Enhanced Detection of Ubiquitin Isopeptides Using Reductive Methylation

Identification of ubiquitination (Ub) sites is of great interest due to the critical roles that the modification plays in cellular regulation. Current methods using mass spectrometry rely upon tryptic isopeptide diglycine tag generation followed by database searching. We present a novel approach to ubiquitin detection based upon the dimethyl labeling of isopeptide N-termini glycines. Ubiquitinated proteins were digested with trypsin and the resulting peptide mixture was derivatized using formaldehyde-D2 solution and sodium cyanoborohydride. The dimethylated peptide mixtures were next separated by liquid chromatography and analyzed on a quadrupole-TOF based mass spectrometer. Diagnostic b2′ and a1′ ions released from the isopeptide N-terminus upon collision-induced dissociation (CID) were used to spectrally improve the identification of ubiquitinated isopeptides. Proof of principle was established by application to a ubiquitinated protein tryptic digest spiked into a six-protein mix digest background. Extracted ion chromatograms of the a1′ and b2′ diagnostic product ions from the diglycine tag resulted in a significant reduction in signal complexity and demonstrated a selectivity towards the identification of diglycine branched isopeptides. The method was further shown to be capable of identifying diglycine isopeptides resulting from in-gel tryptic digests of ubiquitin enriched material from a His-Ub transfected cell line. We envisage that these ions may be utilized in global ubiquitination studies with post-acquisition MS/MS (or MSe) data interrogation on high resolution hybrid mass spectrometers.

Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

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.

Activated Ion ETD Performed in a Modified Collision Cell on a Hybrid QLT-Oribtrap Mass Spectrometer

We describe the implementation and characterization of activated ion electron transfer dissociation (AI-ETD) on a hybrid QLT-Orbitrap mass spectrometer. AI-ETD was performed using a collision cell that was modified to enable ETD reactions, in addition to normal collisional activation. The instrument manifold was modified to enable irradiation of ions along the axis of this modified cell with IR photons from a CO₂ laser. Laser power settings were optimized for both charge (z) and mass to charge (m/z) and the instrument control firmware was updated to allow for automated adjustments to the level of irradiation. This implementation of AI-ETD yielded 1.6-fold more unique identifications than ETD in an nLC-MS/MS analysis of tryptic yeast peptides. Furthermore, we investigated the application of AI-ETD on large scale analysis of phosphopeptides, where laser power aids ETD, but can produce b- and y-type ions because of the phosphoryl moiety’s high IR adsorption. nLC-MS/MS analysis of phosphopeptides derived from human embryonic stem cells using AI-ETD yielded 2.4-fold more unique identifications than ETD alone, demonstrating a promising advance in ETD sequencing of PTM containing peptides.

A New Matrix Assisted Ionization Method for the Analysis of Volatile and Nonvolatile Compounds by Atmospheric Probe Mass Spectrometry

Matrix assisted ionization of nonvolatile compounds is shown not to be limited to vacuum conditions and does not require a laser. Simply placing a solution of analyte dissolved with a suitable matrix such as 3-nitrobenzonitrile (3-NBN) or 2,5-dihydroxyacetophenone on a melting point tube and gently heating the dried sample near the ion entrance aperture of a mass spectrometer using a flow of gas produces abundant ions of peptides, small proteins, drugs, and polar lipids. Fundamental studies point to matrix-mediated ionization occurring prior to the entrance aperture of the mass spectrometer. The method is analytically useful, producing peptide mass fingerprints of bovine serum albumin tryptic digest consuming sub-picomoles of sample. Application of 100 fmol of angiotensin I in 3-NBN matrix produces the doubly and triply protonated molecular ions as the most abundant peaks in the mass spectrum. No carryover is observed for samples containing up to 100 pmol of this peptide. A commercial atmospheric samples analysis probe provides a simple method for sample introduction to an atmospheric pressure ion source for analysis of volatile and nonvolatile compounds without using the corona discharge but using sample preparation similar to matrix-assisted laser desorption/ionization.

ETD Allows for Native Surface Mapping of a 150 kDa Noncovalent Complex on a Commercial Q-TWIMS-TOF Instrument

Top-down approaches for the characterization of intact proteins and macromolecular complexes are becoming increasingly popular, since they potentially simplify and speed up the assignment process. Here we demonstrate how, on a commercially available Q-TWIMS-TOF instrument, we performed top-down ETD of the native form of tetrameric alcohol dehydrogenase. We achieved good sequence coverage throughout the first 81 N-terminal amino acids of ADH, with the exception of a loop located on the inside of the protein. This is in agreement with the exposed parts of the natively folded protein according to the crystal structure. Choosing the right precursor charge state and applying supplemental activation were found to be key to obtaining a high ETD fragmentation efficiency. Finally, we briefly discuss opportunities to further increase the performance of ETD based on our results.

