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The extracellular chlamydial infectious particle, or elementary body (EB), is enveloped by an intra- and intermolecular cysteine cross-linked protein shell called the chlamydial outer membrane complex (COMC). A few abundant proteins, including the major outer membrane protein and cysteine-rich proteins (OmcA and OmcB), constitute the overwhelming majority of COMC proteins. The identification of less-abundant COMC proteins has been complicated by limitations of proteomic methodologies and the contamination of COMC fractions with abundant EB proteins. Here, we used parallel liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analyses ofChlamydia trachomatisserovar L2 434/Bu EB, COMC, and Sarkosyl-soluble EB fractions to identify proteins enriched or depleted from COMC. All well-described COMC proteins were specifically enriched in the COMC fraction. In contrast, multiple COMC-associated proteins found in previous studies were strongly enriched in the Sarkosyl-soluble fraction, suggesting that these proteins are not COMC components or are not stably associated with COMC. Importantly, we also identified novel proteins enriched in COMC. The list of COMC proteins identified in this study has provided reliable information for further understanding chlamydial protein secretion systems and modeling COMC and EB structures.

Singly-protonated proline-containing peptides with N-terminal arginine are photodissociated with vacuum ultraviolet (VUV) light in an ESI linear ion trap/orthogonal-TOF (LIT/o-TOF). When proline is the nth residue from the N-terminus, unusual b n + 2 and a n + 2 ions are observed. Their formation is explained by homolytic cleavage of the Cα− C bond in conjunction with a rearrangement of electrons and an amide hydrogen. The latter is facilitated by a proline-stabilized gas-phase peptide conformation.

Cross sections as a function of reagent collision energy are measured for the reaction of Al + ( 1 S) with NO 2 , N 2 O, and CO 2 by using guided-ion-beam mass spectrometry. In the NO 2 system, NO + +AlO and AlO + +NO are formed in endothermic reactions. The threshold for the former reaction is used to measure the bond energy, D o 0 (AlO)=123.1±1.0 kcal/mol, in good agreement with the presently accepted value of 121.2±2.2 kcal/mol.

The adiabatic ionization energy (IE) of NO2 is measured to be 9.60±0.03 eV by studying the charge‐transfer reactions of Zn+, NO+, and CH3I+ with NO2 and those of NO+2 with α,α,α‐trifluorotoluene and CH3I using guided ion‐beam mass spectrometry. This value confirms the accuracy of a very precise spectroscopic value measured by Haber et al. [J. Chem. Phys. 144, 58 (1988)] and Tanaka and Jursa [J. Chem. Phys. 36, 2493 (1962)], IE(NO2)=9.586±0.002 eV, but is much lower than many other measurements that are limited by very unfavorable Franck–Condon factors. The mechanism that allows the charge‐transfer reactions to occur at the thermodynamic limit is discussed by examining qualitative potential‐energy surfaces for the charge‐transfer processes.

Gas-phase ScO_2, YO_2, LaO_2 and the singly charged cations of these species are formed in endothermic reactions between MO+ (M=Sc, Y, and La) and NO_2 in a guided ion beam mass spectrometer. The cross sections of these reactions are measured as a function of kinetic energy and are interpreted to give the following 0 K bond energies (in eV): D^0(OScO)=3.95±0.33, D^0(OYO)=4.14±0.22, D^0(OLaO)=4.20±0.33, D^0(OSc^+O)=1.72±0.19, D^0(OY^+O)=1.76±0.16, and D^0(OLa^+O)=0.99±0.31. Values for the MO_2 ionization energies (in eV) are determined to be IE(ScO_2)=8.66±0.20, IE(YO_2)=8.23±0.16 and IE(LaO_2)=8.11±0.35. The differences between these values and estimates in the literature are discussed by considering the nature of the bonding in MO_2 and MO^+_2.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and formation of intracytoplasmic Lewy bodies (LBs). Loss-of-function mutations in parkin which encodes an E3 ubiquitin protein ligase contribute to a predominant cause of a familial form of PD termed autosomal recessive juvenile Parkinsonism (AR-JP). Drosophila parkin null mutants display muscle degeneration and mitochondrial dysfunction, providing an animal model to study Parkin-associated molecular pathways in PD. To define protein alterations involved in Parkin pathogenesis, we performed quantitative proteomic analyses of Drosophila parkin null mutants and age-matched controls utilizing both global internal standard technology (GIST) and extracted ion chromatogram peak area (XICPA) label-free approaches. A total of 375 proteins were quantified with a minimum of two peptide identifications from the combination of the XICPA and GIST measurements applied to two independent biological replicates. Sixteen proteins exhibited significant alteration. Seven of the dysregulated proteins are involved in energy metabolism, of which six were down-regulated. All five proteins involved in transporter activity exhibited higher levels, of which larval serum protein 1alpha, larval serum protein 1beta, larval serum protein 1gamma, and fat body protein 1 showed10-fold up-regulation and substantially higher level of fat body protein 1 was confirmed by Western blot analysis. These findings suggest that abnormalities in energy metabolism and protein transporter activity pathways may be associated with the pathogenesis of Parkin-associated AR-JP.

