejection of the charge reduced molecular ion during ECD resulted in significant changes in many fragment ion populations. The ratio of the ion peak intensities in the double resonance (DR)-ECD to that in the normal ECD experiment can be used to calculate the lifetime of the radical intermediates from which these fragments are derived. These lifetimes are often in the ms range, a time sufficiently long to facilitate multiple free radical rearrangements. These ratios correlate with the intramolecular noncovalent interactions in the precursor ion, and can be used to deduce information about the gas-phase conformation of peptide ions. DR-ECD experiments can also provide valuable information on ECD mechanisms, such as the importance of secondary electron capture and the origin of c./z ions.

Cyclosporine↗

PubMed2006

The effect of radical trap moieties on electron capture dissociation spectra of substance P.

To further test the hypothesis that electron capture dissociation (ECD) involves long-lived radical intermediates and radical migration occurs within these intermediates before fragmentation, radical trap moieties were attached to peptides with the assumption that they would reduce fragmentation by decreasing the mobility of the radical. Coumarin labels were chosen for the radical traps, and unlabeled, singly-labeled, and doubly-labeled Substance P were analyzed by ECD. The results demonstrated a correlation between the number and position of tags on the peptide and the intensity of side-chain cleavages observed, as well as an inverse correlation between the number of tags on the peptide and the intensity of backbone cleavages. Addition of radical traps to the peptide inhibits backbone cleavages, suggesting that either radical mobility is required for these cleavages, or new noncovalent interactions prevent separation of backbone cleavage fragments. The enhancement of side-chain cleavages and the observation of new side-chain cleavages associated with aromatic groups suggest that the gas-phase conformation of this peptide is substantially distorted from untagged Substance P and involves previously unobserved interactions between the coumarin tags and the phenylalanine residues. Furthermore, the use of a double resonance (DR)-ECD experiment showed that these side-chain losses are all products of long-lived radical intermediate species, which suggests that steric hindrance prevents the coumarin-localized radical from interacting with the backbone while simultaneously increasing the radical rearrangements with the side chains.

Chromatography, High Pressure Liquid↗

PubMed2006

Detailed map of oxidative post-translational modifications of human p21ras using Fourier transform mass spectrometry.

P21ras, the translation product of the most commonly mutated oncogene, is a small guanine nucleotide exchange protein. Oxidant-induced post-translational modifications of p21ras including S-nitrosation and S-glutathiolation have been demonstrated to modulate its activity. Structural characterization of this protein is critical to further understanding of the biological functions of p21ras. In this study, high-resolution and high mass accuracy Fourier transform mass spectrometry was utilized to map, in detail, the post-translational modifications of p21ras (H-ras) exposed to oxidants by combining bottom-up and top-down techniques. For peroxynitrite-treated p21ras, five oxidized methionines, five nitrated tyrosines, and at least two oxidized cysteines (including C118) were identified by "bottom-up" analysis, and the major oxidative modification of C118, Cys118-SO3H, was confirmed by several tandem mass spectrometry experiments. Additionally, "top-down" analysis was conducted on p21ras S-glutathiolated by oxidized glutathione and identified C118 as the major site of glutathiolation among the four surface cysteines. The present study provides a paradigm for an effective and efficient method not only for mapping post-translational modifications of proteins but also for predicting the relative selectivity and specificity of oxidative post-translational modifications, especially using top-down analysis.

Amino Acid Sequence↗

PubMed2006

Use of the filter diagonalization method in the study of space charge related frequency modulation in fourier transform ion cyclotron resonance mass spectrometry.

The filter diagonalization method (FDM) is a recently developed computational technique capable of extracting resonance frequencies and amplitudes from very short transient signals. Although it requires stable resonance frequencies and is slower than the fast Fourier transform (FFT), FDM has a resolution and accuracy that is unmatched by the FFT or any other comparable techniques. This unique feature of FDM makes it an ideal tool for tracing space charge induced frequency modulations in Fourier transform ion cyclotron resonance (FT-ICR) cells, which are shown to reach +/-400 ppm even for such simple spectra as Substance P.

