Peptide Anion Research Articles

Negative-Ion Electron Capture Dissociation: Radical-Driven Fragmentation of Charge-Increased Gaseous Peptide Anions

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N-C(α) backbone bond cleavage, resulting in predictable c'/z(•)-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M - nH](n-), n ≥ 1) can capture electrons within a rather narrow energy range (~3.5-6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).

Real Time Analysis of Brain Tissue by Direct Combination of Ultrasonic Surgical Aspiration and Sonic Spray Mass Spectrometry

Direct combination of cavitron ultrasonic surgical aspirator (CUSA) and sonic spray ionization mass spectrometry is presented. A commercially available ultrasonic surgical device was coupled to a Venturi easy ambient sonic-spray ionization (V-EASI) source by directly introducing liquified tissue debris into the Venturi air jet pump. The Venturi air jet pump was found to efficiently nebulize the suspended tissue material for gas phase ion production. The ionization mechanism involving solely pneumatic spraying was associated with that of sonic spray ionization. Positive and negative ionization spectra were obtained from brain and liver samples reflecting the primary application areas of the surgical device. Mass spectra were found to feature predominantly complex lipid-type constituents of tissues in both ion polarity modes. Multiply charged peptide anions were also detected. The influence of instrumental settings was characterized in detail. Venturi pump geometry and flow parameters were found to be critically important in ionization efficiency. Standard solutions of phospholipids and peptides were analyzed in order to test the dynamic range, sensitivity, and suppression effects. The spectra of the intact tissue specimens were found to be highly specific to the histological tissue type. The principal component analysis (PCA) and linear discriminant analysis (LDA) based data analysis method was developed for real-time tissue identification in a surgical environment. The method has been successfully tested on post-mortem and ex vivo human samples including astrocytomas, meningeomas, metastatic brain tumors, and healthy brain tissue.

Relation between charge state distributions of peptide anions and pH changes in the electrospray plume. A mass spectrometry and optical spectroscopy investigation

A laser-induced fluorescence set-up profiling physico-chemical parameters of droplets and their changes in an electrospray plume is coupled to an Agilent single quadrupole mass spectrometer equipped with an Agilent Jet Stream electrospray source. This coupling allows investigating the evolution of the pH of droplets as they evaporate in the electrospray plume by measuring emission spectra of a pH-chromic dye. Simultaneous measurement of mass spectra and optical pH profiling permits to study the relation between the charge states of peptide anions in mass spectra and pH changes in the spray plume. In particular, due to changes in droplets evaporation rates, the source parameters (i.e., sheath gas flow and temperature) influence the pH of the droplets and relative intensities of charge states in peptide mass spectra.

Tandem MS Analysis of Selenamide-Derivatized Peptide Ions

Our previous study showed that selenamide reagents such as ebselen and N-(phenylseleno)phthalimide (NPSP) can be used for selective and rapid derivatization of protein/peptide thiols in high conversion yield. This paper reports the systematic investigation of MS/MS dissociation behaviors of selenamide-derivatized peptide ions upon collision induced dissociation (CID) and electron transfer dissociation (ETD). In the positive ion mode, derivatized peptide ions exhibit tag-dependent CID dissociation pathways. For instance, ebselen-derivatized peptide ions preferentially undergo Se-S bond cleavage upon CID to produce a characteristic fragment ion, the protonated ebselen (m/z 276), which allows selective identification of thiol peptides from protein digest as well as selective detection of thiol proteins from protein mixture using precursor ion scan (PIS). In contrast, NPSP-derivatized peptide ions retain their phenylselenenyl tags during CID, which is useful in sequencing peptides and locating cysteine residues. In the negative ion CID mode, both types of tags are preferentially lost via the Se-S cleavage, analogous to the S-S bond cleavage during CID of disulfide-containing peptide anions. In consideration of the convenience in preparing selenamide-derivatized peptides and the similarity of Se-S of the tag to the S-S bond, we also examined ETD of the derivatized peptide ions to probe the mechanism for electron-based ion dissociation. Interestingly, facile cleavage of Se-S bond occurs to the peptide ions carrying either protons or alkali metal ions, while backbone cleavage to form c/z ions is severely inhibited. These results are in agreement with the Utah-Washington mechanism proposed for depicting electron-based ion dissociation processes.

