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To improve the utility of increasingly large numbers of available unannotated and initially poorly annotated genomic sequences for proteome analysis, we demonstrate that effective protein identification can be made on a large and unannotated genome. The strategy developed is to translate the unannotated genome sequence into amino acid sequence encoding putative proteins in all six reading frames, to identify peptides by tandem mass spectrometry (MS/MS), to localize them on the genome sequence, and to preliminarily annotate the protein via a similarity search by BLAST. These tasks have been optimized and automated. Optimization to obtain multiple peptide matches in effect extends the searchable region and results in more robust protein identification. The viability of this strategy is demonstrated with the identification of 223 cilia proteins in the unicellular eukaryotic model organism Tetrahymena thermophila, whose initial genomic sequence draft was released in November 2003. To the best of our knowledge, this is the first demonstration of large-scale protein identification based on such a large, unannotated genome. Of the 223 cilia proteins, 84 have no similarity to proteins in NCBI's nonredundant (nr) database. This methodology allows identifying the locations of the genes encoding these novel proteins, which is a necessary first step to downstream functional genomic experimentation. Keywords: shotgun proteomic analysis • entire unannotated genome-sequence translation • Tetrahymena thermophila cilia • BLAST analysis • 223 cilia proteins • multiple-peptide matches

A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and cICAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15−20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein D0, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-1-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636−643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and cICAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of cICAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors. Keywords: endometrial cancer markers • iTRAQ • cICAT • liquid chromatography • tandem mass spectrometry

Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein “chips”). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses. Keywords: Chaperonin 10 • tumor marker • endometrial carcinoma • mass spectrometry • MALDI • SELDI • protein discovery • protein identification

The binding energies at 0 K of sodium and silver ions to ammonia, methylamine, ethylamine, acetonitrile, and benzonitrile were determined using threshold collision-induced dissociation (CID) and molecular orbital calculations at the ab initio and density functional theory levels. There is good agreement between experimental and calculated binding energies. For the five ligands, threshold CID/CCSD(t)(fu)/6-311++G(2df,p)//MP2(fu)/6-311++G(d,p) Na⁺ binding energies are the following:  ammonia, 25.6 ± 2.8/24.8; methylamine, 27.0 ± 1.4/25.9; ethylamine, 27.7 ± 2.3/27.1; acetonitrile, 30.0 ± 2.3/30.3; and benzonitrile, 32.7 ± 1.4/35.0 (B3LYP/6-311++G(d,p)//B3LYP/6-311++G(d,p)) kcal/mol. Threshold CID and B3LYP/DZVP Ag⁺ binding energies are the following:  ammonia, 40.6 ± 3.0/38.9; methylamine, 41.5 ± 2.3/41.1; ethylamine, 42.9 ± 1.4/43.2; acetonitrile, 40.8 ± 2.0/39.3; and benzonitrile, 41.5 ± 2.8/43.1 kcal/mol. Wherever comparisons with literature data are possible, the Na⁺ binding energies determined in this study are in good agreement with established data. For Ag⁺ binding energies, agreement with the few published theoretical values is not as good. A comparison of Na⁺ and Ag⁺ binding energies for the five N-containing ligands in this study and those for water, methanol, and ethanol published earlier (El Aribi, H.; Shoeib, T.; Ling, Y.; Rodriquez, C. F.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. A 2002, 106, 2908−2914) shows that for every ligand the Ag⁺ binding energy is higher than the Na⁺ binding energy. As a group, the amines exhibit the largest differences between Ag⁺ and Na⁺ binding energies, followed by the nitriles; the alcohols exhibit the smallest differences. These results are in line with previous observations that Ag⁺ prefers binding with nitrogen to binding with oxygen.

The complexes of silver ion, Ag⁺, with the twenty naturally occurring amino acids have been calculated using hybrid density functional theory at the B3LYP/DZVP level. For all of these silver complexes, several possible structures were examined, but as there are remarkable similarities between all the structures at the global minima, only summarized data are reported. All of the complexes, except that with proline, are solvated ions. Amino acids containing only hydrocarbon side chains are bidentate, coordinating through the amino and carbonyl groups and the remaining amino acids (with the exception of proline) are tricoordinate with the same two interactions as in the simpler amino acids and an additional interaction through the side chain. The proline complex contains zwitterionic proline with the Ag⁺ ion attached to the carboxylate anion. Enthalpies (at 298 K) for dissociation of Ag⁺ from the complexes range from 49.3 kcal mol^-1 for glycine to 80.4 kcal mol^-1 for arginine. Free energies for these reactions are in the range of 40.7 kcal mol^-1 for glycine to 70.3 kcal mol^-1 for arginine. Comparison of the calculated free energies (relative to that of glycine) with those measured by the kinetic method shows good agreement, with the largest discrepancy being 3.9 kcal mol^-1 for aspartic acid. There are some systematic trends with theory giving lower values than experiment for amino acids containing aromatic groups in the side chains (phenylalanine, tryptophan, and tyrosine) and higher values for the four amino acids with carbonyl groups in their side chains (aspartic acid, asparagine, glutamic acid, and glutamine).