The ability to separate isotopes by high-resolution ion mobility spectrometry techniques is considered as a direct means for determining mass at ambient pressures. Calculations of peak shapes from the transport equation show that it should be possible to separate isotopes for low mass ions (

The synthesis of increasingly complex polymers has created daunting, sometimes insurmountable problems for their chemical analysis. The importance is magnified by outsourcing of production and their use in consumer products, including medical devices and food storage, and therefore requires a new generation of technology for quality assurance. Here, we report capturing subtle differences at the molecular level in complex polymer mixtures nearly instantaneously using a prototype multidimensional ion mobility spectrometry/mass spectrometry spectrometry instrument. Bulk activation/fragmentation strategies reported here provide signatures of structural characteristics that permit effortless recognition of minor differences in blends and copolymers, even as structural isomers and from a quantitative perspective. The data displayed as a pictorial snapshot provide a visual pattern that is sufficiently distinctive that computer-aided pattern recognition can be used to address process control and regulatory issues.

The homolytic bond dissociation energies of ZnO and ZnO^+ have been determined by using guided ion‐beam mass spectrometry to measure the kinetic‐energy dependence of the endothermic reactions of Zn^+ with nitrogen dioxide. The data are interpreted to yield the bond energy for ZnO, D^0_0=1.61±0.04 eV, a value considerably lower than previous experimental values, but in much better agreement with theoretical calculations. We also obtain D^0_0(ZnO^+)=1.67±0.05 eV, in good agreement with previous results. Other thermochemistry derived in this study is D^0_0(Zn^+–NO)=0.79±0.10 eV and the ionization energies, IE(ZnO)=9.34±0.02 eV and IE(NO_2)=9.57±0.04 eV.

The reactions of Sc+, Ti+, and V+ with CO are studied as a function of translational energy in a guided‐ion‐beam tandem mass spectrometer. Formation of both metal‐carbide and metal‐oxide ions are observed and rationalized by a direct atom abstraction mechanism. At high energies, the ScC+ and ScO+ cross sections exhibit additional features that are unusual but can be explained by an impulsive pairwise mechanism and formation of excited‐state product ions, respectively. Thresholds of the reaction cross sections are interpreted to give the 0 K bond energies (in eV) D0(ScC+)=3.34±0.06, D0(TiC+)=4.05±0.24, D0(VC+)=3.87±0.14, D0(ScO+)=7.11±0.08, D0(TiO+)=6.88±0.07, and D0(VO+)=5.81±0.17. Additional studies are used to help verify the bond energy for ScO+ and yield a recommended value of 7.14±0.11 eV. The nature of the bonding in MO+ and MC+ is discussed and compared for these three metal ions.

The homolytic bond dissociation energy of the titanium neutral hydride D0(Ti–H) is determined experimentally for the first time by using guided ion beam tandem mass spectrometry to measure the kinetic energy dependence of the endothermic hydride abstraction reactions of Ti+ with methylamine, dimethylamine, and trimethylamine. From the thresholds of these reactions, the value of D0(Ti–H)=2.12±0.09 eV (48.9±2.1 kcal/mol) at 298 K is derived. Other 298 K thermodynamic values obtained are D0(Ti+–H−)=8.19±0.09 eV (188.8±2.1 kcal/mol), I.E.(TiH)=6.59±0.14 eV, P.A.(Ti−)=15.64±0.09 eV (360.6±2.1 kcal/mol), and ΔfH(TiH)=116.4±2.3 kcal/mol. This thermochemistry is compared with theoretical values and its relationship to hydride bond energies for the other first row transition metals is discussed.

The alpha-synuclein protein is associated with several neurodegenarative diseases, including Parkinson's disease (PD). In humans, only mutated forms of alpha-synuclein are linked to PD; however, panneural expression of human wild-type (WT) alpha-synuclein induces Parkinson's like-symptoms in Drosophila. Here, we report a quantitative proteomic analysis of WT alpha-synuclein transgenic flies with age-matched controls at the presymptomatic stage utilizing a global isotopic labeling strategy combined with multidimensional liquid chromatographies and tandem mass spectrometry. The analysis includes two biological replicates, in which samples are isotopically labeled in forward and reverse directions. In total, 229 proteins were quantified from assignments of at least two peptide sequences. Of these, 188 (82%) proteins were detected in both forward and reverse labeling measurements. Twelve proteins were found to be differentially expressed in response to the expression of human WT alpha-synuclein; down-regulations of larval serum protein 2 and fat body protein 1 levels were confirmed by Western blot analysis. Gene Ontology analysis indicates that the dysregulated proteins are primarily associated with cellular metabolism and signaling, suggesting potential contributions of perturbed metabolic and signaling pathways to PD. An increased level of the iron (III)-binding protein, ferritin, typically found in the brains of PD patients, is also observed in presymptomatic WT alpha-synuclein expressing animals. The observed alterations in both pathology-associated and novel proteins may shed light on the pathological roles of alpha-synuclein that may lead to the development of diagnostic strategies at the presymptomatic stage.