Peptide Dimethylation: Fragmentation Control via Distancing the Dimethylamino Group

Direct reductive methylation of peptides is a common method for quantitative proteomics. It is an active derivatization technique; with participation of the dimethylamino group, the derivatized peptides preferentially release intense a₁ ions. The advantageous generation of a₁ ions for quantitative proteomic profiling, however, is not desirable for targeted proteomic quantitation using multiple reaction monitoring mass spectrometry; this mass spectrometric method prefers the derivatizing group to stay with the intact peptide ions and multiple fragments as passive mass tags. This work investigated collisional fragmentation of peptides whose amine groups were derivatized with five linear ω-dimethylamino acids, from 2-(dimethylamino)-acetic acid to 6-(dimethylamino)-hexanoic acid. Tandem mass spectra of the derivatized tryptic peptides revealed different preferential breakdown pathways. Together with energy resolved mass spectrometry, it was found that shutting down the active participation of the terminal dimethylamino group in fragmentation of derivatized peptides is possible. However, it took a separation of five methylene groups between the terminal dimethylamino group and the amide formed upon peptide derivatization. For the first time, the gas-phase fragmentation of peptides derivatized with linear ω-dimethylamino acids of systematically increasing alkyl chain lengths is reported. Figure

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Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common ‘capping’ unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in ‘beam-type’ CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was ‘neutralized’—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

Protein-directed in situ synthesis of platinum nanoparticles with superior peroxidase-like activity, and their use for photometric determination of hydrogen peroxide

Platinum nanoparticles (Pt-NPs) with sizes in the range from 10 to 30 nm were synthesized using protein-directed one-pot reduction. The model globular protein bovine serum albumin (BSA) was exploited as the template, and the resulting BSA/Pt-NPs were studied by transmission electron microscopy, energy dispersive X-ray spectroscopy, and resonance Rayleigh scattering spectroscopy. The modified nanoparticles display a peroxidase-like activity that was exploited in a rapid method for the colorimetric determination of hydrogen peroxide which can be detected in the 50 μM to 3 mM concentration range. The limit of detection is 7.9 μM, and the lowest concentration that can be visually detected is 200 μM.

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.

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.

Silica-coated magnetic nanoparticles loaded with Cu(II): preparation and application to the purification of bovine serum albumin

Magnetic nanoparticles (MNPs) coated with silica gel were prepared, then functionalized with a tridentate ligand via a silane coupling agent (3-chloropropyl)triethoxysilane, and finally loaded with Cu(II) ions. The resulting materials were characterized by TEM, SEM, XRD, FTIR and TGA techniques. They display strong affinity for BSA with an adsorption capacity as high as 235 mg g^?1 and with a fast (30 min) establishment of adsorption equilibrium. Repetitive adsorptions (6 times) hardly affect the adsorption capability. The kinetics and isotherm of the adsorption of BSA were also investigated.

On-Plate Self-Desalting and Matrix-Free LDI MS Analysis of Peptides With a Surface Patterned Sample Support

A hydrophobic-hydrophilic-hydrophobic pattern has been produced on the surface of a silicon substrate for selective enrichment, self-desalting, and matrix-free analysis of peptides in a single step. Upon sample application, the sample solution is first confined in a small area by a hydrophobic F-SAM outer area, after which salt contaminants and peptides are selectively enriched in the hydrophilic and hydrophobic areas, respectively. Simultaneously, matrix background noise is significantly reduced or eliminated because of immobilization of matrix molecules. As a result, the detection sensitivity is enhanced 20-fold compared with that obtained using the usual MALDI plate, and interference-free detection is achieved in the low m/z range. In addition, peptide ions can be identified unambiguously in the presence of NH₄HCO₃ (100 mM), urea (1 M), and NaCl (1 M). When the device was applied to the analysis of BSA digests, the peptide recovery and protein identification confidence were greatly improved.

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.

Influence of Metal–Peptide Complexation on Fragmentation and Inter-Fragment Hydrogen Migration in Electron Transfer Dissociation

The use of metal salts in electrospray ionization (ESI) of peptides increases the charge state of peptide ions, facilitating electron transfer dissociation (ETD) in tandem mass spectrometry. In the present study, K⁺ and Ca²⁺ were used as charge carriers to form multiply-charged metal–peptide complexes. ETD of the potassium- or calcium-peptide complex was initiated by transfer of an electron to a proton remote from the metal cation, and a c'-z? fragment complex, in which the c' and z? fragments were linked together via a metal cation coordinating with several amino acid residues, was formed. The presence of a metal cation in the precursor for ETD increased the lifetime of the c'-z? fragment complex, eventually generating c? and z' fragments through inter-fragment hydrogen migration. The degree of hydrogen migration was dependent on the location of the metal cation in the metal-peptide complex, but was not reconciled with conformation of the precursor ion obtained by molecular mechanics simulation. In contrast, the location of the metal cation in the intermediate suggested by the ETD spectrum was in agreement with the conformation of “proton-removed” precursors, indicating that the charge reduction of precursor ions by ETD induces conformational rearrangement during the fragmentation process.