Cyclotrons↗

PubMed2006

Long-lived electron capture dissociation product ions experience radical migration via hydrogen abstraction.

To explore the mechanism of electron capture dissociation (ECD) of linear peptides, a set of 16-mer peptides were synthesized with deuterium labeled on the alpha-carbon position of four glycines. The ECD spectra of these peptides showed that such peptides exhibit a preference for the radical to migrate to the alpha-carbon position on glycine via hydrogen (or deuterium) abstraction before the final cleavage and generation of the detected product ions. The data show c-type fragment ions, ions corresponding to the radical cation of the c-type fragments, c*, and they also show c*-1 peaks in the deuterated peptides only. The presence of the c*-1 peaks is best explained by radical-mediated scrambling of the deuterium atoms in the long-lived, metastable, radical intermediate complex formed by initial electron capture, followed by dissociation of the complex. These data suggest the presence of at least two mechanisms, one slow, one fast. The abundance of H* and -CO losses from the precursor ion changed upon deuterium labeling indicating the presence of a kinetic isotope effect, which suggests that the values reported here represent an underestimation of radical migration and H/D scrambling in the observed fragments.

Amino Acid Sequence↗

PubMed2006

Detecting deamidation products in proteins by electron capture dissociation.

A nonenzymatic posttranslational modification of proteins and peptides is the spontaneous deamidation of asparaginyl residues via a succinimide intermediate to form a varying mixture of aspartyl and isoaspartyl residues. The isoaspartyl residue is generally difficult to detect particularly using mass spectrometry because isoaspartic acid is isomeric with aspartic acid so that there is no mass difference. However, electron capture dissociation has demonstrated the ability to differentiate the two isoforms in synthetic peptides using unique diagnostic ions for each form; the cr. + 58 and z(l-r) - 57 fragment ions for the isoAsp form and the Asp side chain loss ((M + nH)(n-1)+. - 60) for the Asp form. Shown here are three examples of isoaspartyl detection in peptides from proteins; a deamidated tryptic peptide of cytochrome c, a tryptic peptide from unfolded and deamidated ribonuclease A, and a tryptic peptide from calmodulin deamidated in its native state. In all cases, the cr. + 58 and z(l-r) - 57 ions allowed the detection and localization of isoaspartyl residues to positions previously occupied by asparaginyl residues. The (M + nH)(n-1)+. - 60 ions were also detected, indicating the presence of aspartyl residues. Observation of these diagnostic ions in peptides from proteins shows that the method is applicable to defining the isomerization state of deamidated proteins.

Alkylation↗

PubMed2006

Algorithms for automatic interpretation of high resolution mass spectra.

Automated interpretation of high-resolution mass spectra in a reliable and efficient manner represents a highly challenging computational problem. This work aims at developing methods for reducing a high-resolution mass spectrum into its monoisotopic peak list, and automatically assigning observed masses to known fragment ion masses if the protein sequence is available. The methods are compiled into a suite of data reduction algorithms which is called MasSPIKE (Mass Spectrum Interpretation and Kernel Extraction). MasSPIKE includes modules for modeling noise across the spectrum, isotopic cluster identification, charge state determination, separation of overlapping isotopic distributions, picking isotopic peaks, aligning experimental and theoretical isotopic distributions for estimating a monoisotopic peak's location, generating the monoisotopic mass list, and assigning the observed monoisotopic masses to possible protein fragments. The method is tested against a complex top-down spectrum of bovine carbonic anhydrase. Results of each of the individual modules are compared with previously published work.

Algorithms↗

PubMed2005

Differentiation of aspartic and isoaspartic acids using electron transfer dissociation.

Electron-transfer dissociation allows differentiation of isoaspartic acid and aspartic acid residues using the same c + 57 and z - 57 peaks that were previously observed with electron capture dissociation. These peaks clearly define both the presence and the position of isoaspartic acid residues and they are relatively abundant. The lower resolution of the ion trap instrument makes detection of the aspartic acid residue's diagnostic peak difficult because of interference with side-chain fragment ions from arginine residues, but the aspartic acid residues are still clearly observed in the backbone cleavages and can be inferred from the absence of the isoaspartic acid diagnostic ions.

