Biomedical subjects

A G Marshall

Publications and source records attributed to A G Marshall.

At least 19 recordsLinked to original sources

PubMed2004

Characterization of Tetrahymena histone H2B variants and posttranslational populations by electron capture dissociation (ECD) Fourier transform ion cyclotron mass spectrometry (FT-ICR MS).

This work describes the nature and sequence information content of the electron capture dissociation mass spectra for the intact Tetrahymena histone H2B. Two major variants of this protein were present bearing nominal modifications of both +42 and +84 Da. This work describes identification of the nature of these two modifications. For example, using gas-phase selection and isolation of the +42-Da modified species, from a background of two H2B variants each present in six or more posttranslationally modified isoforms, we were able to determine that this +42-Da modification isoform bears trimethylation rather than acetylation. LC-CIDMS analysis was also employed on digested preparations to obtain complementary detail of the nature of site-specific posttranslational modifications. This study establishes that integration of the information from these two datasets provides a comprehensive map of posttranslational occupancy for each particular covalent assemblage selected for structural investigation.

Amino Acid Sequence↗

PubMed2001

Kendrick mass defect spectrum: a compact visual analysis for ultrahigh-resolution broadband mass spectra.

At currently achievable Fourier transform ion cyclotron resonance broadband mass spectrometry resolving power (m/deltam50% > 350,000 for 200 < m/z < 1,000), it would be necessary to spread out a conventional mass spectrum over approximately 200 m in order to provide visual resolution of the most closely resolved peaks. Fortunately, there are natural gaps in a typical mass spectrum, spaced 1 Da apart, because virtually no commonly encountered elemental compositions yield masses at those values. Thus, it is possible to break a broadband mass spectrum into 1-Da segments, rotate each segment by 90 degrees, scale each segment according to its mass defect (i.e., difference between exact and nominal mass), and then compress the spacing between the segments to yield a compact display. For hydrocarbon systems, conversion from IUPAC mass to "Kendrick" mass (i.e., multiplying each mass by 14.00000/14.01565) further simplifies the display by rectilinearizing the peak patterns. The resulting display preserves not only the "coarse" spacings (e.g., approximately 1 Da between odd and even masses, corresponding to either even vs odd number of nitrogens or 12C(c) vs 12C(c-1)13C1 elemental compositions of the same molecule; approximately 2-Da separations, corresponding to a double bond or ring; approximately 14 Da separations, corresponding to one CH2 group) but also the "fine structure" (i.e., different mass defects for different elemental compositions) across each 1-Da segment. The method is illustrated for experimental electrospray ionization FTICR ultrahigh-resolution mass spectra of a petroleum crude oil. Several thousand elemental compositions may be resolved visually in a single one-page two-dimensional display, and various compound families-class (NnOoSs), type (Z in C(c)H2(c+z)NnOoSs), and alkylation series-may be identified visually as well.

Journal Article↗

PubMed2001

Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptic to yield complementary sequence information.

Glycoproteins are a functionally important class of biomolecules for which structural elucidation presents a challenge. Fragmentation of N-glycosylated peptides, employing collisionally activated dissociation, typically yields product ions that result from dissociation at glycosidic bonds, with little occurrence of dissociation at peptide backbone sites. We have applied two dissociation techniques, electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD), in a 7-T Fourier transform ion cyclotron resonance mass spectrometer, in the investigation of an N-glycosylated peptide from an unfractionated tryptic digest of the lectin of the coral tree, Erythrina corallodendron. ECD provided c and z. ions derived from the peptide backbone, with no observed loss of sugars. Cleavage at 11 of 15 backbone amine bonds was observed. The lack of cleavage at sites located close to the glycosylated asparagine residue may result from steric blocking by the glycan. IRMPD provided abundant fragment ions, primarily through dissociation at glycosidic linkages. The monosaccharide composition and the presence of three glycan branch sites could be determined from the IRMPD fragments. The two types of spectra, obtained with the same instrument, thus provide complementary structural information about the glycopeptide. The current result extends the applicability of ECD for glycopeptide analysis to N-glycosylated peptides and to peptides containing branched, highly substituted glycans.

