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Two-dimensional mass spectrometry (2DMS) allows data independent fragmentation of all ions in a sample and correlation of fragment ions to their precursors without isolation prior to fragmentation. Developments in computer capabilities and implementations in Fourier transform ion cyclotron resonance (FTICR) MS over the past decade have allowed the technique to become a useful analytical tool for bottom-up proteomics (BUP) and, more recently, in top-down protein analysis (TDP). In this work, a new method of TDP is developed using 2D FTICR MS, called MS/2D FTICR MS or MS/2DMS. In MS/2DMS, an entire protein is initially fragmented in a hexapole collision cell, e.g., with collisionally activated dissociation (CAD). The primary fragments are then sent to the ICR cell, where 2DMS is performed with infrared multiphoton dissociation (IRMPD) or electron-capture dissociation (ECD). The resulting 2D mass spectra retain information equivalent to a set of TDP MS3 experiments on the selected protein. Up to n - 1 fragmentation steps can be added to the process, as long as an ion of interest can be unambiguously fragmented before the ICR cell, leading to an MSn/2DMS experiment whose output is a 2D mass spectrum retaining information equivalent to MSn. MS/2DMS and MS/MS/2DMS are used in this work for the structural analysis of ubiquitin (Ubi), noting several unique features which aid fragment identification. The use of CAD-MS/IRMPD-2DMS, CAD-MS/ECD-2DMS, and MS2/2DMS using, respectively, in-source dissociation (ISD), CAD, and ECD-2DMS led to 97% cleavage coverage for Ubi. [ABSTRACT FROM AUTHOR]

Gas-phase hydrogen radicals were introduced into a quadrupole ion trap containing singly charged phospholipids to obtain structural fragmentation patterns in tandem mass spectrometry (MS/MS). Saturated and unsaturated phosphatidylcholines were used as a model phospholipid, whose chain-length ranges between 16 and 24. The MS/MS spectrum yielded a continuous series of fragment ions with a mass difference of 14 Da, representing the saturated fatty acyl chains. The fragment ions corresponding to the double-bond position within a single fatty acyl chain showed a characteristic mass difference of 12 Da. The detection of these diagnostic product ions enabled the structural analysis of double-bond isomers of phospholipids. To further investigate the potential of radical-induced dissociation for the isomeric analysis of phospholipids, gas-phase hydroxyl radicals, and triplet oxygen atoms were employed in tandem mass spectrometry. The methylene bridges adjacent to the double-bond positions were selectively dissociated, accompanied by oxidation of the double bonds. Tandem mass spectrometry incorporating multiple radical species facilitates the structural analysis of isomeric phospholipids. [ABSTRACT FROM AUTHOR]

Tandem mass spectrometry has long been established as a corner stone of analytical and structural chemistry. Fast radical-directed dissociation, produced by electron-transfer and electron-capture dissociation (ETD and ECD) has been shown to provide important complementary information to collision-induced dissociation (CID). We report the first application of extremeultraviolet (XUV) lamps to tandem mass spectrometry. These discharge lamps are versatile, robust, and low-cost sources of energetic photons (40-80 nm). The coupling of the discharge lamp with a Waters Synapt G2-Si Q-ToF mass spectrometer is achieved through a specific trapping scheme in the TriWave region of the instrument, allowing efficient irradiation of the precursor ions. Rich radical-directed dissociation was produced for a number of model compounds, providing unique, complementary information to existing dissociation techniques. [ABSTRACT FROM AUTHOR]

When using biosensors, analyte biomolecules of several different concentrations are percolated over a chip with immobilized ligand molecules that form complexes with analytes. However, in many cases of biological interest, e.g., in antibody interactions, complex formation steady-state is not reached. The data measured are so-called sensorgram, one for each analyte concentration, with total complex concentration vs time. Here we present a new four-step strategy for more reliable processing of this complex kinetic binding data and compare it with the standard global fitting procedure. In our strategy, we first calculate a dissociation graph to reveal if there are any heterogeneous interactions. Thereafter, a new numerical algorithm, AIDA, is used to get the number of different complex formation reactions for each analyte concentration level. This information is then used to estimate the corresponding complex formation rate constants by fitting to the measured sensorgram one by one. Finally, all estimated rate constants are plotted and clustered, where each cluster represents a complex formation. Synthetic and experimental data obtained from three different QCM biosensor experimental systems having fast (close to steady-state), moderate, and slow kinetics (far from steady-state) were evaluated using the four-step strategy and standard global fitting. The new strategy allowed us to more reliably estimate the number of different complex formations, especially for cases of complex and slow dissociation kinetics. Moreover, the new strategy proved to be more robust as it enables one to handle system drift, i.e., data from biosensor chips that deteriorate over time. [ABSTRACT FROM AUTHOR]