Ion mobility-mass spectrometry (IMS-MS) and molecular modeling techniques have been used to characterize ovalbumin N-linked glycans. Some glycans from this glycoprotein exist as multiple isomeric forms. The gas-phase separation makes it possible to resolve some isomers before MS analysis. Comparisons of experimental cross sections for selected glycan isomers with values that are calculated for iterative structures generated by molecular modeling techniques allow the assignment of sharp features to specific isomers. We focus here on an example glycan set, each having a m/z value of 1046.52 with formula [H5N4+2Na]2+, where H corresponds to a hexose, and N to a N-acetylglucosamine. This glycan appears to exist as three different isomeric forms that are assignable based on comparisons of measured and calculated cross sections. We estimate the relative ratios of the abundances of the three isomers to be in the range of approximately 1.0:1.35:0.85 to approximately 1.0:1.5:0.80. In total, IMS-MS analysis of ovalbumin N-linked glycans provides evidence for 19 different glycan structures corresponding to high-mannose and hybrid type carbohydrates with a total of 42 distinct features related to isomers and/or conformers.

A simple method for increasing the efficiency of multidimensional ion mobility spectrometry (IMS-IMS) measurements (as defined by the number of two-dimensional data sets necessary to sample all of the ions in a complex mixture) is illustrated. In this approach, components from a packet containing a mixture of ions are introduced into the first IMS drift region where they are separated based on differences in mobility. At the exit of this region, narrow distributions of ions having identical mobilities are selected, subjected to gentle activation conditions that are intended to induce conformational changes, and transmitted into a second IMS drift region where the new conformations are separated. Here, we describe a simple timing sequence associated with selection and activation of multiple distributions at the entrance of the second drift region in a systematic fashion that improves the efficiency of two-dimensional IMS-IMS by a factor of approximately 8. The method is illustrated by examination of a mixture of tryptic peptides from human hemoglobin.

Aberrant glycosylation of human glycoproteins is related to various physiological states, including the onset of diseases such as cancer. Consequently, the search for glycans that could be markers of diseases or targets of therapeutic drugs has been intensive. Here, we describe a high-throughput ion mobility spectrometry/mass spectrometry analysis of N-linked glycans from human serum. Distributions of glycans are assigned according to their m/z values, while ion mobility distributions provide information about glycan conformational and isomeric composition. Statistical analysis of data from 22 apparently healthy control patients and 39 individuals with known diseases (20 with cirrhosis of the liver and 19 with liver cancer) shows that ion mobility distributions for individual m/z ions appear to be sufficient to distinguish patients with liver cancer or cirrhosis. Measurements of glycan conformational and isomeric distributions by IMS-MS may provide insight that is valuable for detecting and characterizing disease states.

Reactions of Ca+, Zn+ and all first‐row atomic transition metal ions with O2 are studied using guided ion beam techniques. While reactions of the ground states of Sc+, Ti+, and V+ are exothermic, the remaining metal ions react with O2 in endothermic processes. Analyses of these endothermic reactions provide new determinations of the M+–O bond energies for these eight elements. Source conditions are varied such that the contributions of excited states of the metal ions can be explicitly considered for Mn+, Co+, Ni+, and Cu+. Results (in eV) at 0 K are D0(Ca+–O)= 3.57±0.05, D0(Cr+–O)=3.72±0.12, D0(Mn+–O)=2.95±0.13, D0(Fe+–O)=3.53±0.06 (reported previously), D0(Co+–O)=3.32±0.06, D0(Ni+–O) =2.74±0.07, D0(Cu+–O)=1.62±0.15, and D0(Zn+–O)=1.65±0.12. These values along with literature data for neutral metal oxide bond energies and ionization energies are critically evaluated. Periodic trends in the ionic metal oxide bond energies are compared with those of the neutral metal oxides and those of other related molecules.

The reactions of Sc' and Ti' with ammonia are studied as a function of translational energy in a guided ion beam tandem mass spectrometer. The effect of electronic energy for the Ti+ reactions is also probed by varying the conditions for forming Ti+. Excited doublet states of Ti' are found to be much more reactive than the a4F ground and b4F first excited states. Both metals form the MH+, MNH', and MNH2+ products, while only Ti+ forms MN+. The results are consistent with reaction that occurs primarily through a covalently bound insertion intermediate, H-M+-NHz, having a singlet and doublet spin state for M = Sc and Ti, respectively. The reactivities of the different electronic states of the reactant ions can be explained by using spin conservation concepts. The thresholds for the cross sections of the endothermic reactions are interpreted to give the 298 K bond energies of Do(Sc+-NH2) = 3.69 i 0.07 eV, Do(Sc+-NH) = 5.16 f 0.10 eV, Do(Ti+-NH2) = 3.69 f 0.13 eV, D"(Ti+-NH) = 4.83 f 0.12 eV, and Do(Ti+-N) = 5.19 f 0.13 eV. The large bond strengths of the M+-NH2 and M+-NH species indicate that the lone pair electrons on the nitrogen atom are involved in the metal-ligand bond.

