Improved Identification and Relative Quantification of Sites of Peptide and Protein Oxidation for Hydroxyl Radical Footprinting

Protein oxidation is typically associated with oxidative stress and aging and affects protein function in normal and pathological processes. Additionally, deliberate oxidative labeling is used to probe protein structure and protein–ligand interactions in hydroxyl radical protein footprinting (HRPF). Oxidation often occurs at multiple sites, leading to mixtures of oxidation isomers that differ only by the site of modification. We utilized sets of synthetic, isomeric “oxidized” peptides to test and compare the ability of electron-transfer dissociation (ETD) and collision-induced dissociation (CID), as well as nano-ultra high performance liquid chromatography (nanoUPLC) separation, to quantitate oxidation isomers with one oxidation at multiple adjacent sites in mixtures of peptides. Tandem mass spectrometry by ETD generates fragment ion ratios that accurately report on relative oxidative modification extent on specific sites, regardless of the charge state of the precursor ion. Conversely, CID was found to generate quantitative MS/MS product ions only at the higher precursor charge state. Oxidized isomers having multiple sites of oxidation in each of two peptide sequences in HRPF product of protein Robo-1 Ig1-2, a protein involved in nervous system axon guidance, were also identified and the oxidation extent at each residue was quantified by ETD without prior liquid chromatography (LC) separation. ETD has proven to be a reliable technique for simultaneous identification and relative quantification of a variety of functionally different oxidation isomers, and is a valuable tool for the study of oxidative stress, as well as for improving spatial resolution for HRPF studies.

Selective and Nonselective Cleavages in Positive and Negative CID of the Fragments Generated from In-Source Decay of Intact Proteins in MALDI-MS

Selective and nonselective cleavages in ion trap low-energy collision-induced dissociation (CID) experiments of the fragments generated from in-source decay (ISD) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) of intact proteins are described in both positive and negative ion modes. The MALDI-ISD spectra of the proteins demonstrate common, discontinuous, abundant c- and z′-ions originating from cleavage at the N–Cα bond of Xxx-Asp/Asn and Gly-Xxx residues in both positive- and negative-ion modes. The positive ion CID of the c- and z′-ions resulted in product ions originating from selective cleavage at Asp-Xxx, Glu-Xxx and Cys-Xxx residues. Nonselective cleavage product ions rationalized by the mechanism of a “mobile proton” are also observed in positive ion CID spectra. Negative ion CID of the ISD fragments results in complex product ions accompanied by the loss of neutrals from b-, c-, and y-ions. The most characteristic feature of negative ion CID is selective cleavage of the peptide bonds of acidic residues, Xxx-Asp/Glu/Cys. A definite influence of α-helix on the CID product ions was not obtained. However, the results from positive ion and negative ion CID of the MALDI-ISD fragments that may have long α-helical domains suggest that acidic residues in helix-free regions tend to degrade more than those in helical regions.

Imidate-Based Cross-Linkers for Structural Proteomics: Increased Charge of Protein and Peptide Ions and CID and ECD Fragmentation Studies

Chemical cross-linking is an attractive low-resolution technique for structural studies of protein complexes. Distance constraints obtained from cross-linked peptides identified by mass spectrometry (MS) are used to construct and validate protein models. Amidinating cross-linkers such as diethyl suberthioimidate (DEST) have been used successfully in chemical cross-linking experiments. In this work, the application of a commercial diimidate cross-linking reagent, dimethyl suberimidate (DMS), was evaluated with model peptides and proteins. The peptides were designed with acetylated N-termini followed by random sequences containing two Lys residues separated by an Arg residue. After cross-linking reactions, intra- and intermolecular cross-linked species were submitted to CID and ECD dissociations to study their fragmentation features in the gas phase. Fragmentation of intramolecular peptides by collision induced dissociation (CID) demonstrates a unique two-step fragmentation pathway involving formation of a ketimine as intermediate. Electron capture and electron transfer dissociation (ECD and ETD) experiments demonstrated that the cyclic moiety is not dissociated. Intermolecular species demonstrated previously described fragmentation behavior in both CID and ECD experiments. The charge state distributions (CSD) obtained after reaction with DMS were compared with those obtained with disuccinimidyl suberate (DSS). CSDs for peptides and proteins were increased after their reaction with DMS, owing to the higher basicity of DMS modified species. These features were also observed in LC-MS experiments with bovine carbonic anhydrase II (BCA) after cross-linking with DMS and tryptic proteolysis. Cross-linked peptides derived from this protein were identified at high confidence and those species were in agreement with the crystal structure of BCA.