A transformation product of trimethoprim, a contaminant of emerging concern in the environment, is generated using an electro-assisted Fenton reaction and analyzed using differential mobility spectrometry (DMS) in combination with MS/MS techniques and quantum chemical calculations to develop a rapid method for identification. DMS is used as a prefilter to separate positional isomers prior to subsequent identification by mass spectrometric analyses. Collision induced dissociation of each DMS separated species is used to reveal fragmentation patterns that can be correlated to specific isomer structures. Analysis of the experimental data and supporting quantum chemical calculations show that methylene-hydroxylated and methoxy-containing phenyl ring hydroxylated transformation products are observed. The proposed methodology outlines a high-throughput technique to determine transformation products of small molecules accurately, in a short time and requiring minimal sample concentrations (<25 ng/mL). [ABSTRACT FROM AUTHOR]

Single-molecule fluorescence methods can count molecules without calibration, measure kinetics at equilibrium, and observe rare events that cannot be detected in an ensemble measurement. We employ total internal reflection fluorescence microscopy to monitor hybridization kinetics between individual spatially resolved target DNA molecules immobilized at a glass interface and fluorescently labeled complementary probe DNA in free solution. Using super-resolution imaging, immobilized target DNA molecules are located with 36 nm precision, and their individual duplex formation and dissociation kinetics with labeled DNA probe strands are measured at site densities much greater than the diffraction limit. The purpose of this study is to evaluate uncertainties in identifying these individual target molecules based on their duplex dissociation kinetics, which can be used to distinguish target molecule sequences randomly immobilized in mixed-target samples. Hybridization kinetics of individual target molecules are determined from maximum likelihood estimation of their dissociation times determined from a sample of hybridization events at each target molecule. The dissociation time distributions thus estimated are sufficiently narrow to allow kinetic discrimination of different target sequences. For example, a single-base thymine-to-guanine substitution on immobilized strands produces a 2.5-fold difference in dissociation rates of complementary probes, allowing for the identification of individual target DNA molecules by their dissociation rates with 95% accuracy. This methodology represents a step toward high-density single-molecule DNA microarray sensors and a powerful tool to investigate the kinetics of hybridization at surfaces at the molecular level, providing information that cannot be acquired in ensemble measurements. [ABSTRACT FROM AUTHOR]

Thanks to comprehensive and unbiased sampling of all precursor ions, the interest to move toward bottom-up proteomic with data-independent acquisition (DIA) is continuously growing. DIA offers precision and reproducibility performances comparable to true targeted methods but has the advantage of enabling retrospective data testing with the hypothetical presence of new proteins of interest. Nonetheless, the chimeric nature of DIA MS/MS spectra inherent to concomitant transmission of a multiplicity of precursor ions makes the confident identification of peptides often challenging, even with spectral library-based extraction strategy. The introduction of specificity at the fragmentation step upon ultraviolet or visible laser-induced dissociation (LID) range targeting only the subset of cysteine-containing peptides (Cys-peptide) has been proposed as an option to streamline and reduce the search space. Here, we describe the first coupling between DIA and visible LID at 473 nm to test for the presence of Cys-peptides with a peptide-centric approach. As a test run, a spectral library was built for a pool of Cys-synthetic peptides used as surrogates of human kinases (1 peptide per protein). By extracting ion chromatograms of query standard and kinase peptides spiked at different concentration levels in an Escherichia coli proteome lysate, DIA-LID demonstrates a dynamic range of detection of at least 3 decades and coefficients of precision better than 20%. Finally, the spectral library was used to search for endogenous kinases in human cellular extract. [ABSTRACT FROM AUTHOR]

Two-dimensional mass spectrometry (2D MS) correlates precursor and fragment ions without ion isolation in a Fourier transform ion cyclotron resonance mass spectrometer (FTICR MS) for tandem mass spectrometry. Infrared activated electron capture dissociation (IR-ECD), using a hollow cathode configuration, generally yields more information for peptide sequencing in tandem mass spectrometry than ECD (electron capture dissociation) alone. The effects of the fragmentation zone on the 2D mass spectrum are investigated as well as the structural information that can be derived from it. The enhanced structural information gathered from the 2D mass spectrum is discussed in terms of how de novo peptide sequencing can be performed with increased confidence. 2D IR-ECD MS is shown to sequence peptides, to distinguish between leucine and isoleucine residues through the production of w ions as well as between C-terminal (b/c) and N-terminal (y/z) fragments through the use of higher harmonics, and to assign and locate peptide modifications. [ABSTRACT FROM AUTHOR]

Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved. Here, we use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the etiology of Parkinson's disease, with negatively charged lipid vesicles. We further demonstrate that this microfluidic approach enables the characterization of size distributions of different binary mixtures of vesicles, which are not readily accessible using conventional light scattering techniques. Finally, the size distributions of the two α-synuclein conformations, free α-synuclein and membrane-bound α-synuclein, were resolved under varying lipid:protein ratios, thus, allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system. The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus, establishing the basis for an approach for the study of molecular interactions of heterogeneous systems under native conditions. [ABSTRACT FROM AUTHOR]

Understanding nanoconfinement phenomena is necessary to develop nanofluidic technology platforms. One example of nanoconfinement phenomena is shifts in reaction equilibria toward reaction products in nanoconfined systems, which have been predicted theoretically and observed experimentally in DNA hybridization. Here we demonstrate a convection-limited nanofluidic immunoassay that achieves total capture of a target analyte and an apparent shift in the antibody-antigen reaction equilibrium due to nanoconfinement. The system exhibits wavefronts of the target analyte that propagate along the length of the nanochannel at a velocity much slower than that of the carrier fluid. We apply an analytical model describing the propagation of these wavefronts to determine the density of capture antibody binding sites in the enclosed nanochannel for a known concentration of the target analyte. We then use this binding site density to estimate the concentration of solutions with 5× and 10× less analyte. Our analysis suggests that nanoconfinement results in a preference toward binding of the target analyte with the surface-grafted capture antibody, as evidenced by an apparent reduction in the equilibrium dissociation constant. Our findings motivate the advancement of new biomedical and chemical synthesis technologies by leveraging nanoconfinement effects, and demonstrate a useful platform for studying the effect of nanoconfinement on chemical systems. [ABSTRACT FROM AUTHOR]

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS/MS) with radical-based fragmentation techniques has proven to be a promising method for phosphoproteomic applications, owing to its ability to determine phosphorylation sites on proteins. The radical-induced fragmentation technique involves the attachment or abstraction of hydrogen to peptides in an ion trap mass spectrometer, in a process called hydrogen attachment/abstraction dissociation (HAD), which has only been recently developed. In the present investigation, we have analyzed model phosphopeptides and phosphoprotein digests using HAD-MS/MS, combined with matrix-assisted laser desorption/ionization (MALDI), in order to demonstrate the usefulness of the HAD-MS/MS-based analytical method. The tryptic peptides were categorized as arginine- and lysine-terminated peptides, and MALDI HAD-MS/MS is found to facilitate the sequencing of arginine-terminated tryptic peptides, because of the selective observation of C-terminal side fragment ions. In contrast, MALDI HAD-MS/MS of lysine-terminated tryptic peptides produced both N- and C-terminal side fragments, such that the mass spectra were complex. The guanidination of peptide converted lysine into homoarginine, which facilitated the interpretation of MALDI HAD-MS/MS mass spectra. The present method was useful for de novo sequencing of tryptic phosphopeptides. [ABSTRACT FROM AUTHOR]

One of the most significant challenges in contemporary lipidomics lies in the separation and identification of lipid isomers that differ only in site(s) of unsaturation or geometric configuration of the carbon-carbon double bonds. While analytical separation techniques including ion mobility spectrometry (IMS) and liquid chromatography (LC) can separate isomeric lipids under appropriate conditions, conventional tandem mass spectrometry cannot provide unequivocal identification. To address this challenge, we have implemented ozone-induced dissociation (OzID) in-line with LC, IMS, and high resolution mass spectrometry. Modification of an IMS-capable quadrupole time-of-flight mass spectrometer was undertaken to allow the introduction of ozone into the high-pressure trapping ion funnel region preceding the IMS cell. This enabled the novel LC-OzID-IMS-MS configuration where ozonolysis of ionized lipids occurred rapidly (10 ms) without prior mass-selection. LC-elution time alignment combined with accurate mass and arrival time extraction of ozonolysis products facilitated correlation of precursor and product ions without mass-selection (and associated reductions in duty cycle). Unsaturated lipids across 11 classes were examined using this workflow in both positive and negative ion modalities, and in all cases, the positions of carbon-carbon double bonds were unequivocally assigned based on predictable OzID transitions. Under these conditions, geometric isomers exhibited different IMS arrival time distributions and distinct OzID product ion ratios providing a means for discrimination of cis/trans double bonds in complex lipids. The combination of OzID with multidimensional separations shows significant promise for facile profiling of unsaturation patterns within complex lipidomes including human plasma. [ABSTRACT FROM AUTHOR]