Electrons↗

PubMed2001

High-sensitivity electron capture dissociation tandem FTICR mass spectrometry of microelectrosprayed peptides.

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/microL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics.

Animals↗

PubMed2001

High sensitivity Fourier transform ion cyclotron resonance mass spectrometry for biological analysis with nano-LC and microelectrospray ionization.

Modifications to a 7 T nano-LC micro-ESI FT-ICR mass spectrometer, including a shorter octopole, approximately 100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 microL, 50 amol/microL) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 microL, 500 amol/microL) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with arg8-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 microL, 300 amol/microL) and a total of 100 amol in water (1 microL, 100 amol/microL). Arg8-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of FT-ICR to detect and identify a true endogenous biological analyte.

Animals↗

PubMed2001

Baseline mass resolution of peptide isobars: a record for molecular mass resolution.

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, approximately 904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of approximately 3 300 000. The elemental compositions of these molecules differ by N40 vs. S2H8 (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.

Amino Acid Sequence↗

PubMed2001

McLeod neuroacanthocytosis: genotype and phenotype.

McLeod syndrome is caused by mutations of XK, an X-chromosomal gene of unknown function. Originally defined as a peculiar Kell blood group variant, the disease affects multiple organs, including the nervous system, but is certainly underdiagnosed. We analyzed the mutations and clinical findings of 22 affected men, aged 27 to 72 years. Fifteen different XK mutations were found, nine of which were novel, including the one of the eponymous case McLeod. Their common result is predicted absence or truncation of the XK protein. All patients showed elevated levels of muscle creatine phosphokinase, but clinical myopathy was less common. A peripheral neuropathy with areflexia was found in all but 2 patients. The central nervous system was affected in 15 patients, as obvious from the occurrence of seizures, cognitive impairment, psychopathology, and choreatic movements. Neuroimaging emphasized the particular involvement of the basal ganglia, which was also detected in 1 asymptomatic young patient. Most features develop with age, mainly after the fourth decade. The resemblance of McLeod syndrome with Huntington's disease and with autosomal recessive chorea-acanthocytosis suggests that the corresponding proteins--XK, huntingtin, and chorein--might belong to a common pathway, the dysfunction of which causes degeneration of the basal ganglia.

Adult↗

PubMed2001

Gas-phase hydrogen/deuterium exchange of positively charged mononucleotides by use of Fourier-transform ion cyclotron resonance mass spectrometry.

The gas-phase structures of protonated (deoxy)nucleoside-5'- and 3'-monophosphates (mononucleotides) have been examined by the use of gas-phase hydrogen/deuterium (H/D) exchange and high-field Fourier-transform ion cyclotron resonance mass spectrometry. These nucleotides were reacted with three different deuterating reagents: ND3, D2O, and D2S, of which ND3 was the most effective. All mononucleotides fully exchanged their labile hydrogen for deuterium with ND3 with the exception of deoxycytidine-3'-monophosphate, deoxyadenosine-5'-monophosphate, adenosine-5'-monophosphate, and adenosine-3'-monophosphate. Semiempirical calculations demonstrate the presence of hydrogen bonding upon protonation of the purine mononucleotides which may lead to incomplete H/D exchange. H/D exchange rates differed between the deoxymononucleotides and the ribomononucleotides, suggesting that the 2'-OH group plays an important role in the exchange process. Reactions of nucleosides and mononucleotides with D2O demonstrate that a structure-specific long-lived ion-molecule complex between D2O and the mononucleotide involving the phosphate group is necessary for exchange to overcome the high-energy activation barrier. In contrast, a structure-specific long-lived ion-molecule complex between the mononucleotides and ND3 is not required for exchange to occur.

Cyclotrons↗

PubMed2001

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: mass-based protein identification without liquid chromatographic separation.

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ("Edman" reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and y(n-1) fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1+ ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.

Amino Acid Sequence↗

PubMed2001

Gas phase RNA and DNA ions 2. Conformational dependence of the gas-phase H/D exchange of nucleotide-5'-monophosphates.