Dissociations of Complexes Between Monovalent Metal Ions and Aromatic Amino Acid or Histidine

The fragmentations of [AA + M]⁺ complexes, where AA = Phe, Tyr, Trp, or His, and M is a monovalent metal (Li, Na, or Ag), have been exhaustively studied through collision-induced dissociation (CID) and through deuterium labeling. Dissociations of the Li- and Ag-containing complexes gave a large number of fragment ions; by contrast, the sodium/amino acid complexes have lower binding energies, and dissociation resulted in much simpler spectra, with loss of the entire ligand dominating. Unambiguous assignments of these fragment ions were made and formation mechanisms are proposed. Of particular interest are fragmentations in which the charge was retained on the organic fragment and the metal was lost, either as a metal hydride (AgH) or hydroxide (LiOH) or as the silver atom (Ag^?).

Exploring Salt Bridge Structures of Gas-Phase Protein Ions using Multiple Stages of Electron Transfer and Collision Induced Dissociation

The gas-phase structures of protein ions have been studied by electron transfer dissociation (ETD) and collision-induced dissociation (CID) after electrospraying these proteins from native-like solutions into a quadrupole ion trap mass spectrometer. Because ETD can break covalent bonds while minimally disrupting noncovalent interactions, we have investigated the ability of this dissociation technique together with CID to probe the sites of electrostatic interactions in gas-phase protein ions. By comparing spectra from ETD with spectra from ETD followed by CID, we find that several proteins, including ubiquitin, CRABP I, azurin, and β-2-microglobulin, appear to maintain many of the salt bridge contacts known to exist in solution. To support this conclusion, we also performed calculations to consider all possible salt bridge patterns for each protein, and we find that the native salt bridge pattern explains the experimental ETD data better than nearly all other possible salt bridge patterns. Overall, our data suggest that ETD and ETD/CID of native protein ions can provide some insight into approximate location of salt bridges in the gas phase.

Evidence for Sequence Scrambling in Collision-Induced Dissociation of y-Type Fragment Ions

Sequence scrambling from y-type fragment ions has not been previously reported. In a study designed to probe structural variations among b-type fragment ions, it was noted that y fragment ions might also yield sequence-scrambled ions. In this study, we examined the possibility and extent of sequence-scrambled fragment ion generation from collision-induced dissociation (CID) of y-type ions from four peptides (all containing basic residues near the C-terminus) including: AAAAHAA-NH₂ (where “A” denotes carbon thirteen (¹³C₁) isotope on the alanine carbonyl group), des-acetylated-α-melanocyte (SYSMEHFRWGKPV-NH₂), angiotensin II antipeptide (EGVYVHPV), and glu-fibrinopeptide b (EGVNDNEEGFFSAR). We investigated fragmentation patterns of 32 y-type fragment ions, including y fragment ions with different charge states (+1 to +3) and sizes (3 to 12 amino acids). Sequence-scrambled fragment ions were observed from ~50 % (16 out of 32) of the studied y-type ions. However, observed sequence-scrambled ions had low relative intensities from ~0.1 % to a maximum of ~12 %. We present and discuss potential mechanisms for generation of sequence-scrambled fragment ions. To the best of our knowledge, results on y fragment dissociation presented here provide the first experimental evidence for generation of sequence-scrambled fragments from CID of y ions through intermediate cyclic “b-type” ions.

The Effect of a Covalent and a Noncovalent Small-Molecule Inhibitor on the Structure of Abg β-Glucosidase in the Gas-Phase

The effects of binding two small-molecule inhibitors to Agrobacterium sp. strain ATCC 21400 (Abg) β-glucosidase on the conformations and stability of gas-phase ions of Abg have been investigated. Biotin-iminosugar conjugate (BIC) binds noncovalently to Abg while 2,4-dinitro-2-deoxy-2-fluoro-β-d-glucopyranoside (2FG-DNP) binds covalently with loss of DNP. In solution, Abg is a dimer. Mass spectra show predominantly dimer ions, provided care is taken to avoid dissociation of dimers in solution and dimer ions in the ion sampling interface. When excess inhibitor, either covalent or noncovalent, is added to solutions of Abg, mass spectra show peaks almost entirely from 2:2 inhibitor-enzyme dimer complexes. Tandem mass spectrometry experiments show similar dissociation channels for the apo-enzyme and 2FG-enzyme dimers. The +21 dimer produces +10 and +11 monomers. The internal energy required to dissociate the +21 2FG-enzyme to its monomers (767?±?30 eV) is about 36 eV higher than that for the apo-enzyme dimer (731?±?6 eV), reflecting the stabilization of the free enzyme dimer by the 2FG inhibitor. The primary dissociation channels for the noncovalent BIC-enzyme dimer are loss of neutral and charged BIC. The internal energy required to induce loss of BIC is 482?±?8 eV, considerably less than that required to dissociate the dimers. For a given charge state, ions of the covalent and noncovalent complexes have about 15 % and 25 % lower cross sections, respectively, compared with the apo-enzyme. Thus, binding the inhibitors causes the gas-phase protein to adopt more compact conformations. Noncovalent binding surprisingly produces the greatest change in protein ion conformation, despite the weaker inhibitor binding.

