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Cysteine is the most reactive naturally occurring amino acid due to the presence of a free thiol, presenting a tantalizing handle for covalent modification of peptides/proteins. Although many mass spectrometry experiments could benefit from site-specific modification of Cys, the utility of direct arylation has not been thoroughly explored. Recently, Spokoyny and co-workers reported a Au(III) organometallic reagent that robustly arylates Cys and tolerates a wide variety of solvents and conditions. Given the chromophoric nature of aryl groups and the known susceptibility of carbon-sulfur bonds to photodissociation, we set out to identify an aryl group that could efficiently cleave Cys carbon-sulfur bonds at 266 nm. A streamlined workflow was developed to facilitate rapid examination of a large number of aryls with minimal sample using a simple test peptide, RAAACGVLK. We were able to identify several aryl groups that yield abundant homolytic photodissociation of the adjacent Cys carbon-sulfur bonds with short activation times (

Sphingolipids are an essential subset of bioactive lipids found in most eukaryotic cells that contribute to membrane biophysical properties and are involved in cellular differentiation, recognition, and mediating interactions. The described nanoHPLC-ESI-Q/ToF methodology utilizes known biosynthetic pathways, accurate mass detection, optimized collision-induced disassociation, and a robust nanoflow chromatographic separation for the analysis of intact sphingolipids found in human tissue, cells, and serum. The methodology was developed and validated with an emphasis on addressing the common issues experienced in profiling these amphipathic lipids, which are part of the glycocalyx and lipidome. The high sensitivity obtained using nanorange flow rates with robust chromatographic reproducibility over a wide range of concentrations and injection volumes results in confident identifications for profiling these low-abundant biomolecules.

Isomeric molecules are important analytes in many biological and chemical arenas, yet their similarity poses challenges for many analytical methods, including mass spectrometry (MS). Tandem-MS provides significantly more information about isomers than intact mass analysis, but highly similar fragmentation patterns are common and include cases where no unique m/z peaks are generated between isomeric pairs. However, even in such situations, differences in peak intensity can exist and potentially contain additional information. Herein, we present a framework for comparing mass spectra that differ only in terms of peak intensity and include calculation of a statistical probability that the spectra derive from different analytes. This framework allows for confident identification of peptide isomers by collision-induced dissociation, higher-energy collisional dissociation, electron-transfer dissociation, and radical-directed dissociation. The method successfully identified many types of isomers including various d/l amino acid substitutions, Leu/Ile, and Asp/IsoAsp. The method can accommodate a wide range of changes in instrumental settings including source voltages, isolation widths, and resolution without influencing the analysis. It is shown that quantification of the composition of isomeric mixtures can be enabled with calibration curves, which were found to be highly linear and reproducible. The analysis can be implemented with data collected by either direct infusion or liquid-chromatography MS. Although this framework is presented in the context of isomer characterization, it should also prove useful in many other contexts where similar mass spectra are generated.

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimers. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Spontaneous chemical modifications play an important role in human disease and aging at the molecular level. Deamidation and isomerization are known to be among the most prevalent chemical modifications in long-lived human proteins and are implicated in a growing list of human pathologies, but the relatively minor chemical change associated with these processes has presented a long standing analytical challenge. Although the adoption of high-resolution mass spectrometry has greatly aided the identification of deamidation sites in proteomic studies, isomerization (and the isomeric products of deamidation) remain exceptionally challenging to characterize. Herein, we present a liquid chromatography/mass spectrometry-based approach for rapidly characterizing the isomeric products of Gln deamidation using diagnostic fragments that are abundantly produced and capable of unambiguously identifying both Glu and isoGlu. Importantly, the informative fragment ions are produced through orthogonal fragmentation pathways, thereby enabling the simultaneous detection of both isomeric forms while retaining compatibility with shotgun proteomics. Furthermore, the diagnostic fragments associated with isoGlu pinpoint the location of the modified residue. The utility of this technique is demonstrated by characterizing the isomeric products generated during in vitro aging of a series of glutamine-containing peptides. Sequence-dependent product profiles are obtained, and the abundance of deamidation-linked racemization is examined. Finally, comparisons are made between Gln deamidation, which is relatively poorly understood, and asparagine deamidation, which has been more thoroughly studied.