Motivation: One of the major problems in shotgun proteomics is the low peptide coverage when analyzing complex protein samples. Identifying more peptides, e.g. non-tryptic peptides, may increase the peptide coverage and improve protein identification and/or quantification that are based on the peptide identification results. Searching for all potential non-tryptic peptides is, however, time consuming for shotgun proteomics data from complex samples, and poses a challenge for a routine data analysis. Results: We hypothesize that non-tryptic peptides are mainly created from the truncation of regular tryptic peptides before separation. We introduce the notion of truncatability of a tryptic peptide, i.e. the probability of the peptide to be identified in its truncated form, and build a predictor to estimate a peptide's truncatability from its sequence. We show that our predictions achieve useful accuracy, with the area under the ROC curve from 76% to 87%, and can be used to filter the sequence database for identifying truncated peptides. After filtering, only a limited number of tryptic peptides with the highest truncatability are retained for non-tryptic peptide searching. By applying this method to identification of semi-tryptic peptides, we show that a significant number of such peptides can be identified within a searching time comparable to that of tryptic peptide identification. Contact: predrag@indiana.edu; rarnold@indiana.edu; hatang@indiana.edu

A survey of the proteome of Drosophila melanogaster at nine time points across the adult lifespan based on several mass-spectrometry-based techniques is presented. In total, there is evidence for 5902 unique peptides corresponding to 1699 different proteins. Of hundreds of relatively abundant components, many appear to be highly dynamic as the adult fly ages. Of those proteins that we observe changing with age, a majority, associated with metabolism, reproduction, and development, are down-regulated. Other biological pathways such as defense response also show variable changes, where some proteins are down-regulated and others are up-regulated. The observed variations are compared with a report of genome-wide changes at the transcriptome level at different ages and the similarities and differences are presented.

A survey of the proteome changes in an A30P alpha-synuclein Drosophila model of Parkinson's disease (PD) in comparison to age-matched controls is presented for seven different ages across the adult lifespan. The data were acquired by a shotgun proteomic approach that involves multidimensional liquid chromatographies coupled to mass spectrometry and database searching techniques. Semiquantitative analysis to assess relative changes in protein expression between the Drosophila PD model and age-matched controls provides evidence that 28, 19, 12, 5, 7, 23, and 17 proteins are significantly differentially expressed at days 1, 10, 20, 30, 40, 50, and 60, respectively. From the experimental approach employed, it appears that most dysregulated proteins are associated with narrow distributions of ages, such that disease-associated differences change substantially across the lifespan. Previous measurements [J. Proteome Res. 2007, 6, 348] at days 1, 10, and 30 showed dysregulation of actin cytoskeletal proteins at day 1 and mitochondrial proteins at day 10, suggesting that defects in the actin cytoskeleton and the mitochondria are associated with dopaminergic neuron degeneration in PD. Analysis of the day 20, 40, 50, and 60 animals supports the finding that these cytoskeletal and mitochondrial changes predominate in the youngest (pre-symtomatic and early disease stages) animals. Although studies across many time points appear to be important for characterizing disease state, an understanding of molecular changes at the youngest ages should be most important for addressing causation.

In numerous studies charge remote fragmentation (CRF) has been shown to be a powerful technique for determination of primary structure by allowing location of double bonds, various functional groups, and branching in a variety of compound types directly by mass spectrometry. Instrumentation and ionization methods traditionally used for CRF, however, are becoming rare, in large part because ESI and MALDI have to a significant extent replaced them. Here we demonstrate that by selecting a matrix that promotes rather than suppresses ionization of fatty acids (FA) by lithium ion adduction, and using a TOF-TOF mass spectrometer for high-energy collisional activation, CRF ions are produced that allow location of double-bond and branching positions. Further, we show that by using solvent-free MALDI sample preparation methods, thus eliminating the inherent segregation of the hydrophobic fatty acid from the hydrophilic LiCl that can occur during the evaporation of solvent, the desired [FA-H+2Li](+) ions are greatly enhanced. Because FAs can be vaporized using laser desorption, matrix assistance in desorption of the fatty acid may occur, but is not necessary. However, the matrix plays a crucial role in enhancing or suppressing ionization. For example, matrix materials with acid (e.g., 2,5-dihydroxybenzoic acid) or hydroxy groups (e.g., dithranol) compete with the FA for Li(+) and because of the high ratio of matrix to analyte, FA lithium adduction is minimized. However, highly electron-deficient matrix materials (e.g., TCNQ) readily donate Li(+) to FAs because of the instability associated with being positively charged.