Resonance Activation and Collision-Induced-Dissociation of Ions Using Rectangular Wave Dipolar Potentials in a Digital Ion Trap Mass Spectrometer

Collision-induced dissociation (CID) of ions by resonance activation in a quadrupole ion trap is usually accomplished by resonance exciting the ions to higher kinetic energy, whereby the high kinetic energy ions collide with a bath gas, such as helium or argon, inside the trap and dissociate to fragments. A new ion activation method using a well-defined rectangular wave dipolar potential formed by dividing down the trapping rectangular waveform is developed and examined herein. The mass-selected parent ions are resonance excited to high kinetic energies by simply changing the frequency of the rectangular wave dipolar potential and dissociation proceeds. A relationship between the ion mass and the activation waveform frequency is also identified and described. This highly efficient (CID) procedure can be realized by simply changing the waveform frequency of the dipolar potential, which could certainly simplify tandem mass spectrometry analysis methods.

Implementation of Dipolar Resonant Excitation for Collision Induced Dissociation with Ion Mobility/Time-of-Flight MS

An ion mobility/time-of-flight mass spectrometer (IMS/TOF MS) platform that allows for resonant excitation collision induced dissociation (CID) is presented. Highly efficient, mass-resolved fragmentation without additional excitation of product ions was accomplished and over-fragmentation common in beam-type CID experiments was alleviated. A quadrupole ion guide was modified to apply a dipolar AC signal across a pair of rods for resonant excitation. The method was characterized with singly protonated methionine enkephalin and triply protonated peptide angiotensin I, yielding maximum CID efficiencies of 44 % and 84 %, respectively. The Mathieu q_x,y parameter was set at 0.707 for these experiments to maximize pseudopotential well depths and CID efficiencies. Resonant excitation CID was compared with beam-type CID for the peptide mixture. The ability to apply resonant waveforms in mobility-resolved windows is demonstrated with a peptide mixture yielding fragmentation over a range of mass-to-charge (m/z) ratios within a single IMS-MS analysis.

Boundaries of Mass Resolution in Native Mass Spectrometry

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)–protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.

Cation-Induced Stabilization of Protein Complexes in the Gas Phase: Mechanistic Insights From Hemoglobin Dissociation Studies

Collision-induced dissociation (CID) of electrosprayed protein complexes usually involves asymmetric charge partitioning, where a single unfolded chain gets ejected that carries a disproportionately large fraction of charge. Using hemoglobin (Hb) tetramers as model system, we confirm earlier reports that bound metal ions can stabilize protein complexes under CID conditions. We examine the mechanism underlying this effect. Nonvolatile salts cause extensive adduct formation. Significant stabilization was observed for Mg²⁺ and Ca²⁺, whereas K⁺, Rb⁺, and Cs⁺ had no effect. Precursor ion selection was used to examine Hb subpopulations with well-defined metal binding levels. K⁺, Rb⁺, and Cs⁺-adducted tetramers eject monomers that carry roughly one-quarter of the metal ions that were bound to the precursor. This demonstrates that charge migration during CID is exclusively due to proton transfer, not metal ion transfer. Also, replacement of highly mobile charge carriers (protons) with less mobile species (metal ions) does not exert a stabilizing influence under the conditions used here. Interestingly, Hb carrying stabilizing ions (Mg²⁺ and Ca²⁺) generates monomeric CID products that are metal depleted. This effect is attributed to a combination of two factors: (1) Me²⁺ binding stabilizes Hb via formation of chelation bridges (e.g., R-COO^– Me^{2+ –}OOC-R); the more Me²⁺ a subunit contains the more stable it is. (2) More than ~90 % of the tetramers contain at least one subunit with a below-average number of Me²⁺. The prevalence of monomeric CID products with depleted Me²⁺ levels is caused by the tendency of these low metal-containing subunits to undergo preferential unfolding/ejection.