Glycans are fundamental biological macromolecules, yet despite their prevalence and recognized importance, a number of unique challenges hinder routine characterization. The multiplicity of OH groups in glycan monomers easily afford branched structures and alternate linkage sites, which can result in isomeric structures that differ by minute details. Herein, radical chemistry is employed in conjunction with mass spectrometry to enable rapid, accurate, and high throughput identification of a challenging series of closely related glycan isomers. The results are compared with analysis by collision-induced dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation (UVPD) at 213 nm. In general, collision-based activation struggles to produce characteristic fragmentation patterns, while UVPD and radical-directed dissociation (RDD) can distinguish all isomers. In the case of RDD, structural differentiation derives from radical mobility and subsequent fragmentation. For glycans, the energetic landscape for radical migration is flat, increasing the importance of the three-dimensional structure. RDD is therefore a powerful and straightforward method for characterizing glycan isomers.

Recent studies suggest that reactions in aqueous microcompartments can occur at significantly different rates than those in the bulk. Most studies have used electrospray to generate a polydisperse source of highly charged microdroplets, leading to multiple confounding factors potentially influencing reaction rates (e.g., evaporation, charge, and size). Thus, the underlying mechanism for the observed enhancement remains unclear. We present a new type of electrodynamic balance-the branched quadrupole trap (BQT)-which can be used to study reactions in microdroplets in a controlled environment. The BQT allows for condensed phase chemical reactions to be initiated by colliding droplets with different reactants and levitating the merged droplet indefinitely. The performance of the BQT is characterized in several ways. Sub-millisecond mixing times as fast as ∼400 μs are measured for low velocity (∼0.1 m/s) collisions of droplets with

In-depth investigations of the kinetics of aqueous chemistry occurring in microdroplet environments require experimental techniques that allow a reaction to be initiated at a well-defined point in time and space. Merging microdroplets of different reactants is one such approach. The mixing dynamics of unconfined (airborne) microdroplets have yet to be studied in detail, which is an essential step toward widespread use and application of merged droplet microreactors for monitoring chemical reactions. Here, we present an on-demand experimental approach for initiating chemical reactions in and characterizing the mixing dynamics of colliding airborne microdroplets (40 ± 5 μm diameter) using a streak-based fluorescence microscopy technique. The advantages of this approach include the ability to generate two well-controlled monodisperse microdroplet streams and collide (and thus mix) the microdroplets with high spatial and temporal control while consuming small amounts of sample ( 1 m/s) off-center collisions, mixing times increased by as much as a factor of 6 (e.g., at ∼6 m/s, mixing times increased from 7 m/s, droplet separation and fragmentation occurred, resulting in incomplete mixing. These results suggest a limited range of collision velocities over which complete and rapid mixing can be achieved when using airborne merged microdroplets to, e.g., study reaction kinetics when reaction times are short relative to typical bulk reactor mixing times. We benchmark our reactor using an aqueous-phase oxidation reaction: iron-catalyzed hydroxyl radical production from hydrogen peroxide (Fenton's reaction) and subsequent aqueous-phase oxidation of organic species in solution. Kinetic simulations of our measurements show that quantitative agreement can be obtained using known bulk-phase kinetics for bimolecular reactions in our colliding-droplet microreactor.

Recent years have seen the development of a large number of electrochemical sandwich assays and reagentless biosensor architectures employing biomolecules modified via the attachment of a redox-active "reporter." Here we survey a large set of potential redox reporters in order to determine which exhibits the best long-duration stability in thiol-on-gold monolayer-based sensors and to identify reporter "sets" signaling at distinct, nonoverlapping redox potentials in support of multiplexing and error correcting ratiometric or differential measurement approaches. Specifically, we have characterized the performance of more than a dozen potential reporters that are, first, redox active within the potential window over which thiol-on-gold monolayers are reasonably stable and, second, are available commercially in forms that are readily conjugated to biomolecules or can be converted into such forms in one or two simple synthetic steps. To test each of these reporters we conjugated it to one terminus of a single-stranded DNA "probe" that was attached by its other terminus via a six-carbon thiol to a gold electrode to form an "E-DNA" sensor responsive to its complementary DNA target. We then measured the signaling properties of each sensor as well as its stability against repeated voltammetric scans and against deployment in and reuse from blood serum. Doing so we find that the performance of methylene blue-based, thiol-on-gold sensors is unmatched; the near-quantitative stability of such sensors against repeated scanning in even very complex sample matrices is unparalleled. While more modest, the stability of sensors employing a handful of other reporters, including anthraquinone, Nile blue, and ferrrocene, is reasonable. Our work thus serves as both to highlight the exceptional properties of methylene blue as a redox reporter in such applications and as a cautionary tale-we wish to help other researchers avoid fruitless efforts to employ the many, seemingly promising and yet ultimately inadequate reporters we have investigated. Finally, we hope that our work also serves as an illustration of the pressing need for the further development of useful redox reporters.