The conformational dependence of the gas-phase hydrogen/deuterium (H/D) exchange of nucleotide-5-monophosphate anions with the H/D exchange reagent D2S is reported here. The electrospray-generated [M-H]- anions of adenosine-5'-monophosphate, adenosine-5'-carboxylic acid, ribitol-5-phosphate, and 2-deoxy-ribitol-5-phosphate were reacted with D2S in the gas phase. Their reactivity (adenosine-5'-monophosphate exchanged 2 of 5 labile hydrogens, adenosine-5'-carboxylic acid exchanged 1 of 4, ribitol-5-phosphate exchanged 2 of 3, and 2-deoxy-ribitol-5-phosphate exchanged 1 of 2) suggests that the hydroxyl group in the 2 position of the ribose sugar and the amino hydrogen on the nucleobase do not exchange readily with D2S. Semiempirical molecular orbital calculations suggest that the labile hydrogens in these positions are thermodynamically facile to exchange but as a conformation inaccessible to the presumed phosphate anion, consistent with a mechanism in which the phosphate anion complexes with the exchange reagent and assists H/D exchange at a neighboring site.

Adenosine↗

PubMed2001

Electrospray ionization Fourier transform mass spectrometric analysis of wine.

Positive- and negative-ion electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has been used for direct analysis of five wines (California Red, Corbiere, Zinfandel, Beaujolais, and Sauvignon Blanc), without any prior separation or purification steps. The high mass resolving power (typically m/Delta m(50%) > or = 80,000, in which Delta m(50%) is mass spectral peak full width at half-maximum peak height) and mass accuracy (< or =1 ppm) of FT-ICR mass spectrometry make it ideal for the study of complex mixtures such as wine, because the components are simultaneously resolved and identified as to elemental composition. Moreover, the high dynamic range of the instrument is advantageous for identifying trace components. The positive-ion mass spectra obtained from the wines were somewhat similar and were dominated by sucrose and (for red wines) anthocyanins. More than 30 compounds (phenolics and carbohydrates) were identified. The negative-ion mass spectra exhibited much greater variation among different wines, with several compounds peculiar to each wine. Elemental compositions could be assigned with high confidence to 76-94% of negative ions of >10% relative abundance. The present results suggest that it may be possible to fingerprint a wine on the basis of its negative-ion ESI FT-ICR mass spectrum.

Carbohydrates↗

PubMed2001

Compositional analysis for identification of arson accelerants by electron ionization Fourier transform ion cyclotron resonance high-resolution mass spectrometry.

Elemental compositions of each of 100 to 500 different constituents (i.e., every peak in a mass-to-charge ratio range, 50 < m/z < 300) of lighter fluid, kerosene, turpatine, gasoline, diesel fuel, and two brands of mineral spirits (and their weathered analogs) make possible direct identification of each accelerant in a experimental fire, based on electron ionization 6.0 Tesla Fourier transform ion cyclotron resonance (EI FT-ICR) ultrahigh resolution mass spectrometry. Septum injection of as little as 500 nL of accelerant into an all-glass heated inlet system yields definitive elemental compositions (molecular formulas) based on accurate (< +/-1 ppm average error) mass measurement alone. Extraction and EI FT-ICR mass analysis of fire debris from a controlled burn of a couch with simple (lighter fluid) and complex (turpatine) ignitable liquid yielded dozens of elemental compositions serving as a unique "fingerprint" for each petroleum product, despite the presence of up to 249 additional extracted matrix and pyrolysis components. Forty-five of 56 lighter fluid constituents and 126 of 133 turpatine constituents (not counting 13C-containing species) were identified in the debris from a fire staged for each respective accelerant.

Journal Article↗

PubMed2000

Stable isotope incorporation triples the upper mass limit for determination of elemental composition by accurate mass measurement.