The advent of on-line multidimensional liquid chromatography-mass spectrometry has significantly impacted proteomic analyses of complex biological fluids such as plasma. However, there is general agreement that additional advances to enhance the peak capacity of such platforms are required in order to enhance the accuracy and coverage of proteome maps of such fluids. Here, we describe the combination of strong-cation-exchange and reversed-phase liquid chromatographies with ion mobility and mass spectrometry as a means of characterizing the complex mixture of proteins associated with the human plasma proteome. The increase in separation capacity associated with inclusion of the ion mobility separation leads to generation of one of the most extensive proteome maps to date. The map is generated by analyzing plasma samples of five healthy humans; we report a preliminary identification of 9087 proteins from 37842 unique peptide assignments. An analysis of expected false-positive rates leads to a high-confidence identification of 2928 proteins. The results are catalogued in a fashion that includes positions and intensities of assigned features observed in the datasets as well as pertinent identification information such as protein accession number, mass, and homology score/confidence indicators. Comparisons of the assigned features reported here with other datasets shows substantial agreement with respect to the first several hundred entries; there is far less agreement associated with detection of lower abundance components.

Multi-dimensional ion mobility spectrometry techniques (IMS-IMS and IMS-IMS-IMS) combined with mass spectrometry are used to study structural transitions of ubiquitin ions in the gas phase. It is possible to select and activate narrow distributions of compact and partially-folded conformation types and examine new distributions of structures that are formed. Different compact conformations unfold, producing a range of new partially-folded states and three resolvable peaks associated with elongated conformers. Under gentle activation conditions, the final populations of the three elongated forms depend on the initial structures of the selected ions. This requires that some memory of the compact state (most likely secondary structure) is preserved along the unfolding pathway. Activation of selected, partially-folded intermediates (formed from specific compact states) leads to elongated state populations that are consistent with the initial selected compact form –evidence that intermediates not only retain elements of initial structure but also are capable of transmitting structure to final states.

A combination of split-field drift tube/mass spectrometry and isotopic labeling techniques is evaluated as a means of identifying single amino acid polymorphisms (SAAPs) in proteins. The method is demonstrated using cytochromec (equine and bovine) and hemoglobin (bovine and sheep). For these studies, proteins from different species are digested with trypsin, and the peptides are labeled at primary amine groups [using either a light (H(3))- or heavy (D(3))-isotopic reagent]. SAAP analysis is carried out by mixing the light-labeled peptides of one species with the heavy-labeled peptides of the other and electrospraying the resulting mixture into a split-field drift tube/mass spectrometer. Peptides having the same sequence in both species appear as doublets in the mass spectrum [shifted in mass-to-charge (m/z) according to the number of incorporated labels]; additionally, these species have identical mobility distributions. Peptides having sequences that differ by one amino acid appear as peaks in the mass spectrum that are shifted in m/z according to the mass difference associated with the SAAP and the number of incorporated labels. The ion mobility distributions for these peptides (differing by only a single amino acid) can often be rationalized by their expected similarities or differences providing additional evidence that they are related. In all, 12 and 26 peptide variants (between species) corresponding to 5 and 11 amino acid polymorphisms have been identified for the cytochrome c and hemoglobin protein samples, respectively.

Ion mobility and mass spectrometry techniques have been used to measure cross sections for 162 tripeptide sequences (27 different sets of six sequence isomers). The isomers have the general forms ABC, ACB, BAC, BCA, CAB, and CBA, where A corresponds to the amino acids Asp, Glu, or Gly, B corresponds to Lys, Arg, or Leu, and C corresponds to Phe, Tyr, or Ser. From these data, we derive a set of size parameters for individual amino acids that reflect the position of the amino acid in the sequence. These sequence-specific intrinsic size parameters (SSISPs) are used to retrodict cross-section values for the 162 measured sequences and to predict cross sections for all remaining tripeptide sequences (567 different sequences) that are comprised of these residues. In several types of peptide compositions, the position of the amino acid in the sequence has a significant impact on the parameter that is derived. For example, the sequence-specific intrinsic size parameter for leucine in the third position of a peptide (SSISP(Leu3)) is approximately 10% larger than SSISP(Leu1). On average, cross sections that are derived using SSISPs provide a better representation of the experimental value than those derived from composition only intrinsic size parameters, derived as described previously (Valentine et al. J. Phys. Chem. 1999, 103, 1203). Finally, molecular modeling techniques are used to derive some insight into the origin of cross-section differences that arise from sequence variation.

A new two-dimensional ion mobility spectrometry approach combined with mass spectrometry has been used to examine ubiquitin ions in the gas phase. In this approach ions are separated in an initial drift tube into conformation types (defined by their collision cross sections) and then a gate is used to introduce a narrow distribution of mobility-separated ions into a second drift tube for subsequent separation. The results show that upon selection a narrow peak shape is retained through the second drift tube. This requires that at 300 K the selected distribution does not interconvert substantially within the broader range of structures associated with the conformation type within the approximately 10-20 ms time scale of these experiments. For the [M + 7H]7+ ion, it appears that many ( approximately 5-10) narrow selections can be made across each of the compact, partially-folded, and elongated conformer types, defined previously (Int. J. Mass Spectrom. 1999, 187, 37-47).