Detailed Study of Cyanobacterial Microcystins Using High Performance Tandem Mass Spectrometry

Microcystins (MC) are a large group of toxic cyclic peptides, produced by cyanobacteria in eutrophic water systems. Identification of MC variants mostly relies on liquid chromatography (LC) combined with collision-induced dissociation (CID) mass spectrometry. Deviations from the essential amino acid complement are a common feature of these natural products, which makes the CID analysis more difficult and not always successful. Here, both CID and electron capture dissociation (ECD) were applied in combination with ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry to study a cyanobacteria strain isolated from the Salto Grande Reservoir in Sao Paulo State, Brazil, without prior LC separation. CID was shown to be an effective dissociation technique for quickly identifying the MC variants, even those that have previously been difficult to characterize by CID. Moreover, ECD provided even more detailed and complementary information, which enabled us to precisely locate metal binding sites of MCs for the first time. This additional information will be important for environmental chemists to study MC accumulation and production in ecosystems.

Use of Doubly Charged Precursors to Validate Dissociation Mechanisms of Singly Charged Poly(Dimethylsiloxane) Oligomers

Collision-induced dissociation of doubly charged poly(dimethylsiloxane) (PDMS) molecules was investigated to provide experimental evidence for fragmentation reactions proposed to occur upon activation of singly charged oligomers. This study focuses on two PDMS species holding trimethylsilyl or methoxy end-groups and cationized with ammonium. In both cases, introduction of the additional charge did not induce significant differences in dissociation behavior, and the use of doubly charged precursors enabled the occurrence of charge-separation reactions, allowing molecules always eliminated as neutrals upon activation of singly charged oligomers to be detected as cationized species. In the case of trimethylsilyl-terminated oligomers, random location of the adducted charge combined with rapid consecutive reactions proposed to occur from singly charged precursors could be validated based on MS/MS data of doubly charged oligomers. In the case of methoxy-terminated PDMS, favored interaction of the adducted ammonium with both end-groups, proposed to rationalize the dissociation behavior of singly charged molecules, was also supported by MS/MS data obtained for molecules adducted with two ammonium cations.

Electron Capture Dissociation and Collision Induced Dissociation of S-Dipalmitoylated Peptides

Here we investigate the effect of S-dipalmitoylation on the electron capture dissociation (ECD) behavior of peptides. The ECD and collision induced dissociation (CID) of peptides modified by covalent attachment of [(RS)-2,3-di(palmitoyloxy)-propyl] (PAM2) group to cysteine residues [C(PAM2)LEYDTGFK and RPPGC(PAM2)SPFK] were examined. The results suggest that ECD of S-dipalmitoylated peptides can provide both primary sequence information and structural information regarding the modification. The structural information provided by CID is complementary to that provided by ECD.

Unblocking the Sink: Improved CID-Based Analysis of Phosphorylated Peptides by Enzymatic Removal of the Basic C-Terminal Residue

A one-step enzymatic reaction for improving the collision-induced dissociation (CID)-based tandem mass spectrometry (MS/MS) analysis of phosphorylated peptides in an ion trap is presented. Carboxypeptidase-B (CBP-B) was used to selectively remove C-terminal arginine or lysine residues from phosphorylated tryptic/Lys-C peptides prior to their MS/MS analysis by CID with a Paul-type ion trap. Removal of this basic C-terminal residue served to limit the extent of gas-phase neutral loss of phosphoric acid (H₃PO₄), favoring the formation of diagnostic b and y ions as determined by an increase in both the number and relative intensities of the sequence-specific product ions. Such differential fragmentation is particularly valuable when the H₃PO₄ elimination is so predominant that localizing the phosphorylation site on the peptide sequence is hindered. Improvement in the quality of tandem mass spectral data generated by CID upon CBP-B treatment resulted in greater confidence both in assignment of the phosphopeptide primary sequence and for pinpointing the site of phosphorylation. Higher Mascot ion scores were also generated, combined with lower expectation values and higher delta scores for improved confidence in site assignment; Ascore values also improved. These results are rationalized in accordance with the accepted mechanisms for the elimination of H₃PO₄ upon low energy CID and insights into the factors dictating the observed dissociation pathways are presented. We anticipate this approach will be of utility in the MS analysis of phosphorylated peptides, especially when alternative electron-driven fragmentation techniques are not available.

