A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.     

Statistical characterization of the charge state and residue dependence of low-energy CID peptide dissociation patterns

Data mining was performed on 28 330 unique peptide tandem mass spectra for which sequences were assigned with high confidence. By dividing the spectra into different sets based on structural features and charge states of the corresponding peptides, chemical interactions involved in promoting specific cleavage patterns in gas-phase peptides were characterized. Pairwise fragmentation maps describing cleavages at all Xxx-Zzz residue combinations for b and y ions reveal that the difference in basicity between Arg and Lys results in different dissociation patterns for singly charged Arg- and Lys-ending tryptic peptides. While one dominant protonation form (proton localized) exists for Arg-ending peptides, a heterogeneous population of different protonated forms or more facile interconversion of protonated forms (proton partially mobile) exists for Lys-ending peptides. Cleavage C-terminal to acidic residues dominates spectra from singly charged peptides that have a localized proton and cleavage N-terminal to Pro dominates those that have a mobile or partially mobile proton. When Pro is absent from peptides that have a mobile or partially mobile proton, cleavage at each peptide bond becomes much more prominent. Whether the above patterns can be found in b ions, y ions, or both depends on the location of the proton holder(s) in multiply protonated peptides. Enhanced cleavages C-terminal to branched aliphatic residues (Ile, Val, Leu) are observed in both b and y ions from peptides that have a mobile proton, as well as in y ions from peptides that have a partially mobile proton; enhanced cleavages N-terminal to these residues are observed in b ions from peptides that have a partially mobile proton. Statistical tools have been designed to visualize the fragmentation maps and measure the similarity between them. The pairwise cleavage patterns observed expand our knowledge of peptide gas-phase fragmentation behaviors and may be useful in algorithm development that employs improved models to predict fragment ion intensities.     

Peptide sequencing and characterization of post-translational modifications by enhanced ion-charging and liquid chromatography electron-transfer dissociation tandem mass spectrometry

We have tested the effect of m-nitrobenzyl alcohol (m-NBA) as a method to increase the average charge state of protonated gas-phase molecular ions generated by ESI from tryptic peptides and phosphopeptides. Various concentrations of m-NBA were added to the mobile phases of a liquid chromatography system coupled to an ESI tandem mass spectrometer. Addition of just 0.1% m-NBA changed the average charge state for the identified tryptic BSA peptides from 2.2+ to 2.6+. As a result, the predominant charge states for BSA peptides were changed from 2+ to > or =3+. To evaluate the benefits of peptide charge enhancement, the ETD fragmentation efficiency and Mascot peptide score were compared for BSA peptides in charge states 2+ and 3+. In all cases but one, triply charged peptides fragmented more efficiently than the analogues 2+ peptide ions. On average, triply charged peptides received a 68% higher Mascot score (24 units) than doubly charged peptides. m-NBA also increased the average charge state of phosphopeptides by up to 0.5 charge unit. The ease of implementation and the analytical benefits of charge enhancement of tryptic peptides by addition of m-NBA to the LC solvents suggest the general application of this reagent in proteomic studies that employ ETD-MS/MS and related techniques.     

Scrubbing ions with molecules: kinetic studies of chemical noise reduction in mass spectrometry using ion-molecule reactions with dimethyl disulfide