Post-translational modifications that do not result in a change in mass are particularly difficult to detect by mass spectrometry. For example, isomerization of aspartic acid or epimerization of any chiral residue within a peptide do not lead to mass shifts but can be identified by examination of independently acquired tandem mass spectra or by combination with another technique. For analysis of a biological sample, this means that liquid chromatography or some other type of separation must be used to first separate the isomers from one another. Furthermore, each specific m/z of interest must be sampled repeatedly to allow for comparison of the tandem mass spectra from each separated isomer, which contrasts with the traditional approach in proteomics where the goal is typically to avoid resampling the same m/z. We illustrate that isomerization and epimerization of peptides can be identified in this fashion by examination of long-lived crystallin proteins extracted from a sheep eye lens. Tandem mass spectrometry relying on a combination of radical directed dissociation (RDD) and collision induced dissociation (CID) following separation by liquid chromatography was used to identify modified peptides. Numerous sites of isomerization and epimerization, including several that have not been previously identified, were determined with peptide specificity. It is demonstrated that the specific sites of amino acid isomerization within each peptide can be identified by comparison with synthetic peptides. For α-crystallin proteins, the sites that undergo the greatest degree of isomerization correspond to disordered regions, which may have important implications on chaperone functionality within the context of aging.

An ambient ionization/desorption technique, namely, easy ambient sonic-spray ionization mass spectrometry (EASI), has been applied to crude oil samples. From a single droplet of the sample placed on an inert surface, EASI([+ or -]) is shown to promote efficient desorption and ionization of a myriad of polar components via the action of its cloud of very minute supersonic bipolar charged droplets. The gaseous [[M + H].sup.+] and [[M - H].sup.-] ions concurrently formed by EASI([+ or -]) were analyzed by Fourier transform mass spectrometry (FT-ICR MS), and a total of ~6000 acidic and basic components have been attributed. EASI(+ or -]) FT-ICR MS of crude oils is show to be almost as fast as ESI(+)/ESI(-) FT-ICR MS, providing similar compositional information of polar components and spectral quality comparable to that of a commercial nonochip-based robotic ESI device. EASI([+ or -]) requires no sample workup thus eliminating risks of contamination during sample manipulation and memory effects because of carry over in pumping ESI lines. More importantly, EASI([+ or -]) is a voltoge-free ionization technique therefore eliminating risks of redox processes or duality of ionization mechanisms that can be observed in voltage-assisted processes. Data visualization via typical petroleomic plots confirms the similarity of the compositional information provided by EASI(+ or -]) compared to ESI(+)/ESI(-). The ambient EASI([+ or -]) FTICR MS method requires no voltage switching in changing the ion polarity mode, offering a workup, heating and voltage-free protocol for petroleomic studies performed at open atmosphere directly on the undisturbed crude oil sample. 10.1021/ac100673v

Here, complementary ion mobility/mass spectrometry (IM/MS) and ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS analyses of light, medium, and heavy petroleum crude oils yielded distributions of the heteroatom-containing hydrocarbons, as well as multiple conformational classes. The IM/MS technique provides unique fingerprints for fast identification of signature conformational/compositional patterns, whereas FTICR MS analysis provides comprehensive heteroatom class distributions. IM/MS and FTICR MS results reveal an increase in compositional complexity in proceeding from light to medium to heavy crude oils. Inspection of the mobility results shows a high structural diversity for the [C.sub.n][H.sub.h]XY (XY = [N.sub.1], [S.sub.1], [N.sub.1], [O.sub.1], NS, S[O.sub.1-2], N[O.sub.1-2], etc.) series, as well as a shift from planar to more compact three-dimensional structures with increasing mass. 10.1021/ac901594f