Controlling Dissociation Channels of Gas-Phase Protein Complexes Using Charge Manipulation

Coarse-grained simulations with charge hopping were performed for a positively charged tetrameric transthyretin (TTR) protein complex with a total charge of +20. Charges were allowed to move among basic amino acid sites as well as N-termini. Charge distributions and radii of gyration were calculated for complexes simulated at two temperatures, 300 and 600 K, under different scenarios. One scenario treated the complex in its normal state allowing charge to move to any basic site. Another scenario blocked protonation of all the N-termini except one. A final scenario used the complex in its normal state but added a basic-site containing tether (charge tag) near the N-terminus of one chain. The differences in monomer unfolding and charging were monitored in all three scenarios and compared. The simulation results show the importance of the N-terminus in leading the unfolding of the monomer units; a process that follows a zipper-like mechanism. Overall, experimentally modifying the complex by adding a tether or blocking the protonation of N-termini may give the potential for controlling the unraveling and subsequent dissociation of protein complexes.

Top-down analysis of 30–80 kDa proteins by electron transfer dissociation time-of-flight mass spectrometry

Electron transfer dissociation (ETD)-based top-down mass spectrometry (MS) is the method of choice for in-depth structure characterization of large peptides, small- and medium-sized proteins, and non-covalent protein complexes. Here, we describe the performance of this approach for structural analysis of intact proteins as large as the 80 kDa serotransferrin. Current time-of-flight (TOF) MS technologies ensure adequate resolution and mass accuracy to simultaneously analyze intact 30–80 kDa protein ions and the complex mixture of their ETD product ions. Here, we show that ETD TOF MS is efficient and may provide extensive sequence information for unfolded and highly charged (around 1 charge/kDa) proteins of ～30 kDa and structural motifs embedded in larger proteins. Sequence regions protected by disulfide bonds within intact non-reduced proteins oftentimes remain uncharacterized due to the low efficiency of their fragmentation by ETD. For serotransferrin, reduction of S–S bonds leads to significantly varied ETD fragmentation pattern with higher sequence coverage of N- and C-terminal regions, providing a complementary structural information to top-down analysis of its oxidized form.

Decay Mechanisms of Protonated 4-Quinolone Antibiotics After Electrospray Ionization and Ion Activation

This study presents a detailed experimental investigation of charge isomers of protonated 4-quinolone antibiotics molecules formed during electrospray ionization (ESI) with proposed dissociation mechanisms after collisional activation. Piperazinyl quinolones have been previously shown to exhibit erratic behavior during tandem MS analyses of biological samples, which originated from varying ratios of two isomeric variants formed during ESI. Here, a combination of ESI-collision-induced dissociation (CID), differential ion mobility spectrometry (DMS), high resolution MS, and density functional theory (DFT) was used to investigate the underlying mechanisms of isomer formation and their individual dissociation behaviors. The study focused on ciprofloxacin; major findings were confirmed using structurally related 4-quinolones. DFT calculations showed a reversal of basicity for piperazinyl quinolones between liquid and gas phase. We provide an experimental comparison and theoretical treatment of factors influencing the formation ratio of the charge isomers during ESI, including solvent pH, protic/aprotic nature of solvent, and structural effects such as pK _a and proton affinity. The actual dissociation mechanisms of the isomers of the protonated molecules were studied by separating the individual isomers via DMS-MS, which allowed type-specific CID spectra to be recorded. Both primary CID reactions of the two charge isomers originated from the same carboxyl group by charge-remote (CO₂ loss) and charge-mediated (H₂O loss) fragmentation of the piperazinyl quinolones, depending on whether the proton resides on the more basic keto or the piperazinyl group, followed by a number of secondary dissociation reactions. The proposed mechanisms were supported by calculated energies of precursors, transition states, and products for competing pathways. Graphical Abstract

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MS/MS Fragmentation Behavior Study of meso-Phenylporphyrinoids Containing Nonpyrrolic Heterocycles and meso-Thienyl-Substituted Porphyrins

Free base and cobalt(II) complexes of six meso-tetraphenylporphyrinoids containing nonpyrrolic heterocycles and of three meso-thienylporphyrins were investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Their fragmentation was studied in a quadrupole ion trap as a function of the porphyrinoid macrocycle structure and compared with the fragmentation behavior of the benchmark compound meso-tetraphenylporphyrin. In situ oxidation of the neutral cobalt(II) complexes under ESI conditions produced singly charged cobalt(III) porphyrinoid ions; the free bases were ionized by protonation. For the porphyrinoids with an intact porphyrin core, the major fragmentation pathways observed were the losses of the meso-substituent (for meso-phenyl groups) and characteristic fragmentations of one or more meso-substituents (for the meso-thienyl group). Complex fragmentation pathways were observed for porphyrinoids with modifications to the porphyrin core but chemically reasonable structures could be assigned to most fragments, thus delineating general patterns for the behavior of pyrrole-modified porphyrins under CID conditions.