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D E Clemmer

Publications and source records attributed to D E Clemmer.

17 recordsLinked to original sources
PubMed2001

Monitoring structural changes of proteins in an ion trap over approximately 10-200 ms: unfolding transitions in cytochrome c ions.

A new technique for studying the time dependence of conformational changes of gas-phase protein ions is described. In this approach, a short pulse of electrosprayed protein ions is introduced into an ion trap and stored. After a defined time period, the distribution of ions is ejected from the trap into an ion mobility/time-of-flight mass spectrometer. Combined measurements of mobilities and flight times in the mass spectrometer provide information about the abundances of different conformer types and charge-state distributions. By varying the storage time in the trap, it is possible to monitor changes in ion conformation that occur over extended time periods (approximately 10-200 ms). The method is demonstrated by examining changes in cytochrome c ion conformations for the +7 to +10 charge states.

Animals↗
PubMed2001

Collision-induced dissociation of mobility-separated ions using an orifice-skimmer cone at the back of a drift tube.

An ion mobility-mass spectrometry technique that incorporates a differentially pumped orifice-skimmer cone (OSC) region at the exit of the drift tube has been developed. The OSC region is similar in design to those used in electrospray ionization sources and offers improvements in ion transmission (by factors of approximately 5-10 compared with previous designs) and the ability to induce fragmentation of mobility-separated ions. The separation of ions prior to dissociation at the skimmer cone allows the origin of fragment ions to be examined. Here, we describe the experimental design and demonstrate the approach by examining fragment ions that are common to multiple charge states and different gas-phase ion conformations of electrosprayed angiotensin II and [Sar1, Val5, Ala8] angiotensin II peptides.

Angiotensin II↗
PubMed2001

Large anhydrous polyalanine ions: evidence for extended helices and onset of a more compact state.

Ion mobility measurements and molecular modeling calculations have been used to examine the conformations of large multiply charged polyalanine peptides. Two series of [Ala(n)+3H](3+) conformations which do not interconvert during the 10 to 30 ms experimental timescales are observed: a family of elongated structures for n = 18 to 39 and a series of more compact conformations for n = 24 to 41. The more compact state becomes the dominant conformer type for n > 32. Molecular modeling studies and comparisons of calculated collision cross sections with experiment indicate that the elongated ions have extended helical conformations. We suggest that the more compact state corresponds to a new conformer type: a folded hinged helix-coil state in which helical and coil regions have similar physical dimensions. The competition between extended and compact states is rationalized by considering differences in charge stabilization and entropy.

Models, Molecular↗
PubMed2001

Assessment of purity and screening of peptide libraries by nested ion mobility-TOFMS: identification of RNase S-protein binders.

Combinatorial peptide synthesis in combination with affinity selection and high-resolution ion mobility/time-of-flight mass spectrometry (IM/TOFMS) analysis has been used to investigate the binding of a series of 96 related eight-residue peptides (with the general sequence NH2-GX1X2FX3X4X5G-CO2H, where X1 = L, F, V, Y; X2 = N, F; X3 = E, V, T; X4 = V, L; X5 = V, L) to the ribonuclease S protein. A key advantage of this strategy is that the IM/ TOFMS approach allows the relative abundances of individual library components (including numerous sequence and structural isomers) to be characterized before and after screening. The relative binding interactions of different sequences are assessed by comparing IM/TOFMS data for those components that pass through the column (as well as those that bind) to data for the library prior to screening. The high-affinity sequences that are found in this study are compared with those selected from much larger combinatorial libraries. The results suggest that many expected sequences in the large libraries may be missing (e.g., due to issues such as failure of specific steps during the synthesis or differences in solubility). Comparison of the binding sequences obtained in these studies and those reported previously indicates that screening results from large libraries should be interpreted with caution.

Amino Acid Sequence↗
PubMed2001

Ion trap/ion mobility/quadrupole/time-of-flight mass spectrometry for peptide mixture analysis.

An ion trap/ion mobility/quadrupole/time-of-flight mass spectrometer has been developed for the analysis of peptide mixtures. In this approach, a mixture of peptides is electrosprayed into the gas phase. The mixture of ions that is created is accumulated in an ion trap and periodically injected into a drift tube where ions separate according to differences in gas-phase ion mobilities. Upon exiting the drift tube, ions enter a quadrupole mass filter where a specific mass-to-charge (m/z) ratio can be selected prior to collisional activation in an octopole collision cell. Parent and fragment ions that exit the collision cell are analyzed using a reflectron geometry time-of-flight mass spectrometer. The overall configuration allows different species to be selected according to their mobilities and m/z ratios prior to collision-induced dissociation and final MS analysis. A key parameter in these studies is the pressure of the target gas in the collision cell. Above a critical pressure, the well-defined mobility separation degrades. The approach is demonstrated by examining a mixture of tryptic digest peptides of ubiquitin.

