Chemical-Mediated Digestion: An Alternative Realm for Middle-down Proteomics?

Protein digestion in mass spectrometry (MS)-based bottom-up proteomics targets mainly lysine and arginine residues, yielding primarily 0.6-3 kDa peptides for the proteomes of organisms of all major kingdoms. Recent advances in MS technology enable analysis of complex mixtures of increasingly longer (>3 kDa) peptides in a high-throughput manner supporting the development of a middle-down proteomics (MDP) approach. Generating longer peptides is a paramount step in launching an MDP pipeline, but the quest for the selection of a cleaving agent that would provide the desired 3-15 kDa peptides remains open. Recent bioinformatics studies have shown that cleavage at the rarely occurring amino acid residues such as methionine (Met), tryptophan (Trp), or cysteine (Cys) would be suitable for MDP approach. Interestingly, chemical-mediated proteolytic cleavages uniquely allow targeting these rare amino acids, for which no specific proteolytic enzymes are known. Herein, as potential candidates for MDP-grade proteolysis, we have investigated the performance of chemical agents previously reported to target primarily Met, Trp, and Cys residues: CNBr, BNPS-Skatole (3-bromo-3-methyl-2-(2-nitrophenyl)sulfanylindole), and NTCB (2-nitro-5-thiobenzoic acid), respectively. Figures of merit such as digestion reproducibility, peptide size distribution, and occurrence of side reactions are discussed. The NTCB-based MDP workflow has demonstrated particularly attractive performance, and NTCB is put forward here as a potential cleaving agent for further MDP development.

Ligand Aspect Ratio as a Decisive Factor for the Self-Assembly of Coordination Cages.

It is possible to control the geometry and the composition of metallasupramolecular assemblies via the aspect ratio of their ligands. This point is demonstrated for a series of iron- and palladium-based coordination cages. Functionalized clathrochelate complexes with variable aspect ratios were used as rod-like metalloligands. A cubic Fe(II)8L12 cage was obtained from a metalloligand with an intermediate aspect ratio. By increasing the length or by decreasing the width of the ligand, the self-assembly process resulted in the clean formation of tetrahedral Fe(II)4L6 cages instead of cubic cages. In a related fashion, it was possible to control the geometry of Pd(II)-based coordination cages. A metalloligand with a large aspect ratio gave an entropically favored tetrahedral Pd(II)4L8 assembly, whereas an octahedral Pd(II)6L12 cage was formed with a ligand of the same length but with an increased width. The aspect ratio can also be used to control the composition of dynamic mixtures of Pd(II) cages. Out of two metalloligands with only marginally different aspect ratios, one gave rise to a self-sorted collection of Pd(II)4L8 and Pd(II)6L12 cages, whereas the other did not.

Molecularly Defined Nanostructures Based on a Novel AAA-DDD Triple Hydrogen-Bonding Motif.

A facile and flexible method for the synthesis of a new AAA-DDD triple hydrogen-bonding motif is described. Polytopic supramolecular building blocks with precisely oriented AAA and DDD groups are thus accessible in few steps. These building blocks were used for the assembly of large macrocycles featuring four AAA-DDD interactions and a macrobicyclic complex with a total of six AAA-DDD interactions.

On the use of electron capture rate constants to describe electron capture dissociation mass spectrometry of peptides.

Electron capture dissociation (ECD) tandem mass spectrometry (MS/MS) is a powerful analytical tool for peptide and protein structure analysis. The product ion abundance (PIA) distribution in ECD MS/MS is known to vary as a function of electron irradiation period. This variation complicates the development of a method of peptide identification by correlation of ECD MS/MS data with experimental and theoretical mass spectra. Here, we first develop a kinetic model to describe primary electron capture by peptide dications leading to product ion formation and secondary electron capture resulting in product ion neutralization. We apply the developed kinetic model to calculate product ion formation rate constants and electron capture rate constants of product ions from ECD mass spectra acquired using various electron irradiation periods. Contrary to ECD PIA distributions, the product ion formation rate constants are shown to be independent of electron irradiation period and, thus, may be employed to characterize ECD product ion formation more universally. The electron capture rate constants of product ions in ECD Fourier transform ion cyclotron resonance MS were found to correlate (with a correlation factor, R(2), of ca 0.9) with ion mobility cross sections of product ions in electron transfer dissociation. Finally, we demonstrate that the electron irradiation period influences the ratio of radical and even-electron c and z product ions.

Producing absorption mode Fourier transform ion cyclotron resonance mass spectra with non-quadratic phase correction functions.