The kinetics and product distributions of the reactions of dimethyl disulfide (DMDS) have been investigated with a group of chemical background ions commonly observed in atmospheric pressure ionization (API) mass spectrometry (MS) in order to assess the value of this molecule in filtering (or "scrubbing") these ions by changing their mass/charge (m/z) ratio. The measurements were taken with a novel electrospray ionization/selected ion flow tube/QqQ tandem mass spectrometer. The background ions studied include those with m/z 42 (protonated acetonitrile, ACN), 83 (protonated ACN dimer), 99 (protonated phosphoric acid), 117 (water cluster of m/z 99), 131 (methanol cluster of m/z 99), 149 (protonated phthalic anhydride, formed from the phthalates), and 327 (protonated triphenyl phosphate). In addition, reactions of DMDS have been studied with two model analytes--protonated caffeine and doubly protonated bradykinin--in order to assess the selectivity of DMDS reactivity. All the measurements were taken at 295 +/- 2 K in helium buffer gas at a pressure of 0.35 +/- 0.01 Torr. DMDS was observed to react efficiently with m/z 42 (ACNH+), 149 (from phthalates), and 99 (protonated phosphoric acid), with k/kc=0.91, 0.47, and 0.38, respectively. Only proton transfer was observed with ACNH+, followed by the secondary reaction of [DMDSH]+ with DMDS to yield [CH3S-S(CH3)-SCH3]+. Ligation of DMDS was the dominant primary channel observed for the reaction of the m/z 149 background ion; however, some proton transfer also was observed. Both of these primary product ions react further with DMDS to yield [CH3S-S(CH3)-SCH3]+, the structure of which we have determined computationally using DFT calculations. Only the sequential ligation with two DMDS molecules was observed for the reaction of the m/z 99 ion. Reactions of DMDS with m/z 117 [H3PO4 + H + H2O]+ and m/z 131 [H3PO4 + H + MeOH]+ were observed to proceed with k/kc=0.71 and 0.058, respectively. Ligand substitution of DMDS for H2O predominated ( approximately 94%) over DMDS ligation ( approximately 6%) in the reaction with m/z 117, while only DMDS ligation was observed for the reaction of m/z 131 with DMDS. In contrast, the reactions of DMDS with ions of m/z 83 (protonated dimer of ACN) and 327 (protonated triphenyl phosphate) were extremely inefficient, with k/kc=0.0042 and 0.0079, respectively. The higher reactivity of DMDS toward ACNH+ (m/z 42) compared to (ACN)2H+ (m/z 83) is attributed to the lower proton affinity of the unsolvated ACN. The reactivity of DMDS toward the two model analyte ions studied-protonated caffeine and doubly protonated bradykinin-was negligible, with k/kc=0.0073 and 0.010, for the respective reactions. These results suggest that, under appropriate reagent pressure conditions, DMDS can be an appropriate reagent for chemically filtering out many common API-MS background ions, without significantly affecting the observed intensity of analyte peaks.     

Ion trap versus low-energy beam-type collision-induced dissociation of protonated ubiquitin ions

The beam-type and ion trap collision-induced dissociation (CID) behaviors of protonated bovine ubiquitin ions were studied for charge states ranging from +6 to +12 on a modified triple quadrupole/linear ion trap tandem mass spectrometer. Both beam-type CID and ion trap CID were conducted in a high-pressure linear ion trap, followed by proton-transfer ion/ion reactions to reduce the charge states of product ions mostly to +1. The product ions observed under each activation condition were predominantly b- and y-type ions. Fragmentation patterns showed a much stronger dependence on parent ion charge state with ion trap CID than with beam-type CID using nitrogen as the collision gas, with preferential cleavages C-terminal to aspartic acid at relatively low charge states, nonspecific fragmentation at moderate charge states, and favored cleavages N-terminal to proline residues at high charge states. In the beam-type CID case, extensive cleavage along the protein backbone was noted, which yielded richer sequence information (77% of backbone amide bond cleavages) than did ion trap CID (52% of backbone amide bond cleavages). Collision gas identity and collision energy were also evaluated in terms of their effects on the beam-type CID spectrum. The use of helium as collision gas, as opposed to nitrogen, resulted in CID behavior that was sensitive to changes in collision energy. At low collision energies, the beam-type CID data resembled the ion trap CID data with preferential cleavages predominant, while at high collision energies, nonspecific fragmentation was observed with increased contributions from sequential fragmentation.     

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.     

Charge-state-dependent sequence analysis of protonated ubiquitin ions via ion trap tandem mass spectrometry

One of the major factors governing the "top-down" sequence analysis of intact multiply protonated proteins by tandem mass spectrometry is the effect of the precursor ion charge state on the formation of product ions. To more fully understand this effect, electrospray ionization coupled to a quadrupole ion trap mass spectrometer, collision-induced dissociation, and gas-phase ion/ion reactions have been employed to examine the fragmentation of the [M + 12H]12+ to [M + H]+ ions of bovine ubiquitin. At low charge states (+1 to +6), loss of NH3 or H2O from the protonated precursor and directed cleavage at aspartic acid residues was observed. At intermediate charge states, (+7, +8, and +9), extensive nonspecific fragmentation of the protein backbone was observed, with 50% sequence coverage obtained from the [M + 8H]8+ ion alone. At high charge states, (+10, +11, +12), the single dominant channel that was observed was the preferential fragmentation of a single proline residue. These data can be readily explained in terms of the current model for intramolecular proton mobilization, that is, the "mobile proton model", the mechanisms for amide bond dissociation developed for protonated peptides, as well as the structures of the multiply charged ions of ubiquitin in the gas phase, examined by ion mobility and hydrogen/deuterium exchange measurements.     

