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Biomedical subjects

F W McLafferty

Publications and source records attributed to F W McLafferty.

At least 55 records · Page 3Linked to original sources
PubMed1995

Surface-induced dissociation of multiply-protonated proteins.

A novel surface design compatible with the open cell geometry allows nonglancing angle collisions of selected ions stored in a Fourier transform mass spectrometer. Dissociation efficiencies of 36%, 22%, and 14% are achieved for gramicidin S, melittin, and carbonic anhydrase (29 kDa), respectively. Ion neutralization by the surface, which is highly competitive for many singly-charged ions, is minimal, and dissociation products of hypervalent neutral species are not detected. Instead, the spectra are similar to those from collisionally activated and infrared multiphoton dissociation; the fragmentation pathways are relatively independent of the method of energy deposition. For carbonic anhydrase, however, the single event excitation inherent to surface-induced dissociation appears to minimize secondary fragmentation, a critical advantage for tandem mass spectrometry of such large ions. Electrically floating the open cell below ground greatly enhances the collection efficiency.

Carbonic Anhydrases↗
PubMed1995

Verification of 50- to 100-mer DNA and RNA sequences with high-resolution mass spectrometry.

Electrospray ionization with Fourier-transform mass spectrometry achieves accurate (< 50-ppm) determination of molecular weights of nucleotides, verifying structures of biological RNA and synthetic single-stranded DNA. High (1o(5)) resolving power makes possible detection of subpicomole impurities and adducts that confuse lower-resolution measurements. Molecular ions in a spectrum of 76-mer tRNA(Phe) had 34-55 Na adducts; when desalted, these show a molecular mass of 24,950.5 Da (expected, 24,950.3 Da) and minor variants at approximately -15 and +15 Da. A 50-mer DNA is characterized with < 10-ppm mass error, with detection of both N + 1 and N - 1 failure sequences. Special electrospray ionization conditions are necessary for a 72-mer to minimize fragmentation in the ion source. Despite the chemical noise from this, as well as failed sequences from automated synthesis, the spectrum of a 100-mer single-stranded DNA yielded a molecular mass of 30,702.4 +/- 1 Da, in good agreement with the expected value, 30,702.1 Da.

Base Sequence↗
PubMed1995

Distinguishing N- and C-terminus ions for mass spectrometry sequencing of proteins without prior degradation.

Identifying complementary pairs of fragment ions, b-type vs y-type, is a key step in mass spectral sequencing of proteins without degradation prior to ionization. Observations that b-type ions tend to be less stable than y-type have been extended to carbonic anhydrase (29 kDa) as a model protein. Infrared multiphoton dissociation (IRMPD) of 24+, 27+, 30+ molecular ions shows y-type ions from complementary pairs require, on average, 27% longer irradiation times for onset of abundance decrease, while in nozzle/skimmer dissociation of all charge states, y-type ions require a 20% higher potential difference. In some cases, b-ion dissociation appears to accompany its formation, as its rate of increase with increasing energy deposition is far less than that of its y-complement.

Acetylation↗
PubMed1994

Collisional activation of large multiply charged ions using Fourier transform mass spectrometry.

For small singly charged ions, Fourier transform mass spectrometry (FTMS) has demonstrated the ability to perform multistage mass spectral experiments (MSn) with high resolution and high mass accuracy using collisionally activated dissociation (CAD). The combination of electrospray ionization (ESI) with the FTMS provides the potential to extend these capabilities for structural characterization of large biomolecules. The standard FTMS-CAD method is problematic in that it is inefficient and produces ions well away from the center of the cell. More efficient collisional methods have been demonstrated for small molecules (sustained off-resonance irradiation (SORI) CAD, very low energy (VLE) CAD, multiple excitation collisional activation (MECA)) that provide the additional benefit of producing product ions closer to the center of the trapped ion cell. The efficiency (> 92%) for producing sequence-informative peaks from large multiply charged ions is far better than standard CAD. Disadvantages, such as blind spots where no product ions are observed, and isotopic distortions which can cause an incorrect mass assignment, must be considered when methods designed for small singly charged ions are applied to large multiply charged ions. SORI provided the highest efficiency, selectivity, and resolving power (9 x 10(5)) for product ion spectra and is recommended for most applications because of its ease of implementation.