The recently developed and commercially available carbonyl-reactive tandem mass tags (aminoxyTMT) enable multiplexed quantification of glycans through comparison of reporter ion intensities. However, challenges still exist for collision activated dissociation (CAD) MS/MS based quantification of aminoxyTMT due to the relatively low reporter ion yield especially for glycans with labile structures. To circumvent this limitation, we utilized the unique structural features of N-glycan molecules, the common core sugar sequence (HexNAc)2(Man)3, and common m/z of Yn ions generated from different types of precursors by MS/MS and designed a Y1 ion triggered, targeted MultiNotch MS[sup 3] relative quantification approach based on aminoxyTMT labeling. This approach was implemented on a nanoHILIC-Tribrid quadrupole-ion trap-Orbitrap platform, which enables prescreening of aminoxyTMT labeled N-glycan precursor ions by Y1ion fragment ion mass in a higher-energy collisional dissociation (HCD) MS/MS scan and coisolation and cofragmentation of multiple Yn fragment ions that carry the isobaric tags from the inclusion list in the MS/MS/ MS scan. Through systematical optimization and evaluation using N-glycans released from several glycoprotein standards and human serum proteins, we demonstrated that the Y1 ion triggered, targeted MultiNotch MS3 approach offers improved accuracy, precision, and sensitivity for relative quantification compared to traditional data-dependent MS2and Y1 ion MS3 quantification methods. [ABSTRACT FROM AUTHOR]

Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ~490 kDa protein complex ~20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin. [ABSTRACT FROM AUTHOR]

Characterizing the structures of glycoconjungates is important because of glycan heterogeneity and structural complexity of aglycon. The presence of relatively weak glycosidic linkages leads to preferential cleavages that limit the acquisition of structural information under typical mass spectrometry dissociation conditions, such as collision-induced dissociation (CID) and infrared multiphoton dissociation. In this paper, we explored the dissociation behaviors of different members of glycoconjugates, including glycopeptides, glycoalkaloids, and glycolipids, under electron-induced dissociation (EID) conditions. Using CID spectra as references, we found that EID is not only a complementary method to CID, but also a method that can generate extensive fragment ions for the structural characterization of all intact glycoconjugates studied. Furthermore, isomeric ganglioside species can be differentiated, and the double bond location in the ceramide moiety of the gangliosides can be identified through the MS3 approach involving sequential CID and EID processes. [ABSTRACT FROM AUTHOR]

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization-ion mobility-mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1. [ABSTRACT FROM AUTHOR]

Two-dimensional Fourier transform ion cyclotron resonance mass spectrometry (2D FTICR MS or 2D MS) allows direct correlation between precursor and fragment ions without isolation prior to fragmentation. The method has been optimized for the analysis of complex mixtures and used so far for the analysis of small molecules and peptides obtained by tryptic digestion of proteins and entire proteins. In this work, a 2D MS method is developed to characterize complex mixtures of polymers using infrared multiphoton decay (IRMPD) and electron capture dissociation (ECD) as fragmentation techniques, and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Polysorbate 80, and poly(methyl methacrylate) (PMMA) as analytes. The use of 2D MS allowed generation of fragment m/z values for all the compounds in the mixture at once and allowed tandem mass spectrometry of species very close in m/z that would have been difficult to isolate with a quadrupole for standard MS/MS. Furthermore, the use of unique features of 2D MS such as the extraction of neutral-loss lines allowed the successful assignment of peaks from low abundant species that would have been more difficult with standard MS/MS. For all the samples, the amount of information obtained with 2D MS was comparable with what obtained with multiple 1D MS/MS experiments targeted on each individual component within each mixture but required a single experiment of about 20-40 min. [ABSTRACT FROM AUTHOR]

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 μm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site. [ABSTRACT FROM AUTHOR]