Aspartic Acid↗

PubMed2005

Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins.

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.

Equipment Design↗

PubMed2005

A minimum thickness gate valve with integrated ion optics for mass spectrometry.

A minimum thickness gate valve design for mass spectrometry is described in detail. The ion optics required to transmit ions from the source to the ICR cell are integrated into the design to minimize fringe field effects on the ions as they travel through the gate valve. The total thickness of the complete gate valve assembly is 1.03 in. (26.2 mm) with a maximum fringe field distance of 0.065 in. (1.7 mm). The gate valve is able to maintain a vacuum of <10(-10) mbar at the ICR cell when the source is vented to atmosphere and the estimated ion transfer efficiency is >95%.

Animals↗

PubMed2005

Ganglioside analysis by thin-layer chromatography matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry.

Metastable decomposition of ions generated in matrix-assisted laser desorption/ionization (MALDI) mass spectrometers complicates analysis of biological samples that have labile bonds. Recently, several academic laboratories and manufacturers of commercial instruments have designed instruments that introduce a cooling gas into the ion source during the MALDI event and have shown that the resulting vibrational cooling stabilizes these labile bonds. In this study, we compared stabilization and detection of desorbed gangliosides on a commercial orthogonal time-of-flight (oTOF) instrument with results we reported previously that had been obtained on a home-built Fourier transform mass spectrometer. Decoupling of the desorption/ionization from the detection steps resulted in an opportunity for desorbing thin-layer chromatography (TLC)-separated gangliosides directly from a TLC plate without compromising mass spectral accuracy and resolution of the ganglioside analysis, thus coupling TLC and oTOF mass spectrometry. The application of a declustering potential allowed control of the matrix cluster and matrix adduct formation, and, thus, enhanced the detection of the gangliosides.

Chromatography, Thin Layer↗

PubMed2005

Deamidation: Differentiation of aspartyl from isoaspartyl products in peptides by electron capture dissociation.

Deamidation of asparaginyl and isomerization of aspartyl residues in proteins proceed through a succinimide intermediate producing a mixture of aspartyl and isoaspartyl residues. Isoaspartic acid is an isomer of aspartic acid with the C(beta) incorporated into the backbone, thus increasing the length of the protein backbone by one methylene unit. This post-translation modification is suspected to contribute to the aging of proteins and to protein folding disorders such as Alzheimer's disease, so that differentiating the two isomers becomes important. This manuscript reports that distinguishing aspartyl from isoaspartyl residues in peptides has been accomplished by electron capture dissociation (ECD) using a Fourier transform mass spectrometer (FTMS). Model peptides with aspartyl residues and their isoaspartyl analogs were examined and unique peaks corresponding to c(n)*+58 and z(l-n)-57 fragment ions (n, position of Asp; l, total number of amino acids in the peptide) were found only in the spectra of the peptides with isoaspartyl residues. The proposed fragmentation mechanism involves cleavage of the C(alpha)-C(beta) backbone bond, therefore splitting the isoaspartyl residue between the two fragments. Also, a complementary feature observed specific to aspartyl residues was the neutral loss of the aspartic acid side chain from the charge reduced species. CAD spectra of the peptides from the same instrument demonstrated the improved method because previously published CAD methods rely on the comparison to the spectra of standards with aspartyl residues. The potential use of the top-down approach to detect and resolve products from the deamidation of asparaginyl and isomerization of aspartyl residues is discussed.

Aspartic Acid↗

PubMed2004

Coupling thin-layer chromatography with vibrational cooling matrix-assisted laser desorption/ionization Fourier transform mass spectrometry for the analysis of ganglioside mixtures.

