Evaluation of Selected Binding Domains for the Analysis of Ubiquitinated Proteomes

Ubiquitination is an abundant post-translational modification that consists of covalent attachment of ubiquitin to lysine residues or the N-terminus of proteins. Mono- and polyubiquitination have been shown to be involved in many critical eukaryotic cellular functions and are often disrupted by intracellular bacterial pathogens. Affinity enrichment of ubiquitinated proteins enables global analysis of this key modification. In this context, the use of ubiquitin-binding domains is a promising but relatively unexplored alternative to more broadly used immunoaffinity or tagged affinity enrichment methods. In this study, we evaluated the application of eight ubiquitin-binding domains that have differing affinities for ubiquitination states. Small-scale proteomics analysis identified ~200 ubiquitinated protein candidates per ubiquitin-binding domain pull-down experiment. Results from subsequent Western blot analyses that employed anti-ubiquitin or monoclonal antibodies against polyubiquitination at lysine 48 and 63 suggest that ubiquitin-binding domains from Dsk2 and ubiquilin-1 have the broadest specificity in that they captured most types of ubiquitination, whereas the binding domain from NBR1 was more selective to polyubiquitination. These data demonstrate that with optimized purification conditions, ubiquitin-binding domains can be an alternative tool for proteomic applications. This approach is especially promising for the analysis of tissues or cells resistant to transfection, of which the overexpression of tagged ubiquitin is a major hurdle.

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.

Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation

The carboxyl groups of tryptic peptides were derivatized with a tertiary or quaternary amine labeling reagent to generate more highly charged peptide ions that fragment efficiently by electron transfer dissociation (ETD). All peptide carboxyl groups—aspartic and glutamic acid side-chains as well as C-termini—were derivatized with an average reaction efficiency of 99 %. This nearly complete labeling avoids making complex peptide mixtures even more complex because of partially-labeled products, and it allows the use of static modifications during database searching. Alkyl tertiary amines were found to be the optimal labeling reagent among the four types tested. Charge states are substantially higher for derivatized peptides: a modified tryptic digest of bovine serum albumin (BSA) generates ~90% of its precursor ions with z? > ?2, compared with less than 40 % for the unmodified sample. The increased charge density of modified peptide ions yields highly efficient ETD fragmentation, leading to many additional peptide identifications and higher sequence coverage (e.g., 70 % for modified versus only 43 % for unmodified BSA). The utility of this labeling strategy was demonstrated on a tryptic digest of ribosomal proteins isolated from yeast cells. Peptide derivatization of this sample produced an increase in the number of identified proteins, a >50 % increase in the sequence coverage of these proteins, and a doubling of the number of peptide spectral matches. This carboxyl derivatization strategy greatly improves proteome coverage obtained from ETD-MS/MS of tryptic digests, and we anticipate that it will also enhance identification and localization of post-translational modifications.

Rapid enrichment of phosphopeptides by BaTiO3 nanoparticles after microwave-assisted tryptic digest of phosphoproteins, and their identification by MALDI-MS

We show that BaTiO₃ nanoparticles (NPs) can be used as a novel substrate for the rapid enrichment of phosphopeptides from microwave tryptic digests of α-casein and non-fat milk prior to their identification by MALDI-MS. Protein digestion is achieved by microwave tryptic digest for 50?s, and the resulting phosphopeptides can be effectively adsorbed on the surfaces of the NPs. The phosphopeptides were selectively detected via MALDI-MS. Digestion, enrichment and detection are accomplished within ～60?min. The method was applied to the indentification of 24 phosphopeptides from α-casein and of 21 phosphopeptides (of the α-casein type) from nonfat milk.

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.

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.

157 nm Photodissociation of Dipeptide Ions Containing N-Terminal Arginine

Twenty singly-charged dipeptide ions with N-terminal arginine were photodissociated using 157 nm light in both a linear ion-trap mass spectrometer and a MALDI-TOF-TOF mass spectrometer. Analogous to previous work on dipeptides containing C-terminal arginine, this set of samples enabled insights into the photofragmentation propensities associated with individual residues. In addition to familiar products such as a-, d-, and immonium ions, m₂ and m₂+13 ions were also observed. Certain side chains tended to cleave between their β and γ carbons without necessarily forming d- or w-type ions, and a few other ions were produced by the high-energy fragmentation of multiple bonds.

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.

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.

Constant-Momentum Acceleration Time-of-Flight Mass Spectrometry with Energy Focusing

Fundamental aspects of constant-momentum acceleration time-of-flight mass spectrometry (CMA-TOFMS) are explored as a means to improve mass resolution. By accelerating all ions to the same momentum rather than to the same energy, the effects of the initial ion spatial and energy distributions upon the total ion flight time are decoupled. This decoupling permits the initial spatial distribution of ions in the acceleration region to be optimized independently, and energy focus, including ion turn-around-time error, to be accomplished with a linear-field reflectron. Constant-momentum acceleration also linearly disperses ions across time according to mass-to-charge (m/z) ratio, instead of the quadratic relationship between flight time and m/z found in conventional TOFMS. Here, CMA-TOFMS is shown to achieve simultaneous spatial and energy focusing over a selected portion of the mass spectrum. An orthogonal-acceleration time-of-flight system outfitted with a reduced-pressure DC glow discharge (GD) ionization source is used to demonstrate CMA-TOFMS with atomic ions. The influence of experimental parameters such as the amplitude and width of the time-dependent CMA pulse on mass resolution is investigated, and a useful CMA-TOFMS focusing window of 2 to 18 Da is found for GD-CMA-TOFMS.