The ability to localize phosphosites to specific amino acid residues is crucial to translating phosphoproteomic data into biological meaningful contexts. In a companion manuscript (Anal. Chem. 2017, DOI: 10.1021/acs.analchem.7b00213), we described a new implementation of activated ion electron transfer dissociation (AI-ETD) on a quadrupole-Orbitrap-linear ion trap hybrid MS system (Orbitrap Fusion Lumos), which greatly improved peptide fragmentation and identification over ETD and other supplemental activation methods. Here we present the performance of AI-ETD for identifying and localizing sites of phosphorylation in both phosphopeptides and intact phosphoproteins. Using 90 min analyses we show that AI-ETD can identify 24,503 localized phosphopeptide spectral matches enriched from mouse brain lysates, which more than triples identifications from standard ETD experiments and outperforms ETcaD and EThcD as well. AI-ETD achieves these gains through improved quality of fragmentation and MS/MS success rates for all precursor charge states, especially for doubly protonated species. We also evaluate the degree to which phosphate neutral loss occurs from phosphopeptide product ions due to the infrared photoactivation of AI-ETD and show that modifying phosphoRS (a phosphosite localization algorithm) to include phosphate neutral losses can significantly improve localization in AI-ETD spectra. Finally, we demonstrate the utility of AI-ETD in localizing phosphosites in a-casein, an ~23.5 kDa phosphoprotein that showed eight of nine known phosphorylation sites occupied upon intact mass analysis. AI-ETD provided the greatest sequence coverage for all five charge states investigated and was the only fragmentation method to localize all eight phosphosites for each precursor. Overall, this work highlights the analytical value AI-ETD can bring to both bottom-up and top-down phosphoproteomics. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS) has emerged as a powerful tool within the growing field of immunoproteomics, which aims to understand antibody-mediated immunity at the molecular-level based on the direct determination of serological antibody repertoire. To date, these methods have relied on the use of high-resolution bottom-up proteomic strategies that require effective sampling and characterization of low abundance peptides derived from the antigen-binding domains of polyclonal antibody mixtures. Herein, we describe a method that uses restricted Lys-C enzymatic digestion to increase the average mass of proteolytic IgG peptides (=4.5 kDa) and produce peptides which uniquely derive from single antibody species. This enhances the capacity to discriminate between very similar antibodies present within polyclonal mixtures. Furthermore, our use of 193-nm ultraviolet photodissociation (UVPD) improves spectral coverage of the antibody sequence relative to conventional collision- and electron-based fragmentation methods. We apply these methods to both a monoclonal and an antibody mixture. By identifying from a database search of approximately 15?000 antibody sequences those which compose the mixture, we demonstrate the analytical potential of middle-down UVPD for MS-based serological repertoire analysis. [ABSTRACT FROM AUTHOR]

Mass resolution (M/ΔM fwhm) is observed to linearly increase with harmonic order in a Fourier transform electrostatic linear ion trap (ELIT) mass spectrometer. This behavior was predicted by Grosshans and Marshall for frequency-multiple detection in a Fourier transform ion cyclotron resonance mass spectrometer only for situations when the prominent mechanism for signal decay is ion ejection from the trap. As the analyzer pressure in our ELIT chamber is relatively high, such that collisional scattering and collision-induced dissociation are expected to underlie much of the ion loss, we sought to explore the relationship between harmonic order and mass resolution. Mass resolutions of 36 900 (fundamental), 75 850 (2nd harmonic), and 108 200 (3rd harmonic) were obtained for GdO+ (avg. m/z 173.919) with a transient length of 300 ms. To demonstrate that the mass resolution was truly increasing with harmonic order, the unresolved isotopes at the fundamental distribution of cytochrome c +8 (m/z ~ 1549) were nearly baseline, resolved at the third harmonic (mass resolution ≈ 23 000) with a transient length of only 200 ms. This experiment demonstrates that, when the ion density is sufficiently low, ions with frequency differences of less than 4 Hz remain uncoalesced. Higher harmonics can be used to increase the effective mass resolution for a fixed transient length and thereby may enable the resolution of closely spaced masses, determination of a protein ion's charge state, and study of the onset of peak coalescence when the resolution at the fundamental frequency is insufficient. [ABSTRACT FROM AUTHOR]

Ozone-induced dissociation (OzID) is a novel ion activation technology that exploits the gas-phase reaction between mass-selected ions and ozone inside a mass spectrometer to assign sites of unsaturation in complex lipids. Since it was first demonstrated [Thomas et al. Anal. Chem. 2008 80 303 ], the method has been widely deployed for targeted lipid structure elucidation but its application to high throughput and liquid chromatography-based workflows has been limited due to the relatively slow nature of the requisite ion-molecule reactions that result in long ion-trapping times and consequently low instrument duty cycle. Here, the implementation of OzID in a high-pressure region, the ion-mobility spectrometry cell, of a contemporary quadrupole time-of-flight mass spectrometer is described. In this configuration, a high number density of ozone was achieved and thus abundant and diagnostic OzID product ions could be observed even on the timescale of transmission through the reaction region (ca. 20-200 ms), representing a 50-1000-fold improvement in performance over prior OzID implementations. Collisional activation applied prereaction was found to yield complementary and structurally informative product ions arising from ozone- and collision-induced dissociation. Ultimately, the compatibility of this implementation with contemporary ultrahigh performance liquid chromatography is demonstrated with the resulting hyphenated approach showing the ability to separate and uniquely identify isomeric phosphatidylcholines that differ only in their position(s) of unsaturation. [ABSTRACT FROM AUTHOR]