Metastable Atom-Activated Dissociation Mass Spectrometry of Phosphorylated and Sulfonated Peptides in Negative Ion Mode

The dissociation behavior of phosphorylated and sulfonated peptide anions was explored using metastable atom-activated dissociation mass spectrometry (MAD-MS) and collision-induced dissociation (CID). A beam of high kinetic energy helium (He) metastable atoms was exposed to isolated phosphorylated and sulfonated peptides in the 3- and 2- charge states. Unlike CID, where phosphate losses are dominant, the major dissociation channels observed using MAD were C(α) - C peptide backbone cleavages and neutral losses of CO(2), H(2)O, and [CO(2) + H(2)O] from the charge reduced (oxidized) product ion, consistent with an electron detachment dissociation (EDD) mechanism such as Penning ionization. Regardless of charge state or modification, MAD provides ample backbone cleavages with little modification loss, which allows for unambiguous PTM site determination. The relative abundance of certain fragment ions in MAD is also demonstrated to be somewhat sensitive to the number and location of deprotonation sites, with backbone cleavage somewhat favored adjacent to deprotonated sites like aspartic acid residues. MAD provides a complementary dissociation technique to CID, ECD, ETD, and EDD for peptide sequencing and modification identification. MAD offers the unique ability to analyze highly acidic peptides that contain few to no basic amino acids in either negative or positive ion mode.

193‐nm photodissociation of singly and multiply charged peptide anions for acidic proteome characterization

193-nm ultraviolet photodissociation (UVPD) was implemented to sequence singly and multiply charged peptide anions. Upon dissociation by this method, a-/x-type, followed by d and w side-chain loss ions, were the most prolific and abundant sequence ions, often yielding 100% sequence coverage. The dissociation behavior of singly and multiply charged anions was significantly different with higher charged precursors yielding more sequence ions; however, all charge states investigated (1- through 3-) produced rich diagnostic information. UVPD at 193 nm was also shown to successfully differentiate and pinpoint labile phosphorylation modifications. The sequence ions were produced with high abundances, requiring limited averaging for satisfactory spectral quality. The intact, charge-reduced radical products generated by UV photoexcitation were also subjected to collision-induced dissociation (termed, activated-electron photodetachment dissociation (a-EPD)), but UVPD alone yielded more predictable and higher abundance sequence ions. With the use of a basic (pH∼11.5), piperidine-modified mobile phase, LC-MS/UVPD was implemented and resulted in the successful analysis of mitogen-activated pathway kinases (MAPKs) using ultrafast activation times (5 ns).

Assessing the native state conformational distribution of ubiquitin by peptide acidity

At equilibrium, every energetically feasible conformation of a protein occurs with a non-zero probability. Quantitative analysis of protein flexibility is thus synonymous with determining the proper Boltzmann-weighting of this conformational distribution. The exchange reactivity of solvent-exposed amide hydrogens greatly varies with conformation, while the short-lived peptide anion intermediate implies an insensitivity to the dynamics of conformational motion. Amides that are well-exposed in model conformational ensembles of ubiquitin vary a million-fold in exchange rates which continuum dielectric methods can predict with an rmsd of 3. However, the exchange rates for many of the more rarely exposed amides are markedly overestimated in the PDB-deposited 2K39 and 2KN5 ubiquitin ensembles, while the 2NR2 ensemble predictions are largely consistent with those of the Boltzmann-weighted conformational distribution sampled at the level of 1%. The correlation between the fraction of solvent-accessible conformations for a given amide hydrogen and the exchange rate constant for that residue provides a useful monitor of the degree of completeness with which a given ensemble has sampled the energetically accessible conformational space. These exchange predictions correlate with the degree to which each ensemble deviates from a set of 46 ubiquitin X-ray structures. Kolmogorov–Smirnov analysis for the distribution of intra- and inter-ensemble pairwise structural rmsd values assisted the identification of a subensemble of 2K39 that eliminates the overestimations of hydrogen exchange rates observed for the full ensemble. The relative merits of incorporating experimental restraints into the conformational sampling process are compared to using these restraints as filters to select subpopulations consistent with the experimental data.

Negative Electron Transfer Dissociation of Deprotonated Phosphopeptide Anions: Choice of Radical Cation Reagent and Competition between Electron and Proton Transfer