Animals↗
PubMed1994

Infrared multiphoton dissociation of large multiply charged ions for biomolecule sequencing.

Characterization and verification of the structures of large biomolecules with high-resolution tandem Fourier transform mass spectrometry with electrospray ionization is critically dependent on the technique used to fragment the multiply charged ions produced. Infrared multiphoton dissociation (IRMPD) of ionized proteins as large as carbonic anhydrase (29 kDa) yields fragment information similar to, but with valuable additions to, that of other dissociation techniques. IRMPD yields product ions on-axis, providing efficient dissociation in further stages; MS3 of ubiquitin (8.6 kDa) yields 11 new sequence ions. Optimum irradiation times for protein ion dissociation vary by more than a factor of 6, with times for oligonucleotides far lower, possibly due to photon resonance with a P-O stretching frequency. IRMPD provides far greater selectivity than collisionally activated dissociation and also appears to yield much less mass discrimination and to dissociate much more stable ions. A technique to remove product ions on formation from the laser beam should improve the present efficiencies of 30-80%.

Animals↗
PubMed1994

High-resolution tandem mass spectrometry of carbonic anhydrase.

Electrospray ionization of carbonic anhydrase with ion dissociation yields a mass spectrum from which the masses of > 100 isotopic clusters are determined accurately, with the number of charges assigned directly from resolved isotopic peaks. Of these clusters, 80% correspond to fragmentation at or near the amino acid proline. The masses of combinations of two, three, and four of these clusters sum to the molecular mass with 0.1-Da accuracy, while further fragment ion dissociation provides additional sequence information. The capability of this methodology to detect sequence variations is illustrated with an isozyme having a single amino acid replacement.

Amino Acids↗
PubMed1994

Mass spectrometry in the development of drugs from traditional medicines.

The mass spectrometer ionizes molecules to separate and weigh the resulting molecular ions and their dissociation products. These product masses indicate directly the sequence of constituents of the original molecule, such as amino acids in proteins or bases in nucleotides. Because single ions can be detected, even subfemtomole sensitivities are possible. In tandem mass spectrometry (MS/MS), molecular ions from a mixture can be separated to isolate ions of a specific component, whose further dissociation can then give structural information on that component. Electrospray ionization and other recently developed methods make possible the ionization of biomolecules even larger than 100 kDa. The Fourier-transfer mass spectrometer has unusual capabilities for measuring ions over a 100 kDa mass range simultaneously at unit resolution for MS/MS applications utilizing subfemtomole sample quantities.

Mass Spectrometry↗
PubMed1993

Coexisting stable conformations of gaseous protein ions.

For further insight into the role of solvent in protein conformer stabilization, the structural and dynamic properties of protein ions in vacuo have been probed by hydrogen-deuterium exchange in a Fourier-transform mass spectrometer. Multiply charged ions generated by electrospray ionization of five proteins show exchange reactions with 2H2O at 10(-7) torr (1 torr = 133.3 Pa) exhibiting pseudo-first-order kinetics. Gas-phase compactness of the S-S cross-linked RNase A relative to denatured S-derivatized RNase A is indicated by exchange of 35 and 135 hydrogen atoms, respectively. For pure cytochrome c ions, the existence of at least three distinct gaseous conformers is indicated by the substantially different values--52, 113, and 74--of reactive H atoms; the observation of these same values for ions of a number--2, 7, and 5, respectively--of different charge states indicates conformational insensitivity to coulombic forces. For each of these conformers, the compactness in vacuo indicated by these values corresponds directly to that of a known conformer structure in the solution from which the conformer ions are produced by electrospray. S-derivatized RNase A ions also exist as at least two gaseous conformers exchanging 50-140 H atoms. Gaseous conformer ions are isometrically stable for hours; removal of solvent greatly increases conformational rigidity. More specific ion-molecule reactions could provide further details of conformer structures.