Mass Recalibration of FT-ICR Mass Spectrometry Imaging Data Using the Average Frequency Shift of Ambient Ions

Achieving and maintaining high mass measurement accuracy (MMA) throughout a mass spectrometry imaging (MSI) experiment is vital to the identification of the observed ions. However, when using FTMS instruments, fluctuations in the total ion abundance at each pixel due to inherent biological variation in the tissue section can introduce space charge effects that systematically shift the observed mass. Herein we apply a recalibration based on the observed cyclotron frequency shift of ions found in the ambient laboratory environment, polydimethylcyclosiloxanes (PDMS). This calibration method is capable of achieving part per billion (ppb) mass accuracy with relatively high precision for an infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) MSI dataset. Comparisons with previously published mass calibration approaches are also presented.

Detection of Amine Impurity and Quality Assessment of the MALDI Matrix α-Cyano-4-Hydroxy-Cinnamic Acid for Peptide Analysis in the amol Range

We have studied sample preparation conditions to increase the reproducibility of positive UV-MALDI-TOF mass spectrometry of peptides in the amol range. By evaluating several α-cyano-4-hydroxy-cinnamic acid (CHCA) matrix batches and preparation protocols, it became apparent that two factors have a large influence on the reproducibility and the quality of the generated peptide mass spectra: (1) the selection of the CHCA matrix, which allows the most sensitive measurements and an easier finding of the “sweet spots,” and (2) the amount of the sample volume deposited onto the thin crystalline matrix layer. We have studied in detail the influence of a contaminant, coming from commercial CHCA matrix batches, on sensitivity of generated peptide mass spectra in the amol as well as fmol range of a tryptic peptide mixture. The structure of the contaminant, N,N-dimethylbutyl amine, was determined by applying MALDI-FT-ICR mass spectrometry experiments for elemental composition and MALDI high energy CID experiments utilizing a tandem mass spectrometer (TOF/RTOF). A recrystallization of heavily contaminated CHCA batches that reduces or eliminates the determined impurity is described. Furthermore, a fast and reliable method for the assessment of CHCA matrix batches prior to tryptic peptide MALDI mass spectrometric analyses is presented.

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.

Differentiation of Diastereoisomers of Protected 1,2-Diaminoalkylphosphonic Acids by EI Mass Spectrometry and Density Functional Theory

Electron ionization mass spectrometry and density functional theory (DFT) calculations have been used to study the fragmentation of diastereoisomers of protected 1,2-diaminoalkylphosphonic acids. The loss of a diethoxyphosphoryl group and the elimination of diethyl phosphonate were found to be competitive fragmentation processes, which can be used to differentiate both stereoisomers. Selective deuterated analogs and product- and precursor-ion mass spectra allowed the elucidation of the fragmentation mechanisms. The structures of the transition states and product ions were optimized using the density functional theory (DFT), and free energy calculations confirmed the observed differences in the formation and relative intensities of specific fragment ions.

Charge Site Mass Spectra: Conformation-Sensitive Components of the Electron Capture Dissociation Spectrum of a Protein

A conventional electron capture dissociation (ECD) spectrum of a protein is uniquely characteristic of the first dimension of its linear structure. This sequence information is indicated by summing the primary c ^m+ and z ^m+? products of cleavage at each of its molecular ion’s inter-residue bonds. For example, the ECD spectra of ubiquitin (M?+?nH)ⁿ⁺ ions, n?=?7–13, provide sequence characterization of 72 of its 75 cleavage sites from 1843 ions in seven c ^(1–7)+ and eight z ^(1–8)+? spectra and their respective complements. Now we find that each of these c/z spectra is itself composed of “charge site (CS)” spectra, the c ^m+ or z ^m+? products of electron capture at a specific protonated basic residue. This charge site has been H-bonded to multiple other residues, producing multiple precursor ion forms; ECD at these residues yields the multiple products of that CS spectrum. Closely similar CS spectra are often formed from a range of charge states of ubiquitin and KIX ions; this indicates a common secondary conformation, but not the conventional α-helicity postulated previously. CS spectra should provide new capabilities for comparing regional conformations of gaseous protein ions and delineating ECD fragmentation pathways.

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.

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.     

Enzyme-linked electrochemical DNA ligation assay using magnetic beads

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5′-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

Reagent Cluster Anions for Multiple Gas-Phase Covalent Modifications of Peptide and Protein Cations

Multiple gas phase ion/ion covalent modifications of peptide and protein ions are demonstrated using cluster-type reagent anions of N-hydroxysulfosuccinimide acetate (sulfo-NHS acetate) and 2-formyl-benzenesulfonic acid (FBMSA). These reagents are used to selectively modify unprotonated primary amine functionalities of peptides and proteins. Multiple reactive reagent molecules can be present in a single cluster ion, which allows for multiple covalent modifications to be achieved in a single ion/ion encounter and at the ‘cost’ of only a single analyte charge. Multiple derivatizations are demonstrated when the number of available reactive sites on the analyte cation exceeds the number of reagent molecules in the anionic cluster (e.g., data shown here for reactions between the polypeptide [K₁₀ + 3H]³⁺ and the reagent cluster [5R_5Na – Na]^–). This type of gas-phase ion chemistry is also applicable to whole protein ions. Here, ubiquitin was successfully modified using an FBMSA cluster anion which, upon collisional activation, produced fragment ions with various numbers of modifications. Data for the pentamer cluster are included as illustrative of the results obtained for the clusters comprised of two to six reagent molecules.