Amino Acid Sequence↗
PubMed2001

Gas-phase separations of complex tryptic peptide mixtures.

High-resolution ion mobility and time-of-flight mass spectrometry techniques have been used to analyze complex mixtures of peptides generated from tryptic digestion of fourteen common proteins (albumin, bovine, dog, horse, pig, and sheep; aldolase, rabbit; beta-casein, bovine; cytochrome c, horse; beta-lactoglobulin, bovine; myoglobin, horse; hemoglobin, human, pig, rabbit, and sheep). In this approach, ions are separated based on differences in mobilities in helium in a drift tube and on differences in their mass-to-charge ratios in a mass spectrometer. From data recorded for fourteen individual proteins (over a m/z range of 405 to 1,000), we observe 428 peaks, of which 205 are assigned to fragments that are expected from tryptic digestion. In a separate analysis, the fourteen mixtures have been combined and analyzed as one system. In the single dataset, we resolve 260 features and are able to assign 168 peaks to unique peptide sequences. Many other unresolved features are observed. Methods for assigning peptides based on the use of m/z information and existing mobilities or mobilities that are predicted by use of intrinsic size parameters are described.

Amino Acid Sequence↗
PubMed2001

Prediction of peptide ion mobilities via a priori calculations from intrinsic size parameters of amino acid residues.

Ion mobility spectrometry (IMS) has recently been established as a powerful tool to separate the protease digest mixtures and identify their peptide components. As accurate calculation of mobilities is critical for this technique, a new rapid method based on intrinsic size parameters (ISPs) of amino acid residues has been devised. However, those parameters had to be obtained by tedious statistical analysis of a large body of experimental data. Here we demonstrate that they can instead be derived a priori, based on the stoichiometry of a residue. Our main finding is that the ISP of a residue is essentially determined by its density, that is, the average mass/size ratio of its constituent atoms. This is in accordance with an interpretation in which peptides assume compact conformations in the gas phase dominated by the solvation of ionic charge.

Algorithms↗
PubMed2000

Mobility labeling for parallel CID of ion mixtures.

An ion mobility/mass spectrometry technique has been developed to record collision-induced dissociation patterns for multiple ions in a parallel fashion. In this approach, a mixture of ions is separated in a drift tube on the basis of differences in mobilities through a buffer gas. As the ions exit the drift tube, they are accelerated into a collision cell and the ensuing fragment ions are dispersed by differences in mass-to-charge (m/z) ratios in a time-of-flight mass spectrometer. Fragment ions that are formed in the collision cell have drift times that are coincident with their antecedent parent ions, allowing the origin of all fragments formed from the mixture of ions to be determined. The approach is demonstrated by examining fragmentation patterns of the [M + H]+ parent and a series of a-, b-, and y-type fragments of [D-Ala2,3]methionine enkephalin.

Enkephalins↗
PubMed2000

Determining synthetic failures in combinatorial libraries by hybrid gas-phase separation methods.

A combinatorial tripeptide library having the general form D-Glu-Xxx-Xxx-CONH2 has been synthesized using a standard mix and split synthetic protocol that is expected to produce 676 components. All components of the mixture were analyzed using a new high-resolution ion mobility/time-of-flight mass spectrometer coupled with an electrospray ionization source. In this approach ions are separated by differences in their gas-phase mobilities prior to being introduced into the mass spectrometer for mass-to-charge analysis. The peptide library includes a wide range of different sequence, structural, and stereo isomers; trends in the number of expected and resolved isomers that are observed at each m/z ratio allow specific synthetic steps that have failed to be identified, even in the presence of other isomers. Information about the relative abundances of different isomers should dramatically improve the reliability of binding affinity studies from direct analysis of mixtures.

Chromatography, Gas↗
PubMed1999

Gas-phase separations of electrosprayed peptide libraries.