Cytochrome c Group↗
PubMed1992

High-resolution tandem mass spectrometry of large biomolecules.

Unit-resolution mass spectra have been obtained for peptides as large as 17 kDa, providing information on impurities and adduct ions, as well as accurate molecular weight values. Electrospray ionization produces many multiply-charged species of the same mass; isotopic peak resolution provides direct charge state assignment from the unit mass spacing of the isotopes. This is of special value when the spectrum also has many masses, such as from precursor ion dissociation or impurities. Mass measuring errors not only are concomitantly lower (less than 0.1 Da) than when the isotopic peaks are unresolved but also are independent of variations in 13C/12C natural isotopic abundances. Also, larger errors are avoided that occur when the measured peak envelope includes impurity or adduct ions. This also benefits tandem mass spectrometry; dissociation of peptide ions as large as 8.5 kDa yields fragment masses consistent (less than 0.1 Da) with their amino acid sequences.

Albumins↗
PubMed1990

Hadamard transform measurement of tandem Fourier-transform mass spectra.

The simultaneous collection of multiple spectra using tandem (MS/MS) and multidimensional (MS/MS/MS) mass spectrometry from multiple precursors is demonstrated to yield correspondingly enhanced sensitivity. This approach utilizes Hadamard transform deconvolution and takes advantage of the multichannel dissociation capability of Fourier-transform mass spectrometry. By application of this to an 11-component mixture, the 11 spectra of the products of dissociating 11 different combinations of six of the component molecular ions are measured; Hadamard transformation yields individual spectra of the precursor ions exhibiting a signal-to-noise improvement of 1.8x over spectra measured separately, as predicted by theory. Precursor ion selection with high specificity and product formation with high abundance reproducibility are critical; spurious peaks resulting from imperfect reproducibility can be minimized by using simultaneous equation coefficients reflecting the degree of precursor dissociation. Extension of this technique to MSn spectra is demonstrated with simultaneous MS/MS/MS monitoring of three precursors and three daughters yielding nine spectra representing the nine possible dissociation pathways. For MSn spectra, coding the product relationships for each additional step (e.g., precursor----daughter, daughter----granddaughter) requires elimination of half of the remaining ions. No ions are lost for coding in an improved Hadamard approach in which the combined daughter spectrum of the selected half of the precursors is subtracted from that of the other half.

Fourier Analysis↗
PubMed1990

Studies of unusual simple molecules by neutralization-reionization mass spectrometry.

Reactive or unstable molecules are key intermediates in many important reactions, but can be difficult to prepare for experimental studies. Species with missing (:CH-OH) or extra (H3) substituents can often be formed conveniently in the gas phase by neutralizing a beam of a more stable ionic counterpart (CH = O+H, H3+). Reionization of the neutral after approximately 10(-6) seconds tests its stability, whereas its unimolecular chemistry can be probed by preparing it with different amounts of internal energy. The resulting neutral products are reionized and mass analyzed. Isomers are then characterized by ion dissociation and a third mass-analysis step. Many unusual molecules have been characterized with this technique, which can also be used to probe complex unimolecular chemistry, such as that of cyclobutadiene and ethylene oxide.

Butadienes↗
PubMed1989

Fourier-transform mass spectrometry of large molecules by electrospray ionization.

The multiply charged ions produced by electrospray ionization of peptides of molecular masses up to 29 kDa have been successfully introduced into a Fourier transform mass spectrometer of unique capabilities for tandem mass spectrometry, large ion dissociation, and resolution. Electrospray ionization places an unusually high number of charges on a peptide yielding mass/charge (m/z) values of 600-1500; in this range at normal operating pressures (approximately 10(-9) torr; 1 torr = 133.3 Pa) Fourier-transform mass spectrometry resolving power is greater than 100,000. Although only 10(-7) torr pressure has been obtained with the initial interface, the resulting resolving power of 5000 makes possible the resolution of isotopic peaks of multiply charged ions. Mass measuring accuracies of a few daltons for molecular masses up to 17 kDa have also been achieved.

Enzymes↗