Despite significant developments in mass spectrometry technology in recent years, no routine proteomics sequencing tool is currently available for peptide anions. The use of a molecular open-shell cation is presented here as a possible reaction partner to induce electron transfer dissociation with deprotonated peptide anions. In this negative electron transfer dissociation (NETD) scheme, an electron is abstracted from the peptide anion and transferred to the radical cation. This is demonstrated for the example of the fluoranthene cation, C(16)H(10)(+*), which is reacted with deprotonated phosphorylated peptides in a 3-D ion trap mass spectrometer. Selective backbone cleavage at the C(alpha)-C bond is observed to yield a and x fragments, similarly to electron detachment dissociation (EDD) of peptide anions. Crucially, the phosphorylation site is left intact in the dissociation process, allowing an identification and localization of the post-translational modification (PTM) site. In contrast, NETD using Xe(+*) as the reagent cation results in sequential neutral losses (CO(2) and H(3)PO(4)) from a/x fragments, which complicate the interpretation of the mass spectra. This difference in dissociation behavior can be understood in the framework of the reduced recombination energy of the electron transfer process for fluoranthene, which is estimated at 2.5-4.5 eV, compared to 6.7-8.7 eV for xenon. Similarly to ETD, proton transfer is found to compete with electron transfer processes in NETD. Isotope fitting of the charge-reduced species shows that in the case of fluoranthene-mediated NETD, proton transfer only accounts for <20%, whereas this process highly abundant for Xe(+*) (43 and 82%). Since proton abstraction from Xe(+*) is not possible, this suggests that Xe(+*) ionizes other transient species in the ion trap, which then engage in proton transfer reactions with the peptide anions.

The Structure of Deprotonated Tri-Alanine and Its a3− Fragment Anion by IR Spectroscopy

We present the first infrared spectra of a mass-selected deprotonated peptide anion (AlaAlaAla) and its decarboxylated fragment anion formed by collision induced dissociation. Spectra are obtained by IRMPD spectroscopy using an FTICR mass spectrometer in combination with the free electron laser FELIX. Spectra have been recorded over the 800-1800 cm(-1) spectral range and compared with density functional theory calculated spectra at the B3LYP/6-31++G(d,p) level for different isomeric structures. These experiments suggest a carboxylate anion for [M-H](-) and an amide deprotonated (amidate) structure for the a(3) fragment anion [M-H-CO(2)](-). The frequency for the amidate carbonyl stretch occurring around 1555 +/- 5 cm(-1) has been confirmed by additional spectroscopic studies of the conjugated base of N-methylacetamide, which serves as a simple model system for the deprotonated amide linkage in a peptide anion.

Conformational Electrostatics in the Stabilization of the Peptide Anion

Conformational Electrostatics in the Stabilization of the Peptide Anion

Intrinsic Neutral and Anionic Structures of Glutathione

Gas-phase intrinsic structures of intact neutral and anionic glutathione (GSH) have been determined by means of a combination of negative ion photo-electron spectroscopy and quantum chemistry calculations. The inferred structures of the neutral parents of those peptide anions are canonical (non-zwitterionic). These intrinsic structures are compared to those already known in aqueous solution or determined by crystallography in binding sites of enzymes.

Polarization and Polarizability Assessed by Protein Amide Acidity

Hydroxide-catalyzed exchange rate constants were determined for those amides of FK506-binding protein (FKBP12), ubiquitin, and chymotrypsin inhibitor 2 (CI2) that are solvent-accessible in the high-resolution X-ray structures. When combined with previous hydrogen exchange results for the rubredoxin from Pyrococcus furiosus, the acidity of these amides was calculated by continuum dielectric methods as a function of the nonpolarizable electrostatic parameter set, internal dielectric, and the charge distribution of the peptide anion. The CHARMM22 parameter set with an internal dielectric value of 3 and an ab initio-derived anion charge distribution yielded an rmsd value of 7 for the 56 amide exchange rate constants ranging from 10(0.67) to 10(9.0) M(-1) s(-1). The OPLS-AA parameter set yielded comparably robust predictions, while that of PARSE, AMBER parm99, and AMBER ff03 performed more poorly. The small value for the optimal internal dielectric, combined with the brief lifetime of the peptide anion intermediate and the uniformity of the correlation between predicted and observed amide acidities, is consistent with electronic polarizability providing the dominant contribution to dielectric shielding. By construction, nonpolarizable force fields do not model electric field attenuation by electronic polarizability. Accurate prediction of the total electrostatic energy by such force fields necessitates the hyperpolarization of the atomic charge values in order to match the average electric field energy density (1/2)epsilon(tau)E(2)(tau) when epsilon(tau) is set to the in vacuo dielectric value of 1. The resulting predictions of the experimental hydrogen exchange data demonstrate the substantial systematic errors in the predicted electrostatic potential that can arise when dielectric shielding due to electronic polarizability is neglected.