High-resolution ion mobility spectrometry has been combined with time-of-flight mass spectrometry for analysis of a combinatorial peptide library that is expected to contain 676 components. In this approach, the components of a mixture of three residue peptides, having the general form (D)Phe-Xxx-Xxx-CONH2 (where Xxx is randomized over 26 residues including 10 naturally occurring amino acids and 16 synthetic forms) were ionized by electrospray ionization. Ion mobility/time-of-flight distributions have been recorded for all ions using a nested drift(flight) time technique. The improvement in resolving power [(t/delta t) = 100-150 for singly charged ions] was illustrated by analysis of a mixture of tryptic digest peptides using high- and low-resolution instruments. The approach allows many components of the library (e.g., structural, sequence, and stereo isomers) that cannot be distinguished by mass spectrometry alone to be resolved. Impurities due to side reactions appear to be minimal, comprising < 10% of the total ion signal. Direct evidence for approximately 60-70% of the expected peptides is found. Variation in ion abundance for different components indicates that there are differences in solution concentrations or ionization efficiencies for the components.

Amino Acid Sequence↗
PubMed1999

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures.

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.

Cytochrome c Group↗
PubMed1999

A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections.

An ion trap/ion mobility/time-of-flight mass spectrometry technique has been used to measure collision cross sections for 660 peptide ions generated by tryptic digestion of 34 common proteins. Measured cross sections have been compiled into a database that contains peptide molecular weight and sequence information. The database is used to generate average intrinsic contributions to cross section (size parameters) for different amino acid residues by solving systems of equations that relate the unknown contributions of individual residues to the sequences and cross sections of database peptides. Size parameters are combined with information about amino acid composition to calculate cross sections for database peptides. Bona fide cross section predictions (made prior to measurement) for peptides observed in tryptic digests of sperm whale myoglobin and yeast enolase are made. Eight of 10 predicted cross sections are within 2% of the experimental values and all 10 are within 3.2%. The utility of size parameters for cross section prediction is explored and discussed.

Algorithms↗
PubMed1998

Three-dimensional ion mobility/TOFMS analysis of electrosprayed biomolecules.

An ion mobility/mass spectrometry technique has been developed to record mass-resolved ion mobility distributions for multiple ions simultaneously. The approach involves a new instrument that couples an electrospray ion source to an injected-ion drift tube/time-of-flight mass spectrometer. Individual components in a mixture of ions are separated by mobility differences in a drift tube and subsequently dispersed by mass-to-charge ratios in a time-of-flight instrument. Flight times in the mass spectrometer are much shorter than residence times in the drift tube, making it possible to record mass-resolved ion mobilities for all ions simultaneously. The result is a three-dimensional spectrum that contains collision cross section, mass-to-charge, and ion abundance information. The instrument and data acquisition system are described. Examples of combined ion mobility/time-of-flight data are presented for distributions of electrosprayed bradykinin and ubiquitin ions.

Bradykinin↗
PubMed1998

High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra.

Injected-ion mobility and high-pressure ion mobility techniques have been used to examine the conformations of bradykinin, insulin chain A, and several other peptide ions in the gas phase. Under the experimental conditions employed, evidence for multimer formation in the mass spectra of peptides is minimal or absent altogether. However, ion mobility distributions show that aggregates of peptides (containing a single charge per monomer unit) are observed at the same mass-to-charge ratios as the singly charged parent ions. Collision cross sections for these clusters show that they have tightly packed roughly spherical conformations. We have bracketed the average density as 0.87 < p < 1.00 g cm-3. In some cases, specific stable aggregate forms within a cluster size can be distinguished indicating that some high order structures are favored in the gas phase. Multimer formation between different sizes of polyalanine peptides shows no evidence for size specificity in aggregate formation. Collisional and thermal excitation studies have been used to examine structural transitions and dissociation of the multimers. Aggregates appear to dissociate via loss of singly charged monomers. The observation that peptide multimers can be concealed in mass spectral data requires that fragmentation patterns and reactivity studies of singly charged monomers be undertaken with care.

Algorithms↗
PubMed1998

Gas-phase separations of protease digests.

A mixture of peptides from a complete tryptic digest of ubiquitin has been analyzed by ion mobility/time-of-flight mass spectrometry techniques. All components of the mixture were electrosprayed and ions were separated in the gas phase based on differences in their mobilities through helium before being dispersed into a time-of-flight mass spectrometer for mass-to-charge analysis. The data show that ions separate into families primarily according to differences in their charge states and, to a lesser extent, differences in conformation. This approach reduces spectral congestion typically associated with electrosprayed mixtures and provides charge assignments for mass-to-charge ratio data. Gas-phase separations of ions appear to provide a new physical basis for characterizing components of biological mixtures.

Endopeptidases↗