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.     

Determination of deamidation artifacts introduced by sample preparation using (18)o-labeling and tandem mass spectrometry analysis

The sites and levels of Asn deamidation in proteins are often determined by LC-MS analysis of peptides obtained from enzymatic digestion. However, deamidation that occurs during sample preparation steps results in overestimation of the original level of deamidation. The inherent deamidation and those introduced by sample preparation can be differentiated by preparing samples in (18)O water. When using H(2)(18)O, the formation of isoAsp and Asp by Asn deamidation during sample preparation results in a molecular weight increase of 3 Da due to the incorporation of the (18)O atom to the side chains of isoAsp or Asp; in contrast, inherent deamidation only results in a molecular weight increase of 1 Da. In addition, up to two (18)O atoms can also be incorporated into the peptide C-terminal carboxyl group during enzymatic digestion. Therefore, the 2 Da molecular weight difference at the deamidation sites can only be used to differentiate deamidation that occurs prior to or during sample preparation under conditions that a fixed number of (18)O atoms are incorporated into the peptide C-terminal carboxyl groups. Otherwise, it is challenging to apply this procedure because of the resulting complicated isotopic distributions. Here, a new procedure of using (18)O-water for sample preparation coupled with tandem mass spectrometry (MS/MS) was established to calculate the deamidation artifacts. In this method, b ions were used for the calculation of Asn deamidation that occurred prior to or during sample preparation, which eliminated the complicated factor of various number of (18)O-atoms to the peptide carboxyl groups. This procedure has the potential to be applied under the general peptide mapping conditions.     

Screening for disulfide bonds in proteins by MALDI in-source decay and LIFT-TOF/TOF-MS

An automated screening method is presented that uses MALDI in-source decay (MALDI-ISD) of disulfide bonds for identification of disulfide-linked peptides in MALDI mass spectra. Peptides released by ISD of a disulfide bond can be detected at an m/z ratio that corresponds to the singly protonated peptide with a reduced cysteine residue. Therefore, screening of peak lists for signal patterns that fulfill the equation, m/z (peak A) + m/z (peak B) - m/z (H2 + H+) = m/z (peak C), facilitated identification of putative ISD fragments of disulfide-linked peptides (peaks A and B) and their precursors (peak C). Signals (peak C) from putatively disulfide-linked peptides were subjected to LIFT-TOF/TOF-MS to confirm the existence of a disulfide bond. Using this method, we identified all 4 disulfide bonds in RNAseA and 8 two-disulfide clusters comprising 16 out of the 17 disulfide bonds in BSA. The presented screening method accelerated the identification of disulfide bonds in RNAseA and BSA, because the number of MS/MS spectra to be acquired was reduced by 1 order of magnitude. Less than 5% of the signals selected for LIFT-TOF/TOF-MS did not correspond to disulfide-linked peptides. Furthermore, the number of possible assignments for disulfide-linked peptides was reduced by 2-3 orders of magnitude, because knowledge of the mechanism of disulfide bond fragmentation by ISD permitted use of stricter rules for the interpretation of mass spectra. Therefore, interpretation of MS/ MS spectra of disulfide-linked peptides was considerably simplified in comparison to conventional approaches.     

Mining a tandem mass spectrometry database to determine the trends and global factors influencing peptide fragmentation

A database of 5500 unique peptide tandem mass spectra acquired in an ion trap mass spectrometer was assembled for peptides derived from proteins digested with trypsin. Peptides were identified initially from their tandem mass spectra by the SEQUEST algorithm and subsequently validated manually. Two different statistical methods were used to identify sequence-dependent fragmentation patterns that could be used to improve fragmentation models incorporated into current peptide sequencing and database search algorithms. The currently accepted "mobile proton" model was expanded to derive a new classification scheme for peptide mass spectra, the "relative proton mobility" scale, which considers peptide ion charge state and amino acid composition to categorize peptide mass spectra into peptide ions containing "nonmobile", "partially mobile", or "mobile" protons. Quantitation of amide bond fragmentation, both N- and C-terminal to any given amino acid, as well as the positional effect of an amino acid in a peptide and peptide length on such fragmentation, has been determined. Peptide bond cleavage propensities, both positive (i.e., enhanced) and negative (i.e., suppressed), were determined and ranked in order of their cleavage preferences as primary, secondary, or tertiary cleavage effects. For example, primary positive cleavage effects were observed for Xaa-Pro and Asp-Xaa bond cleavage for mobile and nonmobile peptide ion categories, respectively. We also report specific pairwise interactions (e.g., Asn-Gly) that result in enhanced amide bond cleavages analogous to those observed in solution-phase chemistry. Peptides classified as nonmobile gave low or insignificant scores, below reported MS/MS score thresholds (cutoff filters), indicating that incorporation of the relative proton mobility scale classification would lead to improvements in current MS/MS scoring functions.     