Atmospheric pressure photoionization (APPI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) has significantly contributed to the molecular speciation of petroleum. However, a typical APPI source operates at 50 [micro]L/min flow rate and thus causes a considerable mass load to the mass spectrometer. The recently introduced microchip APPI ([micro]APPI) operates at much lower flow rates (0.05-10 [micro]L/min) providing decreased mass load and therefore decreased contamination in analysis of petroleum by FT-ICR MS. In spite of the 25 times lower flow rate, the signal response with [micro]APPI was only 40% lower than with a conventional APPI source. It was also shown that [micro]APPI provides very efficient vaporization of higher molecular weight components in petroleum analysis.

We describe automation of liquid injection field desorption/ionization (LIFDI) for reproducible sample application, improved spectral quality, and high-throughput analyses. A commercial autosampler provides reproducible and unattended sample application. A custom-built field desorption (FD) controller allows data station or front panel control of source parameters including high-voltage limit/ramp rate, emitter heating current limit/ramp rate, and feedback control of emitter heating current based on ion current measurement. Automated LIFDI facilitates ensemble averaging of hundreds of Fourier transform ion cyclotron resonance mass spectra for increased dynamic range, mass accuracy, and S/N ratio relative to single-application FD experiments, as shown here for a South American crude oil. This configuration can be adapted to any mass spectrometer with an LIFDI probe.

We have coupled atmospheric pressure photoionization (APPI) to a home-built 9.4-T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Analysis of naphtho[2,3-a]pyrene and crude oil mass spectra reveals that protonated molecules, deprotonated molecules, and radical molecular ions are formed simultaneously in the ion source, thereby complicating the spectra (>12 000 peaks per mass spectrum and up to 63 peaks of the same nominal mass), and eliminating the 'nitrogen rule' for nominal mass determination of number of nitrogens. Nevertheless, the ultrahigh mass resolving power and mass accuracy of FT-ICR MS enable definitive elemental composition assignments, even for doublets as closely spaced as 1.1 mDa (S[H.sub.3][sup.13]C vs [sup.12][C.sub.4]). APPI efficiently ionizes nonpolar compounds that are unobservable by electrospray and allows nonpolar sulfur speciation of petrochemical mixtures.

We describe the construction and application of a 9.4-T FT-ICR mass spectrometer interfaced to a commercial field desorption ion source for high-resolution, high-mass accuracy measurements of nonpolar species. The FT-ICR MS instrument includes a liquid injection field desorption ionization source, octopole ion guides, external octopole ion trap capable of an axial potential gradient for ion ejection, capacitively coupled open cylindrical ion trap, and pulsed gas valve for ion cooling. Model compound responses with regard to various source and instrument conditions provide a basis for interpretation of broadband mass spectra of complex mixtures. As an example, we demonstrate broadband speciation of a Gulf Coast crude oil, with respect to numerous heteroatomic classes, compound types (rings plus double bonds), and carbon number distributions.

Ultrahigh-resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry has resolved and identified the elemental compositions of over 10 000 organic constituents of coal and petroleum crude oil. A plot of Kendrick mass defect versus Kendrick nominal mass sorts compounds into homologous series according to compound class (i.e., numbers of N, O, and S heteroatoms), type (number of rings plus double bonds), and degree of alkylation (number of C[H.sub.2] groups), to yield unique elemental assignments from ultrahigh-resolution mass measurements in the 200-900 Da range. Interpretation of such a vast compilation requires a simple (preferably graphical) means to differentiate between complex organic mixtures of different origin or processing. In an extension of the recently revived van Krevelen plot, each elemental composition is projected onto two or three axes according to its H/C, O/C, and/or N/C atomic ratios. The H/C ratio separates compounds according to degree of saturation, whereas O/C or N/C ratios separate according to O and N classes. We show that the three-dimensional van Krevelen diagram can completely separate different classes in pyridine-extracted coal or petroleum samples and can also graphically distinguish fossil fuels according to their nature (coal vs petroleum), maturation (coals of different rank), and processing (the same coal at two stages of liquefaction). The van Krevelen diagram thus appears well suited to amplifying and exposing compositional differences within and between complex organic mixtures.