When a packet of ions in a buffer gas is exposed to a weak electric field, the ions will separate according to differences in their mobilities through the gas. This separation forms the basis of the analytical method known as ion mobility spectroscopy and is highly efficient, in that it can be carried out in a very short time frame (micro- to milliseconds). Recently, efforts have been made to couple the approach with liquid-phase separations and mass spectrometry in order to create a high-throughput and high-coverage approach for analyzing complex mixtures. This article reviews recent work to develop this approach for proteomics analyses. The instrumentation is described briefly. Several multidimensional data sets obtained upon analyzing complex mixtures are shown in order to illustrate the approach as well as provide a view of the limitations and required future work.

Multidimensional separations combined with mass spectrometry are used to study the proteins that are present in two states of Drosophila melanogaster: the whole embryo and the adult head. The approach includes the incorporation of a gas-phase separation dimension in which ions are dispersed according to differences in their mobilities and is described as a means of providing a detailed analytical map of the proteins that are present. Overall, we find evidence for 1133 unique proteins. In total, 780 are identified in the head, and 660 are identified in the embryo. Only 307 proteins are in common to both developmental stages, indicating that there are significant differences in these proteomes. A comparison of the proteome to a database of mRNAs that are found from analysis by cDNA approaches (i.e., transcriptome) also shows little overlap. All of this information is discussed in terms of the relationship between the predicted genome, and measured transcriptomes and proteomes. Additionally, the merits and weaknesses of current technologies are assessed in some detail.

The cross sections of five different protonated trimers consisting of two base molecules and trifluoroacetic acid were measured by using ion mobility spectrometry. The gas-phase basicities of these five base molecules span an 8-kcal/mol range. These cross sections are compared with those determined from candidate low-energy salt-bridge and charge-solvated structures identified by using molecular mechanics calculations using three different force fields: AMBER*, MMFF, and CHARMm. With AMBER*, the charge-solvated structures are all globular and the salt-bridge structures are all linear, whereas with CHARMm, these two forms of the protonated trimers can adopt either shape. Globular structures have smaller cross sections than linear structures. Conclusions about the structure of these protonated trimers are highly dependent on the force field used to generate low-energy candidate structures. With AMBER*, all of the trimers are consistent with salt-bridge structures, whereas with MMFF the measured cross sections are more consistent with charge-solvated structures, although the assignments are ambiguous for two of the protonated trimers. Conclusions based on structures generated by using CHARMm suggest a change in structure from charge-solvated to salt-bridge structures with increasing gas-phase basicity of the constituent bases, a result that is most consistent with structural conclusions based on blackbody infrared radiative dissociation experiments for these protonated trimers and theoretical calculations on the uncharged base-acid pairs.

Chirality plays an important role in the formation and stability of noncovalent clusters which are made of chiral molecules. It is shown that clusters can exhibit a preference for both homochirality and heterochirality. Serine cluster formation is dominated by the formation of heterochiral aggregates, with the exception of the previously observed homochiral serine octamer. Thus, the majority of serine clusters lead to chiral anti-resolution, or the racemic mixing of enantiomers.

Chiral enrichment of serine is achieved in experiments that involve formation of serine octamers starting from non-racemic serine solutions. Serine octamers were generated by means of electrospray and sonic spray ionization of aqueous solutions of d(3)-L-serine (108 Da) and D-serine (105 Da) having different molar ratios of enantiomers. A cyclic process involving the formation of chirally-enriched octameric cluster ions and their dissociation, viz. Ser(1) --Ser(8) --Ser(1), allows serine monomers to be regenerated with increased enantiomeric excess as shown in two types of experiments: (1) Chiral enrichment in serine was observed in MS/MS/MS experiments in a quadrupole ion trap in which the entire distribution of serine octamers formed from non-racemic solutions was isolated, collisionally activated, and fragmented. Monomeric serine was regenerated with increased enantiomeric excess upon dissociation of octamers when compared with the enantiomeric composition of the original solution. (2) Chiral enrichment was observed in the products of soft-landing of mass-selected protonated serine octamers. These ions were generated by means of electrospray or sonic spray ionization, mass selected, and collected on a gold surface using ion soft-landing. Chiral enrichment of the soft-landed serine was established by redissolving the recovered material and comparing the intensities of protonated molecular ions of d(3)-L-serine and D-serine after APCI-MS analysis. Both of these experiments showed comparable results, suggesting that formation of serine octamers depends only on the enantiomeric composition of the serine solution and that the magnitude of the chiral preference is intrinsic to octamers formed from solutions of given chiral composition.