Tandem mass spectrometry of large biomolecule ions by blackbody infrared radiative dissociation

A new method for the dissociation of large ions formed by electrospray ionization is demonstrated. Ions trapped in a Fourier transform mass spectrometer at pressures below 10(-)(8) Torr are dissociated by elevating the vacuum chamber to temperatures up to 215 °C. Rate constants for dissociation are measured and found to be independent of pressure below 10(-)(7) Torr. This indicates that the ions are activated by absorption of blackbody radiation emitted from the chamber walls. Dissociation efficiencies as high as 100% are obtained. There is no apparent mass limit to this method; ions as large as ubiquitin (8.6 kDa) are readily dissociated. Thermally stable ions, such as melittin 3+ (2.8 kDa), did not dissociate at temperatures up to 200 °C. This method is highly selective for low-energy fragmentation, from which limited sequence information can be obtained. From the temperature dependence of the dissociation rate constants, Arrhenius activation energies in the low-pressure limit are obtained. The lowest energy dissociation processes for the singly and doubly protonated ions of bradykinin are loss of NH(3) and formation of the b(2)/y(7) complementary pair, with activation energies of 1.3 and 0.8 eV, respectively. No loss of NH(3) is observed for the doubly protonated ion; some loss of H(2)O occurs. These results show that charge-charge interactions not only lower the activation energy for dissociation but also can dramatically change the fragmentation, most likely through changes in the gas-phase conformation of the ion. Dissociation of ubiquitin ions produces fragmentation similar to that obtained by IRMPD and SORI-CAD. Higher charge state ions dissociate to produce y and b ions; the primary fragmentation process for low charge state ions is loss of H(2)O.     

锂离子电池正极材料Li3V2-2x/3Mnx（PO4）3的溶胶凝胶法合成和电化学性能

以CH3COOLi·2H2O、V2O5、Mn(CH3COO)2·4H2O、(NH4)2HPO4和蔗糖为原料,采用溶胶–凝胶法合成了掺锰磷酸钒锂/碳(Li3V2-2x/3Mnx(PO4)3/C)复合正极材料,用XRD、XPS、SEM、电化学性能对样品进行了表征.测试结果表明,少量锰的掺杂并未改变Li3V2(PO4)3/C的单斜结构,Li3V1.94Mn0.09(PO4)3中的Mn和V分别以+2和+3价存在,其颗粒类似球形,直径比较均匀且小于200 nm,并表现出良好的电化学性能.在0.1C倍率和3.0～4.8 V电压内,该样品的首次充、放电容量分别为182.1和168.8 mAh/g,放电效率高达92.69%,而且100次循环后,其放电比容量仍是首次放电容量的77.4%.     

Determination of lysergic acid diethylamide (LSD), iso-LSD, and N-demethyl-LSD in body fluids by gas chromatography/tandem mass spectrometry.

Procedures for detection and quantitation of lysergic acid diethylamide (LSD), iso-LSD, and N-demethyl-LSD by capillary chromatography/tandem mass spectrometry (GC/MS/MS) are presented. Several methods for derivatization, sample introduction, and ionization, in combination with mass spectrometry/mass spectrometry (MS/MS), have been evaluated for overall ionization efficiency and product-ion sensitivity and specificity. Fragmentation pathways derived from low-energy collision-induced dissociation (CID) spectra of protonated LSD, and the protonated trimethylsllyl derivatives of LSD (LSD-TMS) and deuterium-labeled analogs of LSD, have been proposed. Principal dissociations primarily involve the amide and piperidine-ring moieties in which losses of CH3 radical, CH3NH2, CH3NCH2, diethylamine, diethylformamide, and N,N-diethylpropenamide from MH+ are observed. Positive-ion ammonia chemical ionization and subsequent MS/MS analysis of the protonated molecules (MH+) of the trimethylsilyl (TMS) derivatives of LSD, iso-LSD, and N-demethyl-LSD provide a high degree of specificity for identification of these compounds in urine or blood at low-pg/mL concentrations. Negative-ion chemical ionization and GC/MS/MS analysis of the molecular anion (M-) of the trifluoroacetyl (TFA) derivative is well suited for trace-level identification of N-demethyl-LSD, a metabolite of LSD.     