The binding enthalpies at 0 K of the silver ion to water, methanol, ethanol, diethyl ether, and acetone were calculated using density functional theory (DFT) using the hybrid B3LYP level of theory with the DZVP basis set; they were also measured using the threshold collision-induced dissociation (CID) method. There is good agreement between the two sets of data. For the five ligands, the DFT/threshold CID values are:  water, 28.1/31.6 ± 2.5; methanol, 30.1/33.0 ± 3.7; ethanol, 32.0/33.9 ± 3.5; diethyl ether, 33.3/33.2 ± 1.5; and acetone, 36.2/38.0 ± 1.4 kcal/mol. The average of the absolute differences between the DFT and threshold CID results is 2.0 kcal/mol, a value smaller than the average experimental uncertainty of 2.5 kcal/mol. For identical ligands, the silver ion binding energies are lower than the lithium ion binding energies, but higher than the sodium ion binding energies.

Gas-phase collision-induced dissociation of the Ag⁺-proline complex shows six major product ions. Tandem mass spectrometry reveals that at least three of the fragment ions are formed directly from the complex. These are a cyclic immonium ion, formed after elimination of AgH from the Ag⁺-proline complex, another cyclic immonium ion formed after the elimination of both AgH and CO2, and finally an ion formed as the product of a reductive-elimination reaction in which H2 is lost as a neutral. Selective and nonselective deuterium labeling experiments and hybrid density functional calculations have been employed to probe fragmentation mechanisms that account for all experimental results. The mechanisms for the competitive losses of AgH and H2 from the Ag⁺-proline complex have been calculated at B3LYP/DZVP.

Density functional calculations at B3LYP/DZVP are used to obtain the binding enthalpies and free energies for the reaction Ag⁺ + XCN → AgNCX⁺, where X = H, CH3, NH2, OH, F, CF3, CN, NO2, N(CH3)2, C6H5, p-C6H4N(CH3)2, p-C6H4NO2, and p-C6H4NH2. The calculated binding enthalpies at 298 K range from 52.2 kcal mol^-1 for X = p-C6H4N(CH3)2 to 21.3 kcal mol^-1 for X = NO2. Calculations at this level of theory are also used to optimize the structures of Ag(NCCH3)n⁺ and Ag(NCH)n⁺ ions, where n = 1−6. The binding enthalpies for the addition of the first and second molecules of CH3CN are 40.1 and 35.3 kcal mol^-1, whereas for HCN, they are calculated to be 31.2 and 28.3 kcal mol^-1, respectively. The binding enthalpies of the third and fourth ligands are much smaller at 15.9 and 10.8 kcal mol^-1 for CH3CN and 13.5 and 9.7 kcal mol^-1 for HCN. The 5- and 6-coordinate structures have positive free energies of formation with both ligands. Electrospraying a solution of AgNO3 and acetonitrile in water shows the dominant ions to be Ag⁺, AgNCCH3⁺, and Ag(NCCH3)2⁺, with the Ag(NCCH3)3⁺ ion being observed only in very small amounts and only under relatively mild conditions. Energy-resolved collision-induced dissociation (CID) experiments confirm the Ag(NCCH3)3⁺ ion to be a loosely bound species, while the Ag(NCCH3)2⁺ and AgNCCH3⁺ ions have substantially higher and comparable binding energies. Using the threshold method, we determined the binding energies at 0 K of NCCH3 to Ag⁺ and of NCCH3 to AgNCCH3⁺ to be 38.7 and 34.6 kcal mol^-1, respectively; the corresponding energies at 298 K are 39.4 and 34.7 kcal mol^-1.

Ion mobility spectrometry (IMS) assumes increasing prominence among the tools for characterization of gas-phase ions and analysis of complex mixtures. The assignment of features observed in IMS experiments to specific structures necessitates the calculation of mobilities for plausible candidate geometries. All previous methods for these calculations have assumed that the ion−buffer gas collisions are fully elastic and that the drifting ions cannot rotate during a collisional event. This paradigm does not mesh well with the fact that the measured quantity is the orientationally averaged collision integral. Here we model the effect of the thermal rotation of drifting ions on their mobility. Simulations show that the cross sections for rotating objects are greater than those for static ones because a molecular image “blurs out” over the duration of collision. This increase is particularly significant for light and elongated ions. For a given ion, the effect is dramatically larger in heavy buffer gases, in some cases exceeding 20%. Present findings reveal the importance of accounting for the nonelasticity of scattering in ion mobility calculations.

A recently developed method to calculate gas-phase mobilities by scattering on electron density isosurfaces (SEDI) has been applied to carbon cluster ions. The investigation has covered species belonging to all major structural families identified in drift tube studies (chains, monocyclic and bicyclic rings, graphite sheets, and fullerenes). Relative cross sections of Cn^- and Cn⁺ predicted by SEDI are in excellent agreement with the measurements across a wide range of cluster sizes and shapes. However, absolute values could not be fit for either charge state. This happens because SEDI ignores the long-range ion−buffer gas interaction known to be important for many systems including carbon clusters. To overcome this problem, we propose a new technique to evaluate mobilities by coupling SEDI with trajectory calculations. This approach allows one to introduce the repulsive interaction accurately and still account for the attractive part of the potential. This hybrid SEDI−TC treatment has been found substantially superior to all models previously described in reproducing both absolute and relative mobilities of Cn anions and cations.