RATIONALE: Previously described methods for producing absorption mode Fourier transform ion cyclotron resonance (FTICR) mass spectra have all relied on the phase correction function being quadratic. This assumption has been found to be invalid for some instruments and spectra and so it has not been possible to produce absorption mode spectra for these cases. METHODS: The Autophaser algorithm has been adapted to allow nth order polynomial phase correction functions to be optimized. The data was collected on a modified Thermo LTQ FTICR mass spectrometer, using electrospray ionization and a novel ICR cell design (NADEL). Peak assignment and mass calibration were undertaken using the pyFTMS framework. RESULTS: An nth-order phase correction function has been used to produce an absorption mode mass spectrum of the maltene fraction of a crude oil sample which was not possible using the previous assumption that the phase correction function must be quadratic. Data processing for this spectrum in absorption mode has shown the expected benefits in terms of increasing the number of assigned peaks and also improving the mass accuracy (i.e. confidence) of the assignments. CONCLUSIONS: It is possible to phase-correct time-domain data in FTICRMS to yield absorption mode mass spectra representation even when the data does not correspond to the theoretical quadratic phase correction function predicted by previous studies. This will allow a larger proportion of spectra to be processed in absorption mode.

A Functional Group Approach for Prediction of APPI Response of Organic Synthetic Targets.

Atmospheric pressure photoionization (APPI) is a technique of choice for ionization of non-polar molecules in mass spectrometry (MS). Reported APPI-based studies tend to focus on a selected compound class, which may contain a variety of functional groups. These studies demonstrate that APPI response frequently differs substantially, indicating a certain dependence on the functional group present. Although this dependence could be employed for APPI response prediction, its systematic use is currently absent. Here, we apply APPI MS to a judiciously-compiled set of 63 compounds containing a number of diverse functional groups commonly utilized in synthesis, reactive functional groups, as well as those containing boron and silicon. Based on the outcome of APPI MS of these compounds, we propose and evaluate a simple guideline to estimate the APPI response for a novel compound, the key properties of which have not been characterized in the gas phase. Briefly, we first identify key functional groups in the compound and gather knowledge on the known ionization energies from the smallest analogues containing said functional groups. We then consider local inductive and resonance effects on said ionization energies for the compounds of interest to estimate the APPI response. Finally, application of APPI MS to compounds of interest considered herein demonstrated extended upper mass ionization limit of 3.5 kDa for non-polymeric compounds.

Self-assembly of a giant molecular Solomon link from 30 subcomponents.

The synthesis of topologically complex structures, such as links and knots, is one of the current challenges in supramolecular chemistry. The so-called Solomon link consists of two doubly interlocked rings. Despite being a rather simple link from a topological point of view, only few molecular versions of this link have been described so far. Here, we report the quantitative synthesis of a giant molecular Solomon link from 30 subcomponents. The highly charged structure is formed by assembly of 12 cis-blocked Pt(2+) complexes, six Cu(+) ions, and 12 rigid N-donor ligands. Each of the two interlocked rings is composed of six repeating Pt(ligand) units, while the six Cu(+) ions connect the two rings. With a molecular weight of nearly 12 kDa and a diameter of 44.2 Å, this complex is the largest non-DNA-based Solomon link described so far. Furthermore, it represents a molecular version of a "stick link".

Conference report CH analysis 2013.

Two PhD students reflect on their experience and impressions of the latest CH analysis 2013 conference, held at Beatenberg this past November.

Distinguishing analyte from noise components in mass spectra of complex samples: where to cut the noise?

Fourier transform mass spectrometry (FTMS) enables comprehensive analysis of complex molecular mixtures. Given the broad intensity ranges of components in the mass spectra, it is imperative to accurately determine a noise threshold level above which peak assignments will be made. Conventionally, to find the threshold level, the "N sigma" approach or an equivalent rule is used. However, the "N sigma" approach cannot be applied to mass spectra stored with partially removed noise (reduced-profile mode). It is also not directly applicable to mass spectra acquired in the absorption mode with removed negative spectral amplitudes. Moreover, N value selection is normally made based on a rule of thumb, meaning that the calculated threshold level may be biased. Here, we present a noise thresholding method which addresses these limitations for analysis of mass spectra of complex molecular mixtures. The introduced data-dependent thresholding method involves analysis of the distribution of logarithmic intensity of all peaks, including noise and analyte, for a given mass spectrum. Selected method applications include FTMS analysis of crude oil fractions as well as tandem MS analysis of intact proteins.

Ping-pong protons: how hydrogen-bonding networks facilitate heterolytic bond cleavage in peptide radical cations.