By comparing electrospray ionization Fourier-transform ion cyclotron resonance (FT-ICR) mass spectra and collision-induced dissociation (CID) FT-ICR mass spectra of a phospholipid (851 Da) extracted from natural abundance and 99% 13C bacterial growth media, we are able to reduce its number of possible elemental compositions (based on +/-10 ppm externally calibrated mass accuracy and biologically relevant compositional constraints) from 394 to 1. The basic idea is simply that the mass of a molecule containing N carbon atoms increases by N Da when 12C is replaced by 13C. Once the number of carbons is known, the number of possible combinations of other atoms in the molecule is greatly reduced. We demonstrate the method for a stored-waveform inverse Fourier transform-isolated phospholipid from an extract of membrane lipids from Rhodococcus rhodochrous hydrocarbon-degrading bacteria grown on either natural abundance or 99% 13C-enriched mixtures of n-hexadecane and n-octadecane. We project that this method raises the upper mass limit for unique determination of elemental composition from accurate mass measurement by a factor of at least 3, thereby extending "chemical formula" determination to identification and sequencing of larger synthetic and bio-polymers: phospholipids, oligopeptides of more than three to four amino acids, DNA or RNA of more than two nucleotides, oligosaccharides of more than three sugars, etc. The method can also be extended to determination of the number of other atoms for which heavy isotopes are available (e.g., 15N, 34S, 18O, etc.).

Carbon Isotopes↗

PubMed2000

Competitive binding to the oligopeptide binding protein, OppA: in-trap cleanup in an Fourier transform ion cyclotron resonance mass spectrometer.

This communication demonstrates that gentle infrared laser heating can remove unwanted buffer adducts from a gas-phase protein complex without dissociating the complex itself. Specifically, noncovalent complexes of the oligopeptide-binding protein, OppA, bound to either (Ala)3 or LysTrpLys were electrosprayed from aqueous buffer solution into a 9.4 tesla Fourier transform ion cyclotron resonance mass spectrometer. In addition to the intact complexes, several additional buffer adduct species were produced under the conditions of the experiment. Irradiation of the trapped ion population with a continuous-wave infrared CO2 laser at relatively low power (2.5 W) for 1 s dissociated the buffer adducts but retained the intact protein:peptide complexes. Adduct-free complex(es) were then readily identified, and signal-to-noise ratio also increased by an order of magnitude because the same number of protein ions are distributed over fewer species. Higher IR power (5 W for 1 s) dissociated the adduct-free complex(es) without internal fragmentation. The present in-trap clean-up technique may prove especially useful for identifying and screening the combinatorial library ligands most strongly bound to a receptor in the gas phase.

Bacterial Proteins↗

PubMed2000

Unequivocal determination of metal atom oxidation state in naked heme proteins: Fe(III)myoglobin, Fe(III)cytochrome c, Fe(III)cytochrome b5, and Fe(III)cytochrome b5 L47R.

Unambiguous determination of metal atom oxidation state in an intact metalloprotein is achieved by matching experimental (electrospray ionization 9.4 tesla Fourier transform ion cyclotron resonance) and theoretical isotopic abundance mass distributions for one or more holoprotein charge states. The ion atom oxidation state is determined unequivocally as Fe(III) for each of four gas-phase unhydrated heme proteins electrosprayed from H2O: myoglobin, cytochrome c, cytochrome b5, and cytochrome b5 L47R (i.e., the solution-phase oxidation state is conserved following electrospray to produce gas-phase ions). However, the same Fe(III) oxidation state in all four heme proteins is observed after prior reduction by sodium dithionite to produce Fe(II) heme proteins in solution: thus proving that oxygen was present during the electrospray process. Those results bear directly on the issue of similarity (or lack thereof) of solution-phase and gas-phase protein conformations. Finally, infrared multiphoton irradiation of the gas-phase Fe(III)holoproteins releases Fe(III)heme from each of the noncovalently bound Fe(III)heme proteins (myoglobin, cytochrome b5 and cytochrome b5 L47R), but yields Fe(II)heme from the covalently bound heme in cytochrome c.

Animals↗

PubMed1999

Identification of intact proteins in mixtures by alternated capillary liquid chromatography electrospray ionization and LC ESI infrared multiphoton dissociation Fourier transform ion cyclotron resonance mass spectrometry.