A simple ion trap/ion mobility/time-of-flight (TOF) mass spectrometer has been coupled with nanoflow liquid chromatography to examine the feasibility of analyzing mixtures of intact proteins. In this approach proteins are separated using reversed-phase chromatography. As components elute from the column, they are electrosprayed into the gas phase and separated again in a drift tube prior to being dispersed and analyzed in a TOF mass spectrometer. The mobilities of ions through a buffer gas depend upon their collision cross sections and charge states; separation based on these gas-phase parameters provides a new means of simplifying mass spectra and characterizing mixtures. Additionally it is possible to induce dissociation at the exit of the drift tube and examine the fragmentation patterns of specific protein ion charge states and conformations. The approach is demonstrated by examining a simple three-component mixture containing ubiquitin, cytochrome c, and myoglobin and several larger prepared protein mixtures. The potential of this approach for use in proteomic applications is considered.

A linear octopole trap interface for an ion mobility time-of-flight mass spectrometer has been developed for focusing and accumulating continuous beams of ions produced by electrospray ionization. The interface improves experimental efficiencies by factors of approximately 50-200 compared with an analogous configuration that utilizes a three-dimensional Paul geometry trap (Hoaglund-Hyzer, C. S.; Lee, Y. J.; Counterman, A. E.; Clemmer, D. E. Anal. Chem. 2002, 74, 992-1006). With these improvements, it is possible to record nested drift (flight) time distributions for complex mixtures in fractions of a second. We demonstrate the approach for several well-defined peptide mixtures and an assessment of the detection limits is given. Additionally, we demonstrate the utility of the approach in the field of proteomics by an on-line, three-dimensional nano-LC-ion mobility-TOF separation of tryptic peptides from the Drosophila proteome.

A prototype linear octopole ion trap/ion mobility/tandem mass spectrometer has been coupled with a nanoflow liquid chromatography separation approach and used to separate and characterize a complicated peptide mixture from digestion of soluble proteins extracted from human urine. In this approach, two dimensions of separation (nanoflow liquid chromatography and ion mobility) are followed by collision induced dissociation (CID) and mass spectrometry (MS) analysis. From a preliminary analysis of the most intense CID-MS features in a part of the dataset, it is possible to assign 27 peptide ions which correspond to 13 proteins. The data contain many additional CID-MS features for less intense ions. A limited discussion of these features and their potential utility in identifying complicated peptide mixtures required for proteomics study is presented.

Recent improvements in ion mobility/time-of-flight mass spectrometry techniques have made it possible to incorporate nano-flow liquid chromatography and collision induced dissociation techniques. This combination of approaches provides a new strategy for detailed characterization of complex systems—such as, combinatorial libraries. Our work uses this technology to provide a detailed analysis of a tetrapeptide library having the general form Xxx1-Xxx2-Xxx3-Xxx4 where Xxx1 = Glu, Phe, Val, Asn; Xxx2 = Glu, Phe, Val, Tyr; Xxx3 = Glu, Phe, Val, Thr; and Xxx4 = Glu, Phe, Val, Leu—a system that is expected to contain 256 different peptide sequences. The results corroborate the presence of many expected peptide sequences and indicate that some synthetic steps appear to have failed. Particularly interesting is the observation of a t-butyl protecting group on the tyrosine (Tyr) residue. It appears that most Tyr containing peptides that have this t-butyl group attached favor formation of [2M + 2H]2+ dimers, which can be readily distinguished from [M + H]+ monomers based on differences in their gas-phase mobilities. In this case, we demonstrate the use of the mobility differences between [2M + 2H]2+ and [M + H]+ ions as a signature for a failure of a synthetic step.

Recent developments in combinatorial synthesis and high-throughput screening (HTS) have emphasized parallel strategies for the generation and characterization of large libraries of synthetic molecules. In the era following the sequencing of the human genome [1], proteomics has been developed as a high-throughput approach to the discovery of biological target molecules that are up- or downregulated in diseased cells [2]. The identification of such targets will certainly increase the demand for combinatorial libraries of potential binding ligands that are chemically diverse, well characterized, and appropriate for screening against multiple target molecules.

Injected ion drift tube techniques have been used to probe LaC[sub n][sup +] clusters in the size range around LaC[sub 36][sup +] where the fullerene cage becomes too small to accommodate a La atom. Mobility measurements show that as the fullerene cage shrinks, the La atom moves out of the cage into the carbon network, stabilizing clusters with an odd number of carbon atoms. Annealing studies demonstrate that in this size range the presence of the La atom dramatically enhances the efficiency of conversion of bicyclic and monocyclic LaC[sub n][sup +] rings into fullerenes and graphite sheets. 20 refs., 4 figs.