Sequencing of argentinated peptides by means of matrix-assisted laser desorption/ionization tandem mass spectrometry

Argentinated peptide ions are formed in abundance under matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) conditions in the presence of Ag+ ions. These argentinated peptide ions are fragmented facilely under MALDI-MS/MS conditions to yield [b(n) + OH + Ag]+, [b(n) - H + Ag]+ and [a(n) - H + Ag]+ ions that are indicative of the C-terminal sequence. These observations parallel those made earlier under electrospray MS conditions (Chu, I. K; Guo, X.; Lau, T.-C.; Siu, K W. M. Anal. Chem. 1999, 71, 2364-2372). A mixed protonated and argentinated tryptic peptide map was generated from 37 fmol of bovine serum albumin (BSA) using MALDI-MS. MALDI-MS/MS data from four argentinated peptides at a protein amount of 350 fmol unambiguously identified the protein as BSA. Sequence-tag analysis of two argentinated tryptic peptides was used to identify unambiguously myocyte enhancer factor 2A, which had been recombinantly expressed in a bacterial cell line.     

High temperature oxidation of C2Cl4/CH4 mixtures

Experiments on high temperature oxidation of multi-chlorinated hydrocarbons, tetrachloroethylene (C2Cl4), with hydrocarbon fuels, CH4, were performed in a 15 mm i.d. tubular flow reactor. Temperatures ranged from 700 to 850 degrees C, with the average residence time in the range from 0.3 to 1.5s. Three equivalence ratios, phi=0.87 (fuel-lean (FL)), phi=1 (stoichiometry (S)), and phi=1.3 (fuel-rich (FR)), were studied. The global Arrhenius equations for the decomposition of C(2)Cl(4) for each reactant set ratio are: k(lean)=5.77 x 10(15) exp(-30447/RT), k(stoi)=5.15 x 10(15) exp(-30421/RT), and k(rich)=6.32 x 10(14) exp(-28879/RT). The important reactions for destruction of parent C2Cl4 include: C2Cl4 --> C2Cl3 + Cl, C2Cl4 + H--> C2Cl3 + HCl and C2Cl4 + H --> C2HCl3 + Cl. The resulting reactant loss, and intermediate and final product profiles were determined. C2HCl3, C2Cl2, CO, CO2 and HCl are the major products for the reaction of C2Cl4/CH4/O2 mixtures for these three reaction systems. Minor intermediates include C2H3Cl, C2HCl, COCl2, CH3CHCl2, C2H4, C2H6, CCl2CHCH3 , trans-CHClCHCl, cis-CHClCHCl, trans-ClHC=CClCH(3), C6H6, and Cl2. The experimental data showed that as the oxygen concentration increased, the temperature needed to detect the resulting products decreased.     

Use of a designed peptide array to infer dissociation trends for nontryptic peptides in quadrupole ion trap and quadrupole time-of-flight mass spectrometry

Observed peptide gas-phase fragmentation patterns are a complex function of many variables. To systematically probe this phenomenon, an array of 40 peptides was synthesized for study. The array of sequences was designed to hold certain variables (peptide length) constant and randomize or balance others (peptide amino acid distribution and position). A high-quality tandem mass spectrometry (MS/MS) data set was acquired for each peptide for all observed charge states on multiple MS instruments, quadrupole-time-of-flight and quadrupole ion trap. The data were analyzed as a function of total charge state and number of mobile protons. Previously known dissociation trends were observed, validating our approach. In addition, the general influence of basic amino acids on dissociation could be determined because, in contrast to the more widely studied tryptic peptides, the amino acids H, K, and R were positionally distributed. Interestingly, our results suggest that cleavage at all basic amino acids is suppressed when a mobile proton is available. Cleavage at H becomes favored only under conditions where a partially mobile proton is present, a caveat to the previously reported trend of enhanced cleavage at H. Finally, all acquired data were used as a benchmark to determine how well these sequences would have been identified in a database search using a common algorithm, Mascot.     