Meteorin and Cometin (Meteorin-like) are secreted proteins belonging to a newly discovered growth factor family. Both proteins play important roles in neural development and may have potential as therapeutic targets or agents. Meteorin and Cometin are homologues and contain ten evolutionarily conserved Cys residues across a wide variety of species. However, the status of the Cys residues has remained unknown. Here, we have successfully determined the disulfide structure for murine Meteorin by LC-MS analysis of fragments generated by trypsin plus endoprotease-Asp-N. For proteolytic fragments linked by more than one disulfide bond, we used electron transfer dissociation (ETD) to partially dissociate disulfide bonds followed by high-energy collisional dissociation (HCD) to determine disulfide linkages. Our analysis revealed that the ten Cys residues in murine Meteorin form five disulfide bonds with Cys7 (C1) linked to Cys28 (C2), Cys59 (C3) to Cys95 (C4), Cys148 (C5) to Cys219 (C8), Cys151 (C6) to Cys243 (C9), and Cys161 (C7) to Cys266 (C10). Since the ten Cys residues are highly conserved in Meteorin and Cometin, it is likely that the disulfide linkages are also conserved. This disulfide structure information should facilitate structure-function relationship studies on this new class of neurotrophic factors and also assist in evaluation of their therapeutic potentials. [ABSTRACT FROM AUTHOR]

This work provides the first use of helium charge transfer dissociation (He-CTD) tandem mass spectrometry (MS/MS) in negative polarity mode. Three sulfated oligosaccharides of natural origin were chosen as representative structures that are difficult to solve by conventional MS/MS approaches. Negative polarity He-CTD provided a full set of structurally informative fragments, which permitted the unambiguous determination of the complete structures of these molecules, including the characterization of labile sulfated functional groups. Despite close structural features, the three molecules led to distinct fragmentation patterns depending on the position of the sulfate group in the heterocycle. The observed fragments showed a consistent radical-initiated mechanism of dissociation, which shares similarities with fragment types produced in electron detachment dissociation (EDD), negative electron transfer dissociation (NETD), or extreme UV photodissociation (XUV-PD). Short times of data collection and the fact that the technique can be affordably implementable in any standard laboratory and with a classical ion trap mass spectrometer were other remarkable characteristics of negative polarity He-CTD. [ABSTRACT FROM AUTHOR]

Surface plasmon resonance (SPR) has been well established as a method of choice for label-free kinetic measurements of biomolecular interactions. The conventional approach involves multiple injections of an analyte of different concentrations into a fluidic channel covered with a fixed ligand density. Optimization of the experimental conditions and assessment of the data quality can be complicated by issues such as disruption of the ligand structure by the regeneration step and the limited availability of the sample solution. By sequentially closing fluidic channels on a five-channel SPR instrument, different densities of a ligand can be immobilized and determined in one step. With a subsequent injection of a single sample solution, SPR sensorgrams can be simultaneously collected to yield binding and dissociation rate constants (ka and kd) and dissociation constant (KD) between the ligand and analyte. For biomolecular interactions that obey the Langmuir isotherm, we show that the fidelity of the kinetic data can only be reliably confirmed when there exists a strong linear correlation between the SPR signals and the ligand densities. The use of a multichannel SPR instrument also obviates the regeneration step, allowing the binding kinetics between the green fluorescent protein and its antibody to be measured. In comparison to the conventional approach, the method simplifies the experimental procedure, reduces costs associated with sensor chips and biological samples, expedites kinetic measurements, and allows affinity constants to be determined more straightforwardly. [ABSTRACT FROM AUTHOR]

Tandem mass spectrometry (MS/MS) has emerged as the core technology for identification of post-translational modifications (PTMs). Here, we report the mass spectrometry analysis of serine and threonine pyrophosphorylation, a protein modification that has eluded detection by conventional MS/MS methods. Analysis of a set of synthesized, site-specifically modified peptides by different fragmentation techniques shows that pyrophosphorylated peptides exhibit a characteristic neutral loss pattern of 98, 178, and 196 Da, which enables the distinction between isobaric pyro- and diphosphorylated peptides. In addition, electron-transfer dissociation combined with higher energy collision dissociation (EThcD) provides exceptional data-rich MS/MS spectra for direct and unambiguous pyrophosphosite assignment. Remarkably, sufficient fragmentation of doubly charged precursors could be achieved by electron-transfer dissociation (ETD) with increased supplemental activation, without losing the labile modification. By exploiting the specific fragmentation behavior of pyrophosphorylated peptides during collision-induced dissociation (CID), a data dependent neutral-loss-triggered EThcD acquisition method was developed. This strategy enables reliable pyrophosphopeptide identification in complex samples, without compromising speed and sensitivity. [ABSTRACT FROM AUTHOR]