The presence of an interacting water or methanol molecule has been shown to catalyze the 1,3-proton shift in a peptide linkage between the tautomers of protonated formamide and glycylglycylglycine. Density functional theory calculations at the B3LYP/6-31++G(d,p) level of theory show that, for glycylglycylglycine, the forward barrier of this shift decreases from a free energy at 298 K of 39.6 kcal/mol in the absence of solvent to 26.7 kcal/mol in the presence of water and to 22.0 kcal/mol in the presence of methanol. Protonation at the amide nitrogen of the second residue results in a large increase in the C−N bond distance from 1.336 to 1.519 Å, whereas protonation at the carbonyl oxygen leads to a decrease in the C−N bond distance from 1.336 to 1.321 Å. Solvent-catalyzed tautomerism may play an important role in the fragmentation of electrosprayed, protonated peptides in the gas phase.

Molecular orbital calculations at B3LYP/6-31++G(d,p) are reported for bases 2-(aminomethyl)-5-oxazolone, 2-(aminomethyl)-4-methyl-5-oxazolone, 2-phenyl-5-oxazolone, and 2-phenyl-4 methyl-5-oxazolone and for the cations formed by protonation of these bases on their imino nitrogens. Structures and relative energies for isomers generated by protonation at each of the four heteroatoms of 2-(aminomethyl)-5-oxazolone are reported. Lithium and silver cations both add to 2-(aminomethyl)-5-oxazolone, but unlike the proton, they bind with two heteroatoms simultaneously. For both the lithiated and argentinated 2-(aminomethyl)-5-oxazolone cations the lowest energy isomers have the metal coordinated with the two nitrogen atoms. Proton affinities of these bases are in the range 217.0−221 kcal mol^-1, with the methyl group at C4 increasing the proton affinity by ~3 kcal mol^-1. Single-point calculations were performed at MP4(fc)/6-311++G(2df,p)//B3LYP/6-31++G(d,p) for 2-(aminomethyl)-5-oxazolone, diketopiperazine, glycine, and alanine and their conjugate acids. The proton affinities from this level of theory are lower by as much as 2.7 kcal mol^-1 than those calculated at B3LYP/6-31++G(d,p). Enthalpies of formation calculated at B3LYP/6-31++G(d,p) from isodesmic reactions for glycine, alanine, and their conjugate acids are all within 1 kcal mol^-1 of the experimental values, but those calculated at MP4 deviate by as much as 4.8 kcal mol^-1. Enthalpies of formation from atomization reactions at the MP4 level are in larger disagreement with experimental values.

An unprecedented method of producing molecular radical cations of oligopeptides in the gas phase has been discovered. Electrospraying a methanolic mixture of a Cu(II)−amine complex, e.g., Cu^II(dien)(NO3)2 (where dien = diethylenetriamine), and an oligopeptide (M) yields the [Cu^II(dien)M]^•2+ ion, whose collision-induced dissociation (CID) produces [Cu^I(dien)]⁺ and M^•+, the molecular cation of the oligopeptide. Abundant M^•+ is apparent when the oligopeptide contains both a tyrosyl and a basic residue&sbd;arginyl, lysyl, or histidyl. These structural requirements are similar to those in the metalloradical enzyme process in photosystem II. Tandem mass spectrometry of M^•+ produces fragment ions that are both common to and also different from [M + H]⁺. The fragmentation chemistry of M^•+ and of its products appear to be radical driven.

The effectiveness of the kinetic method for estimating the proton affinities of bases that di-coordinate the proton is evaluated using α,ω-diaminoalkanes as model bases. The proton affinities of these diamines have previously been examined using the equilibrium method and critically evaluated. Calculations using density functional theory at the B3LYP/6-31++G(d,p) level confirm that protonated α,ω-diaminoalkanes have cyclic structures with the proton covalently bound to one of the amino nitrogen atoms and hydrogen-bonded to the other. Furthermore, this cyclic structure persists in the protonated heterodimer ion between an α,ω-diaminoalkane and ammonia (the model reference base); binding of the two bases takes place via a second hydrogen bond between the RNH3⁺ and ammonia. Measuring the proton affinities under several collision energies and extrapolating to zero collision energy yields proton affinities that are smaller than the reference values by −2.8 kcal/mol, on average. Application of the Fenselau correction gives proton affinities that differ from the reference values by ±1.0 kcal/mol. These results indicate that the kinetic method is effective for estimating the proton affinities of molecules that tend to have more than one potential protonation site. Application of this method is particularly suited to biological molecules, such as peptides, where application of the equilibrium method is impossible due to low sample volatility.