Here we propose a novel method for rapidly identifying proteins in complex mixtures. A list of candidate proteins (including provision for posttranslational modifications) is obtained by database searching, within a specified mass range about the accurately measured mass (e.g., +/- 0.1 Da at 10 kDa) of the intact protein, by capillary liquid chromatography electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (LC ESI FT-ICR MS). On alternate scans, LC ESI infrared multiphoton dissociation (IRMPD) FT-ICR MS yields mostly b and y fragment ions for each protein, from which the correct candidate is identified as the one with the highest "hit" score (i.e., most b and y fragments matching the candidate database protein amino acid sequence masses) and sequence "tag" score (based on a series of fragment sequences differing in mass by 1 or 2 amino acids). The method succeeds in uniquely identifying each of a mixture of five proteins treated as unknowns (melittin, ubiquitin, GroES, myoglobin, carbonic anhydrase II), from more than 1000 possible database candidates within a +/- 500 Da mass window. We are also able to identify posttranslational modifications of two of the proteins (mellitin and GroES). The method is simple, rapid, and definitive and is extendable to a mixture of affinity-selected proteins, to identify proteins with a common biological function.

Algorithms↗

PubMed1999

Phase correction for collision model analysis and enhanced resolving power of fourier transform ion cyclotron resonance mass spectra.

Phase correction of FT-ICR data yields an absorption spectrum that offers a gain by up to a factor of 2 in mass resolving power (at half-maximum peak height), compared to conventional magnitude-mode display. That improvement is equivalent to doubling the applied magnetic field strength, without loss in signal-to-noise (S/N) ratio, provided that the time-domain data are padded with an equal number of zeroes before FFT. Our simple, visual, user-interactive algorithm quickly corrects for zero-order and first-order variation of phase with frequency. We find that the theoretical mass resolving power enhancement for pressure-limited absorption-mode over magnitude-mode line shape depends on the collision mechanism: factor of 1.40 for hard sphere vs 3(1/2) for Langevin (ion: induced dipole). Thus, the experimental enhancement in mass resolving power (factor of 1.43 +/- 0.09) for isotopically resolved peaks in the FT-ICR mass spectra of electrosprayed bovine carbonic anhydrase (approximately 29 kDa) directly supports the hard-sphere collision model. Optimal implementation of phasing requires the following: (a) a delay between excitation and detection of less than half of one sampling interval to avoid baseline "roll" and Gibb's oscillations; (b) accurate analog-to-digital conversion; (c) a sufficiently long acquisition period to yield several data points per absorption-mode peak width at half-maximum peak height; and (d) avoidance of FT-ICR apodization functions (e.g., Hamming and Hanning) that suppress the initial time-domain data. Pulsed single-frequency excitation (duration much less than the reciprocal of the Nyquist bandwidth) can eliminate higher than first-order variation of phase with frequency. Phased FT-ICR spectra should prove especially desirable for analysis of complex mixtures, for resolving isotopic distributions in electrosprayed multiply charged macromolecules and for characterizing ion collisions (and thus ion size and shape).

Animals↗

PubMed1999

Fourier transform ion cyclotron resonance mass spectrometric detection of small Ca(2+)-induced conformational changes in the regulatory domain of human cardiac troponin C.

Troponin C (TnC), a calcium-binding protein of the thin filament of muscle, plays a regulatory role in skeletal and cardiac muscle contraction. NMR reveals a small conformational change in the cardiac regulatory N-terminal domain of TnC (cNTnC) on binding of Ca2+ such that the total exposed hydrophobic surface area increases very slightly from 3090 +/- 86 A2 for apo-cNTnC to 3108 +/- 71 A2 for Ca(2+)-cNTnC. Here, we show that measurement of solvent accessibility for backbone amide protons by means of solution-phase hydrogen/deuterium (H/D) exchange followed by pepsin digestion, high-performance liquid chromatography, and electrospray ionization high-field (9.4 T) Fourier transform Ion cyclotron resonance mass spectrometry is sufficiently sensitive to detect such small ligand binding-induced conformational changes of that protein. The extent of deuterium incorporation increases significantly on binding of Ca2+ for each of four proteolytic segments derived from pepsin digestion of the apo- and Ca(2+)-saturated forms of cNTnC. The present results demonstrate that H/D exchange monitored by mass spectrometry can be sufficiently sensitive to detect and identify even very small conformational changes in proteins, and should therefore be especially informative for proteins too large (or too insoluble or otherwise intractable) for NMR analysis.

Amino Acid Sequence↗