Full scan field asymmetric waveform ion mobility spectrometry (FAIMS) combined with liquid chromatography and mass spectrometry (LC-FAIMS-MS) is shown to enhance peak capacity for omics applications. A miniaturized FAIMS device capable of rapid compensation field scanning has been incorporated into an ultrahigh performance liquid chromatography (UHPLC) and time-of-flight mass spectrometry analysis, allowing the acquisition of full scan FAIMS and MS nested data sets within the time scale of a UHPLC peak. Proof of principle for the potential of scanning LC-FAIMS-MS in omics applications is demonstrated for the nontargeted profiling of human urine using a HILIC column. The high level of orthogonality between FAIMS and MS provides additional unique compound identifiers with detection of features based on retention time, FAIMS dispersion field and compensation field (DF and CF), and mass-to-charge (m/z). Extracted FAIMS full scan data can be matched to standards to aid the identification of unknown analytes. The peak capacity for features detected in human urine using LC-FAIMS-MS was increased approximately threefold compared to LC-MS alone due to a combination of the reduction of chemical noise and separation of coeluting isobaric species across the entire analytical space. The use of FAIMS-selected in source collision induced dissociation (FISCID) yields fragmentation of ions, which reduces sample complexity associated with overlapping fragmentation patterns and provides structural information on the selected precursor ions. [ABSTRACT FROM AUTHOR]

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications. [ABSTRACT FROM AUTHOR]

Protein adenosine diphosphate (ADP)-ribosylation is a physiologically and pathologically important post-translational modification. Recent technological advances have improved analysis of this complex modification and have led to the discovery of hundreds of ADP-ribosylated proteins in both cultured cells and mouse tissues. Nevertheless, accurate assignment of the ADP-ribose acceptor site(s) within the modified proteins identified has remained a challenging task. This is mainly due to poor fragmentation of modified peptides. Here, using an Orbitrap Fusion Tribrid mass spectrometer, we present an optimized methodology that not only drastically improves the overall localization scores for ADP-ribosylation acceptor sites but also boosts ADP-ribosylated peptide identifications. First, we systematically compared the efficacy of higher-energy collision dissociation (HCD), electron-transfer dissociation with supplemental collisional activation (ETcaD), and electron-transfer/higher-energy collision dissociation (EThcD) fragmentation methods when determining ADP-ribose acceptor sites within complex cellular samples. We then tested the combination of HCD and EThcD fragmentation, which were employed in a product-dependent manner, and the unique fragmentation properties of the ADP-ribose moiety were used to trigger targeted fragmentation of only the modified peptides. The best results were obtained with a workflow that included initial fast, high-energy HCD (Orbitrap, FT) scans, which produced intense ADP-ribose fragmentation ions. These potentially ADP-ribosylated precursors were then selected and analyzed via subsequent high-resolution HCD and EThcD fragmentation. Using these resulting high-quality spectra, we identified a xxxxxxKSxxxxx modification motif where lysine can serve as an ADP-ribose acceptor site. Due to the appearance of serine within this motif and its close presence to the lysine, further analysis revealed that serine serves as a new ADP-ribose acceptor site across the proteome. [ABSTRACT FROM AUTHOR]

Fluorescence anisotropy measurements of reagents compartmentalized into individual nanoliter droplets are shown to yield high-resolution binding curves from which precise dissociation constants (Kd) for protein-peptide interactions can be inferred. With the current platform, four titrations can be obtained per minute (based on ∼100 data points each), with stoichiometries spanning more than 2 orders of magnitude and requiring only tens of microliters of reagents. In addition to affinity measurements with purified components, Kd values for unpurified proteins in crude cell lysates can be obtained without prior knowledge of the concentration of the expressed protein, so that protein purification can be avoided. Finally, we show how a competition assay can be set up to perform focused library screens, so that compound labeling is not required anymore. These data demonstrate the utility of droplet compartments for the quantitative characterization of biomolecular interactions and establish fluorescence anisotropy imaging as a quantitative technique in a miniaturized droplet format, which is shown to be as reliable as its macroscopic test tube equivalent. [ABSTRACT FROM AUTHOR]