The parent anion of the RGD tripeptide: Photoelectron spectroscopy and quantum chemistry calculations

The gas-phase conformation of the intact (parent) unprotected RGD(-) peptide anion has been investigated using a combination of anion photoelectron spectroscopy and quantum chemistry calculations of its low-energy stable structures. The experimentally observed RGD(-) species correspond to a conformation in which the guanidinium group is protonated, the C-terminus is neutral, the aspartic acid carboxyl is deprotonated, and the anion's excess electron orbital is localized on the protonated guanidinium. This structure is reminiscent of the RGD loop, which is the peptide motif recognized by trans-membrane integrins. The parent RGD(-) radical anion was generated using a unique infrared desorption-photoemission-helium jet ion source, whose ability to produce radical anions of peptides may also have analytical mass spectrometric implications.

Transition Metal Complex Cations as Reagents for Gas-Phase Transformation of Multiply Deprotonated Polypeptides

Triply deprotonated DGAILDGAILD was reacted in the gas-phase with doubly charged copper, cobalt, and iron metal complexes containing either two or three phenanthroline ligands. Reaction products result from two major pathways. The first pathway involves the transfer of an electron from the negatively charged peptide to the transition-metal complex. The other major pathway consists of the displacement of the phenanthroline ligands by the peptide resulting in the incorporation of the transition-metal into the peptide to form [M - 3H + X(II)](-) ions, where X is Cu, Co, or Fe, respectively. The extent to which each pathway contributes is dependent on the nature of transition-metal complex. In general, bis-phen complexes result in more electron-transfer than the tris-phen complexes, while the tris-phen complexes result in more metal insertion. The metal in the complex plays a large role as well, with the Cu containing complexes giving rise to more electron transfer than the corresponding complexes of Co and Fe. The results show that a single reagent solution can be used to achieve two distinct sets of products (i.e., electron-transfer products and metal insertion products). These results constitute the demonstration of novel means for the gas-phase transformation of peptide anions from one ion type to another via ion/ion reactions using reagents formed via electrospray ionization.

Backbone conformational dependence of peptide acidity

Electrostatic interactions at the protein surface yield over a billion-fold range of amide hydrogen exchange rates. This range is equivalent to the maximal degree of attenuation in exchange rates that have been shown to occur for amides buried within the protein interior. Continuum dielectric analysis of Ala-Ala, Ala-Gly, Gly-Ala and trans-Pro-Ala peptide conformer acidities predicts that the relative orientation of the two neighboring peptide groups can account for a million-fold variation in hydroxide-catalyzed hydrogen exchange rates. As in previous protein studies, an internal dielectric value of 3 was found to be applicable to simple model peptides, presumably reflecting the short lifetime of the peptide anion intermediate. Despite the million-fold range in conformer acidities, the small differences in the experimental exchange rates for these peptides are accurately predicted. Ala-Ala conformers with an extended N-terminal residue and the C-terminal residue in the α conformation are predicted to account for over 60% of the overall hydrogen exchange reaction, despite constituting only 12% of the protein coil population.

A Billion-fold Range in Acidity for the Solvent-Exposed Amides of Pyrococcus furiosus Rubredoxin

The exchange rates of the static solvent-accessible amide hydrogens of Pyrococcus furiosus rubredoxin range from near the diffusion-limited rate to a billion-fold slower for the non-hydrogen-bonded Val 38 (eubacterial numbering). Hydrogen exchange directly monitors the kinetic acidity of the peptide nitrogen. Electrostatic solvation free energies were calculated by Poisson-Boltzmann methods for the individual peptide anions that form during the hydroxide-catalyzed exchange reaction to examine how well the predicted thermodynamic acidities match the experimentally determined kinetic acidities. With the exception of the Ile 12 amide, the differential exchange rate constant for each solvent-exposed amide proton that is not hydrogen bonded to a backbone carbonyl can be predicted within a factor of 6 (10 (0.78)) root-mean-square deviation (rmsd) using the CHARMM22 electrostatic parameter set and an internal dielectric value of 3. Under equivalent conditions, the PARSE parameter set yields a larger rmsd value of 1.28 pH units, while the AMBER parm99 parameter set resulted in a considerably poorer correlation. Either increasing the internal dielectric value to 4 or reducing it to a value of 2 significantly degrades the quality of the prediction. Assigning the excess charge of the peptide anion equally between the peptide nitrogen and the carbonyl oxygen also reduces the correlation to the experimental data. These continuum electrostatic calculations were further analyzed to characterize the specific structural elements that appear to be responsible for the wide range of peptide acidities observed for these solvent-exposed amides. The striking heterogeneity in the potential at sites along the protein-solvent interface should prove germane to the ongoing challenge of quantifying the contribution that electrostatic interactions make to the catalytic acceleration achieved by enzymes.