Studies on the electrochemistry of rutin and its interaction with bovine serum albumin using a glassy carbon electrode modified with carbon-coated nickel nanoparticles

We report on a glassy carbon electrode modified with carbon-coated nickel nanoparticles (C-Ni/GCE) that can be used to study the electrochemical properties of rutin and its interaction with bovine serum albumin (BSA) via cyclic voltammetry and differential pulse voltammetry. The effects of pH value, accumulation potential, accumulation time and reaction time were optimized. A pair of reversible peaks is found in the potential range of 0 to around 0.6 V at pH?5.0. Two linear response ranges (with different slopes) are found, one in the 2 to 210 nM concentration range, the other between 0.21 and 1.72 μM. The detection limit is as low as 0.6 nM. On addition of BSA to the rutin solution, a decrease of the current is observed that is proportional to the concentration of BSA. The binding constant and stoichiometric ratio were calculated.

Electron Transfer Dissociation of Photolabeled Peptides. Backbone Cleavages Compete with Diazirine Ring Rearrangements

Gas-phase conformations and electron transfer dissociations of pentapeptide ions containing the photo-Leu residue (L*) were studied. Exhaustive conformational search including molecular dynamics force-field, semi-empirical, ab initio, and density functional theory calculations established that the photo-Leu residue did not alter the gas-phase conformations of (GL*GGK? + ?2H)²⁺ and (GL*GGK-NH₂?+?H)⁺ ions, which showed the same conformer energy ranking as the unmodified Leu-containing ions. This finding is significant in that it simplifies conformational analysis of photo-labeled peptide ions. Electron transfer dissociation mass spectra of (GL*GGK? + ?2H)²⁺, (GL*GGK-NH₂?+?2H)²⁺,(GL*GGKK?+?2H)²⁺, (GL*GLK?+?2H)²⁺, and (GL*LGK?+?2H)²⁺ showed 16 %–21 % fragment ions originating by radical rearrangements and cleavages in the diazirine ring. These side-chain dissociations resulted in eliminations of N₂H₃, N₂H₄, [N₂H₅], and [NH₄O] neutral fragments and were particularly abundant in long-lived charge-reduced cation-radicals. Deuterium labeling established that the neutral hydrazine molecules mainly contained two exchangeable and two nonexchangeable hydrogen atoms from the peptide and underwent further H/D exchange in an ion–molecule complex. Electron structure calculations on the charge-reduced ions indicated that the unpaired electron was delocalized between the diazirine and amide π* electronic systems in the low electronic states of the cation-radicals. The diazirine moiety in GL*GGK-NH₂was calculated to have an intrinsic electron affinity of 1.5 eV, which was further increased by the Coulomb effect of the peptide positive charge. Mechanisms are proposed for the unusual elimination of hydrazine from the photo-labeled peptide ions.

Electron Transfer and Collision Induced Dissociation of Non-Derivatized and Derivatized Desmosine and Isodesmosine

Electron transfer dissociation (ETD) has attracted increasing interest due to its complementarity to collision-induced dissociation (CID). ETD allows the direct localization of labile post-translational modifications, which is of main interest in proteomics where differences and similarities between ETD and CID have been widely studied. However, due to the fact that ETD requires precursor ions to carry at least two charges, little is known about differences in ETD and CID of small molecules such as metabolites. In this work, ETD and CID of desmosine (DES) and isodesmosine (IDS), two isomers that due to the presence of a pyridinium group can carry two charges after protonation, are studied and compared. In addition, the influence of DES/IDS derivatization with propionic anhydride and polyethyleneglycol (PEG) reagents on ETD and CID was studied, since this is a common strategy to increase sensitivity and to facilitate the analysis by reversed-phase chromatography. Clear differences between ETD and CID of non-derivatized and derivatized-DES/IDS were observed. While CID is mainly attributable to charge-directed fragmentation, ETD is initiated by the generation of a hydrogen atom at the initial protonation site and its subsequent transfer to the pyridinium ring of DES/IDS. These differences are reflected in the generation of complex CID spectra dominated by the loss of small, noninformative molecules (NH₃, CO, H₂O), while ETD spectra are simpler and dominated by characteristic side-chain losses. This constitutes a potential advantage of ETD in comparison to CID when employed for the targeted analysis of DES/IDS in biological samples.

Efficient sample proteolysis based on a microchip containing a glass fiber core with immobilized trypsin

Trypsin was immobilized on cellulose-coated glass fibers via a condensation reaction between the aldehyde groups of the oxidized cellulose and the primary amino groups of trypsin. A piece of the modified fiber was inserted into the main channel of a poly(methyl methacrylate) microchip to form a microfluidic proteolytic bioreactor. Scanning electron microscopy of the cross section of the fiber revealed a rough film on the surface of the fiber glass. The performance of the bioreactor was demonstrated by the tryptic digestion of hemoglobin and cytochrome c, where the time for digestion was reduced to <10?s. The digests were identified by MALDI-TOF-MS to obtain peptide mass fingerprint spectra. The results indicated that the digestion in the microfluidic bioreactor is comparable to that of a 12-h solution tryptic digest and thus provides a promising platform for the high throughput identification of proteins.