Mass spectrometry continues to develop as a valuable tool in the analysis of proteins and protein complexes. In protein complex mass spectrometry studies, surface-induced dissociation (SID) has been successfully applied in quadrupole time-of-flight (Q-TOF) instruments. SID provides structural information on noncovalent protein complexes that is complementary to other techniques. However, the mass resolution of Q-TOF instruments can limit the information that can be obtained for protein complexes by SID. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides ultrahigh resolution and ultrahigh mass accuracy measurements. In this study, an SID device was designed and successfully installed in a hybrid FT-ICR instrument in place of the standard gas collision cell. The SID-FT-ICR platform has been tested with several protein complex systems (homooligomers, a heterooligomer, and a protein-ligand complex, ranging from 53 to 85 kDa), and the results are consistent with data previously acquired on Q-TOF platforms, matching predictions from known protein interface information. SID fragments with the same m/z but different charge states are well-resolved based on distinct spacing between adjacent isotope peaks, and the addition of metal cations and ligands can also be isotopically resolved with the ultrahigh mass resolution available in FT-ICR. [ABSTRACT FROM AUTHOR]

Phosphorylation is the most abundant protein modification, and tandem mass spectrometry (MS²) with electron transfer dissociation (ETD) has proven to be a promising method for phosphoproteomic applications owing to its ability to determine phosphorylation sites on proteins. However, low precursor charge states hinder the ability to obtain useful information through peptide sequencing by ETD, and the presence of acidic phosphate groups contributes to a low charge state of peptide ions. In the present report, we used a dinuclear zinc complex, (Zn2L)3+ (L = alkoxide form of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol) for electrospray ionization (ESI), followed by ETD-MS² analysis. Since (Zn2L)3+ selectively bound to phosphopeptide with addition of a positive charge per phosphate group, the use of (Zn2L)3+ for ESI improved the ionization yield of phosphopeptides in phosphoprotein digest. Additionally, an increase in the charge state of phosphopeptides were observed by addition of (Zn2L)3+, facilitating phosphopeptide sequencing by ETD-MS². Since the binding between (Zn2L)3+ and the phosphate group was retained during the ETD process, a comparison between the ETD mass spectra obtained using two dinuclear zinc complex derivatives containing different zinc isotopes, namely (64Zn2L)3+ and (68Zn2L)3+, provided information about the number of phosphate groups in each fragment ion, allowing the phosphorylation site to be unambiguously determined. The details of the fragmentation processes of the (Zn2L)3+-phosphopeptide complex were investigated using a density functional theory calculation. As in the case of protonated peptides, ETD induced peptide backbone dissociation in the (Zn2L)3+-phosphopeptide complex proceeded through an aminoketyl radical intermediate. [ABSTRACT FROM AUTHOR]

The growing use of mass spectrometry in the field of structural biology has catalyzed the development of many new strategies to examine intact proteins in the gas phase. Native mass spectrometry methods have further accelerated the need for methods that can manipulate proteins and protein complexes while minimizing disruption of noncovalent interactions critical for stabilizing conformations. Proton-transfer reactions (PTR) in the gas phase offer the ability to effectively modulate the charge states of proteins, allowing decongestion of mass spectra through separation of overlapping species. PTR was combined with ultraviolet photodissociation (UVPD) to probe the degree of structural changes that occur upon charge reduction reactions in the gas phase. For protein complexes myoglobin⋅heme (17.6 kDa) and dihydrofolate reductase⋅methotrexate (19.4 kDa), minor changes were found in the fragmentation patterns aside from some enhancement of fragmentation near the N- and C-terminal regions consistent with slight fraying. After finding little perturbation was caused by charge reduction using PTR, homodimeric superoxide dismutase/CuZn (31.4 kDa) was subjected to PTR in order to separate overlapping monomer and dimer species of the protein that were observed at identical m/z values. [ABSTRACT FROM AUTHOR]

The recent approval of covalent inhibitors for multiple clinical indications has reignited enthusiasm for this class of drugs. As interest in covalent drugs has increased, so too has the need for analytical platforms that can leverage their mechanism-of-action to characterize modified protein targets. Here we describe novel gas phase dissociation pathways which yield predictable fragment ions during MS/MS of inhibitor-modified peptides. We find that these dissociation pathways are common to numerous cysteine-directed probes as well as the covalent drugs, Ibrutinib and Neratinib. We leverage the predictable nature of these fragment ions to improve the confidence of peptide sequence assignment in proteomic analyses and explore their potential use in selective mass spectrometry-based assays. [ABSTRACT FROM AUTHOR]