SINCE the discovery of fullerenes1, efforts have been made to trap metal atoms inside fullerene cages2, and both endohedral3,4 and exohedral5,6 metallofullerenes have been synthesized. There is, however, a third possibility: a 'networked' metallofullerene, where the metal atom is incorporated into the carbon cage. Here we report the results of experiments to study the structure and reactivity of gas-phase fullerenes doped with niobium (NbCn+ with n = 28–50). These experiments, which use injected-ion drift-tube tech-niques, indicate that for fullerenes containing an even number of carbon atoms the metal is endohedral, but for fullerenes with an odd number of carbon atoms, the niobium metal is bound as a part of the carbon cage. Thus, networked metallofullerenes appear to be a stable class of metallofullerene. We suggest that such metallo-fullerenes can form if the metal atom retains sufficient electron density to form several strong covalent metal–carbon bonds.

METALLOFULLERENES1,2consist of metal atoms trapped inside closed fullerene cages. Virtually nothing is known about the mechanism of metallofullerene synthesis, although the possibility of a condensed-phase mechanism has been suggested in recent studies3,4. Here we show that laser vaporization of a La2O3/graphite rod produces a number of LaC60+ isomers, including the endohedral metallofullerene and a variety of different isomers in which lanthanum seems to be bound to polycyclic polyyne rings. When heated, nearly all of the different ring isomers convert spontaneously into metallofullerenes, trapping the metal atom inside the fullerene cage with remarkably high efficiency (>98%). We suggest that in the first step of this annealing process the lanthanum atom acts as a nucleation centre and the carbon rings arrange themselves around the lanthanum atom before converting into a fullerene cage.

The development of a multidimensional approach involving high-performance liquid chromatography (LC), ion mobility spectrometry (IMS) and tandem mass spectrometry is described for the analysis of complex peptide mixtures. In this approach, peptides are separated based on differences in their LC retention times and mobilities (as ions drift through He) prior to being introduced into a quadrupole/octopole/time-of-flight mass spectrometer. The initial LC separation and IMS dispersion of ions is used to label ions for subsequent fragmentation studies that are carried out for mixtures of ions. The approach is demonstrated by examining a mixture of peptides generated from tryptic digestion of 18 commercially available proteins. Current limitations of this initial study and potential advantages of the experimental approach are discussed.

The fragmentation pathways of different conformations of three charge states of ubiquitin ions are examined using ion mobility/collisional activation/time-of-flight techniques. Mass spectra for fragments for different conformers of a single charge state appear to be identical (within the experimental reproducibility). These results are consistent with a mechanism in which different conformers of each charge state rearrange to similar dissociation transition states prior to fragment formation.

High-resolution ion mobility/time-of-flight techniques were used to measure collision cross sections for 968 tryptic digest peptide ions obtained from digestion of common proteins. Here, we report a mobility signature that aids in identifying proline-containing peptides containing 4-10 residues. Of 129 peptides (or = 10 residues in length) in the database that contain proline residues, 57% show multiple resolved features in the ion mobility distribution for at least one of the [M + H]+ or [M + 2H]2+ ions. These multiple features are attributed to different conformations that arise from populations of cis and trans forms of proline. The number of resolved peaks in the ion mobility distribution appears to be correlated with the peptide ion charge state and the number of proline residues in the peptide.

Quantitative structure-property relationships (QSPRs) have been developed to predict the ion mobility spectrometry (IMS) collision cross sections of singly protonated lysine-terminated peptides using information derived from topological molecular structure and various amino acid parameters. The primary amino acid sequence alone is sufficient to accurately predict the collision cross section. The models were built using multiple linear regression (MLR) and computational neural networks (CNNs). The best MLR model found contains six descriptors and predicts 94 of 113 peptides (83%) to within 2% of their experimentally determined values. The best CNN model using the same six descriptors predicts 105 of the 113 peptides (93%) to within 2% of their experimentally determined values. The best overall CNN model, using a different set of six descriptors, predicts 109 of the 113 peptides (96%) to within 2% of their experimentally determined values. In addition, this model can discriminate among peptides having identical amino acid composition, but differing in primary amino acid sequence. This represents a capability not found in previously described models. The descriptors used in the models presented may provide some insight into the nature of peptide ion folding in the gas phase.

An ion trap/ion mobility/quadrupole/collision cell/time-of-flight mass spectrometer that incorporates a differentially pumped orifice-skimmer cone region at the back of the drift tube has been developed for the analysis of peptide mixtures. The combined approach allows a variety of strategies to be employed for collisionally activating ions, and fragments can be monitored by subsequent stages of mass spectrometry in a parallel fashion, as described previously (Anal. Chem. 2000, 72, 2737). Here, we describe the overall experimental approach in detail. Applications involving different aspects of the initial mobility separation and various collisional activation and parallel sequencing strategies are illustrated by examining several simple peptide mixtures and a mixture of tryptic peptides from beta-casein. Detection limits associated with various experimental configurations and the utility for analysis of complex systems are discussed.

A polyalanine-based peptide which forms a stable, negatively-charged α-helix in the gas phase is reported. Addition of an N-terminal acidic residue forms a stabilizing hydrogen bond network and an electrostatic interaction with the helical dipole. Formation of this secondary structure was demonstrated using ion mobility-mass spectrometry and molecular modelling techniques.

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.