Effects of charge state and cationizing agent on the electron capture dissociation of a peptide

Electron capture dissociation (ECD) is a promising method for de novo sequencing proteins and peptides and for locating the positions of labile posttranslational modifications and binding sites of noncovalently bound species. We report the ECD of a synthetic peptide containing 10 alanine residues and 6 lysine residues uniformly distributed across the sequence. ECD of the (M + 2H)(2+) produces a limited range of c (c(7)-c(15)) and z (z(9)-z(15)) fragment ions, but ECD of higher charge states produces a wider range of c (c(2)-c(15)) and z (z(2)-z(6), z(9)-z(15)) ions. Fragmentation efficiency increases with increasing precursor charge state, and efficiencies up to 88% are achieved. Heating the (M + 2H)(2+) to 150 degrees C does not increase the observed range of ECD fragment ions, indicating that the limited products are due to backbone cleavages occurring near charges and not due to effects of tertiary structure. ECD of the (M + 2Li)(2+) and (M + 2Cs)(2+) produces di- and monometalated analogues of the same c and z ions observed from the (M + 2H)(2+), with the abundance of dimetalated fragment ions increasing with fragment ion mass, a result consistent with the metal cations being located near the peptide termini to minimize Coulombic repulsion. In stark contrast to the ECD results, collisional activation of cesiated dications overwhelmingly results in ejection of Cs(+). The abundance of cesiated fragment ions formed from ECD of the (M + Cs + Li)(2+) exceeds that of lithiated fragment ions by 10:1. ECD of the (M + H + Li)(2+) results in exclusively lithiated c and z ions, indicating an overwhelming preference for neutralization and cleavage at protonated sites over metalated sites. These results are consistent with preferential neutralization of the cation with the highest recombination energy.     

Electrochemiluminescence from Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene)

The electrochemiluminescence (ECL) of Os(phen)2(dppene)2+ (phen = 1,10-phenanthroline and dppene = bis(diphenylphosphino)ethene) is reported in mixed CH3CN/H2O (50:50 v/v) and aqueous (0.1 M KH2PO4) solutions with tri-n-propylamine (TPrA) as an oxidative-reductive coreactant. ECL efficiencies (phi(ecl) = photons emitted/redox event) of 2.0 in aqueous, and 0.95 in mixed for Os(phen)2(dppene)2+ were obtained using Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) as a relative standard (phi(ecl) = 1). Photoluminescence (PL) efficiencies of 0.094 and 0.053 were obtained in aqueous and mixed solutions, respectively, as compared to Ru(bpy)3(2+) (phi(em) = 0.042). The ECL spectra were identical to photoluminescence spectra (lambda(max) approximately 584 nm), indicating formation of the same metal-to-ligand (MLCT) excited states in both ECL and PL. The ECL is linear over several orders of magnitude in aqueous and mixed solution, with theoretical detection limits (blank plus three times the standard deviation of the noise) of 16.9 nM in H2O and 0.29 nM in CH3CN/H2O (50:50 v/v).     

Tandem mass spectrometry of very large molecules: serum albumin sequence information from multiply charged ions formed by electrospray ionization.

Serum albumin proteins, Mr approximately 66 kDa, from 10 different species (bovine, human, rat, horse, sheep, goat, rabbit, dog, porcine, and guinea pig) have been studied by electrospray ionization mass spectrometry (ESI-MS) and tandem MS using a triple-quadrupole instrument. The effectiveness of collisional activation for the multiply charged albumin ions greatly exceeds that for singly charged ions, allowing an extension by a factor of at least 20 to the molecular mass range for obtaining sequence-specific product ions by tandem MS. Efficient dissociation is largely attributed to "preheating" in the interface Coulombic instability and the large number of collisions. Increasing the electric field in the intermediate pressure region, between the nozzle-skimmer elements of the atmospheric pressure/vacuum interface, allows fragmentation of the multiply protonated (to 96+) molecules produced by ESI. The most abundant dissociation product ions assigned have a low charge state (2+ to 5+) and are attributed to "bn" mode species from cleavage of the -CO-N- peptide backbone bonds. Particularly abundant dissociation products originate from regions near residues n = 20-25 from the NH2 terminus for parent ions of moderate charge (approximately 50+). Collisionally activated dissociation (CAD) mass spectra from porcine serum albumin, in contrast to the other albumins, also gave prominent singly charged "yn" fragments formed from cleavages near the COOH terminus. Tandem mass spectrometry (MS/MS) of the multiply charged molecular ions, and of fragment species produced by dissociation in the interface (i.e., effective MS/MS/MS), produced similar "bn" species and served to confirm spectral assignments. We also show that ESI mass spectra allow a qualitative assessment of protein microheterogeneity and, in some cases, resolution of major contributions. The physical and analytical implications of the results are discussed, including the identification of possible errors in previously published sequences.