Charge inversion of polypeptide anions using protein and dendrimer cations as charge inversion reagents

The conversion of deprotonated bradykinin, as well as anions derived from other polypeptides, to protonated species has been demonstrated via ion/ion reactions with multiply protonated polypropylenimine diaminobutane (DAB) dendrimers and proteins in a linear ion trap. The charge inversion characteristics of both the proteins and the dendrimers were examined with emphasis on the extent of charging of the analyte and the tendency for adduct formation. Multiply protonated proteins, for example, tended to result in significant adduct formation whereas the multiply protonated amino-terminated dendrimers showed essentially no adduct formation. Ions derived from five dendrimer generations were examined as charge inversion reagents with deprotonated bradykinin serving as a peptide model. Both the size and the charge state of the dendrimer reagent ion played a role in determining the number of protons transferred from the reagent cation to the peptide anion. For a given dendrimer generation, the tendency is for increasing numbers of protons to transfer with increasing dendrimer charge. For a given charge state, the numbers of protons transferred tends to be inversely related to dendrimer size. All of the observed data are consistent with charge inversion taking place via a long-lived intermediate complex with the ultimate products being determined by the fate of the complex (i.e., stabilization of the complex versus break-up into one of several competitive dissociation channels).

Coupling of ion‐molecule reactions with liquid chromatography on a quadrupole ion trap mass spectrometer

We report for the first time a coupling of gas-phase ion-molecule reactions with chromatographic separations on a quadrupole ion trap mass spectrometer. The interface was accomplished by using a pulsed valve for the introduction of a volatile neutral into the ion trap. The pulsed valve controller is synchronized with the mass spectrometer software. The setup requires some minor modifications to the vacuum system of the commercial quadrupole ion trap but most of the modifications are external to the mass spectrometer. Two applications of this interface are described: differentiation between two phosphoglucose positional isomers and detection of a phosphopeptide in a peptide mixture. Both applications are using the reactivity of trimethoxyborate towards a phosphate moiety in the negative ion mode. The detection of phosphopeptides hinges on our findings that non-phosphorylated peptide anions do not react with trimethoxyborate. This LC/MS detection can be easily visualized in terms of selected reaction monitoring.

Electrostatic Stabilization and General Base Catalysis in the Active Site of the Human Protein Disulfide Isomerase a Domain Monitored by Hydrogen Exchange

The nucleophilic Cys36 thiol of the human protein disulfide isomerase a domain is positioned over the N terminus of the alpha(2) helix. Amides in the active site exhibit diffusion-limited, hydroxide-catalyzed exchange, indicating that the local positive electrostatic potential decreases the pK value for peptide anion formation by at least 2 units so as to equal or exceed the acidity of water. In stark contrast to the pH dependence of exchange for simple peptides, the His38 amide in the reduced enzyme exhibits a maximum rate of exchange at pH 5 due to efficient general base catalysis by the neutral imidazole of its own side chain and suppression of its exchange by the ionization of the Cys36 thiol. Ionization of this thiol and deprotonation of the His38 side chain suppress the Cys39 amide hydroxide-catalyzed exchange by a million-fold. The electrostatic potential within the active site monitored by these exchange experiments provides a means of stabilizing the two distinct transition states that lead to substrate reduction and oxidation. Molecular modeling offers a role for the conserved Arg103 in coordinating the oxidative transition-state complex, thus providing further support for mechanisms of disulfide isomerization that utilize enzymatic catalysis at each step of the overall reaction.

Spatial distribution of dielectric shielding in the interior of Pyrococcus furiosus rubredoxin as sampled in the subnanosecond timeframe by hydrogen exchange

Experimental p K values of ionizable sidechains provide the most direct test for models representing dielectric shielding within the interior of a protein. However, only the strongly shifted p K values are particularly useful for discriminating among models. NMR titration studies have usually found only one or two such shifted p K values in each protein, so that the fitting of the experimental data to a uniform internal dielectric ( ε int) model is not well constrained. The observed variation among proteins for such ε int estimates may reflect nonuniformity of dielectric shielding within each protein interior or qualitative differences between individual proteins. The differential amide kinetic acidities for a series of metal-substituted rubredoxins are shown to be consistent with Poisson–Boltzmann predictions of dielectric shielding that is relatively uniform for all of the amides that are sensitive to the metal charge, a region which corresponds to roughly 1 / 3 of the internal volume. The effective ε int values near 6 that are found in this study are significantly lower than many such estimates derived from sidechain p K measurements. The differing timeframes in which dielectric relaxation can respond to the highly transient peptide anion as compared to the longer lived states of the charged sidechains offers an explanation for the lower apparent dielectric constant deduced from these measurements.