Thin-layer chromatography (TLC), which is widely used for separation of glycolipids, oligosaccharides, lipids, and compounds of environmental and pharmaceutical interest, can be readily coupled to matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometers, but this arrangement usually compromises mass spectral resolution due to the irregularity of the TLC surface. However, TLC can be coupled to an external ion source MALDI-Fourier transform (FT) MS instrument without compromising mass accuracy and resolution of the spectra. Furthermore, when the FTMS has a vibrationally cooled MALDI ion source, fragile glycolipids can be desorbed from TLC plates without fragmentation, even to the point that desorption of intact molecules from "hot"matrixes such as alpha-cyano-4-hydroxycinnamic acid is possible. In this work, whole brain gangliosides are separated using TLC; the TLC plates are attached directly to the MALDI target, where the gangliosides are desorbed, ionized, and detected in the FTMS with >70 000 resolving power.

Carbohydrate Sequence↗

PubMed2004

Use of statistical methods for estimation of total number of charges in a mass spectrometry experiment.

Estimation of the number of ions in a mass spectrometry experiment is needed to determine instrumentation parameters such as ionization efficiency, collision-induced dissociation efficiency, ion-transfer efficiency, ion trapping efficiency, and preamplifier detection limit. This work aims at analyzing the statistical characteristics (primarily variance) in the intensities of the isotopic distributions, which depend on the number of ions in the cell. A mathematical derivation was developed based on the maximum likelihood estimation method, which estimates the most likely number of ions in the cell using a method known as nonrandom parameter estimation. The performance of the method improves with increase in the number of observed distributions. The method works well provided the spectra show isotopic resolution and is independent of the instrument or method used to arrive at the spectra.

Carbon Isotopes↗

PubMed2004

T cell activation by lipopeptide antigens.

Unlike major histocompatibility proteins, which bind peptides, CD1 proteins display lipid antigens to T cells. Here, we report that CD1a presents a family of previously unknown lipopeptides from Mycobacterium tuberculosis, named didehydroxymycobactins because of their structural relation to mycobactin siderophores. T cell activation was mediated by the alphabeta T cell receptors and was specific for structure of the acyl and peptidic components of these antigens. These studies identify a means of intracellular pathogen detection and identify lipopeptides as a biochemical class of antigens for T cells, which, like conventional peptides, have a potential for marked structural diversity.

Antigen Presentation↗

PubMed2004

A high pressure matrix-assisted laser desorption ion source for Fourier transform mass spectrometry designed to accommodate large targets with diverse surfaces.

A new design for a high pressure MALDI-FTMS instrument is described and initial data are shown. The instrument incorporates a large, 10 cm x 10 cm, sample translation stage to accommodate and position the MALDI target. The new instrument allows coupling to a wide variety of surface techniques such as gel electrophoresis or surface plasmon resonance. Coupling to thin layer chromatography is shown. Furthermore, a new nozzle design allows high pressure collisional cooling sufficient to stabilize gangliosides while minimizing the gas load on the system.

Chromatography, Thin Layer↗

PubMed2003

Attomole peptide analysis by high-pressure matrix-assisted laser desorption/ionization Fourier transform mass spectrometry.

A new high-pressure matrix-assisted laser desorption/ionization (HP-MALDI) source for FTMS has recently been described (O'Connor et al. J. Am. Soc. Mass Spectrom., in press). Improvements to the source design, including the incorporation of a new high-pressure gas channel plate, resulted in ions devoid of metastable fragmentation and also in increased sensitivity compared to the HP-MALDI prototype source design. The focus of this contribution is the evaluation of the current HP-MALDI FTMS configuration. The use of nonconductive sample surfaces, such as Parafilm and Teflon, was explored, and spectra from 30 amol of peptide applied to these surfaces were routinely obtained. In addition, the current limit of detection for this configuration is demonstrated to be 300 zmol for the phosphopeptide RRREEE(pS)EEEAA using multishot accumulation of the ions from 15 laser shots in the hexapole and 1 scan. In addition, the performance of the new HP-MALDI FTMS configuration and its potential application for high-throughput proteomics analyses are discussed.

Peptides↗