Electron capture and electron transfer dissociation (ECD/ETD) tandem mass spectrometry (MS/MS) are commonly employed techniques for biomolecular analysis. The ECD/ETD process predominately cleaves N-Cα peptide backbone bonds, leading to primary sequence information complementary to other mass spectrometry techniques. Despite frequent laboratory use, the mechanistic underpinnings surrounding N-Cα bond cleavage remain debated. While the majority of mechanisms assume a homolytic bond rupture, we recently showed that heterolytic cleavage is also thermodynamically viable. For a cleavage of this type to be feasible, the charge separation created upon breaking of the N-Cα backbone bond must be quickly annihilated. In this work, we show, using density functional computations, that specific hydrogen-bonding motifs and structural rearrangements involving proton transfers stabilize the transition state associated with heterolytic cleavage and eliminate the ensuing charge separation from the final product fragments. The movement of protons can occur either directly from the z- to c-fragment or in a more complex manner including a ping-pong-type mechanism. The nature of these diverse hydrogen-bonding motifs reveals that not only those functional groups proximate to the bond rupture site, but also the entire global chemical environment, play important roles in backbone cleavage characteristic of ECD/ETD MS/MS. For doubly charged systems, both conformation and electron localization site dictate which of the two fragments retains the final positive charge.

On the viability of heterolytic peptide N-C(α) bond cleavage in electron capture and transfer dissociation mass spectrometry.

While frequently employed as an experimental technique, the mechanistic picture surrounding the gas-phase dissociation of peptides carrying multiple positive charges during electron capture and electron transfer dissociation tandem mass spectrometry remains incomplete. Despite this mechanistic uncertainty, most proposals agree that the peptide backbone N-Cα bond located to the C-terminal (right) side of an aminoketyl radical formed in a peptide backbone during the electron capture process is homolytically cleaved. Recently, we introduced the "enol" mechanism, which proposes that a backbone N-Cα bond located to the N-terminal (left) side of an aminoketyl radical is cleaved heterolytically. Here, we further validate this mechanism using replica-exchange molecular dynamics to create unbiased representative sets of low-energy conformers for several model tryptic peptide systems (H-Alax-Lys-OH(2+), x = 3-5). Transition state barrier enthalpies for the cleavage of N-Cα bonds proceeding via the homolytic (right-side) and heterolytic (left-side) pathways, determined by density functional computations, identify the preferred cleavage route for each conformer. These findings support our original hypothesis that heterolytic N-Cα cleavage can exist in a competitive balance with homolytic cleavages, independent of the relative energy of the precursor dication species. Smaller peptide systems see decreased heterolytic N-Cα cleavage probabilities, likely resulting from an insufficient hydrogen-bonding network needed to stabilize and ultimately annihilate the transition state zwitterion. This observation may explain the early dismissal of left-side cleavage pathways based on computational studies employing small model systems.

Iterative method for mass spectra recalibration via empirical estimation of the mass calibration function for Fourier transform mass spectrometry-based petroleomics.

We describe a mass spectra recalibration method, which enables analysis of petroleum samples with Orbitrap FTMS. In this method, the mass calibration function is estimated on the basis of mass-to-charge ratios and abundances of internal calibrants without a need for theoretical description of residual mass errors. Importantly, to maximize the estimation accuracy of the mass calibration function, an iterative approach is implemented to obtain sufficiently high number of internal calibrants covering the entire ranges of mass-to-charge ratios and abundances of interest. For petroleomic samples, the method routinely provides root-mean-square (RMS) mass accuracies at sub-ppm level and hence allows for reliable assignment of elemental compositions. Moreover, since the achieved mass accuracies are normally limited only by random errors of low-abundance analytes, the method maximizes the range of abundances of assignable species for a given signal-to-noise ratio of experimental data. Additionally, despite being initially developed for Orbitrap FTMS, the method is likewise applicable for ion cyclotron resonance FTMS.

Hexagonal class representation for fingerprinting and facile comparison of petroleomic samples.

Because of the high complexity of petroleomic-type samples, there is a need for efficient ways of visualizing and interpreting the resulting data in mass spectrometry-based petroleomics. Over the years, several graphing approaches have become widespread, yet they mostly deal with a particular subset of compounds detected within a given sample. Here, we present an alternative and complementary sample visualization method, the hexagonal class representation, based on relative abundance vs compound classes plot. The representation can be used to "fingerprint" a petroleomic-type sample, provide a simple means of sample comparison, as well as allow for a fast overview and detection of any compound of interest based on its elemental composition and chemical properties.

Principles of electron capture and transfer dissociation mass spectrometry applied to peptide and protein structure analysis.

This tutorial review describes the principles and practices of electron capture and transfer dissociation (ECD/ETD or ExD) mass spectrometry (MS) employed for peptide and protein structure analysis. ExD MS relies on interactions between gas phase peptide or protein ions carrying multiple positive charges with either free low-energy (~1 eV) electrons (ECD), or with reagent radical anions possessing an electron available for transfer (ETD). As a result of recent implementation on sensitive, high resolution, high mass accuracy, and liquid chromatography timescale-compatible mass spectrometers, ExD, more specifically, ETD MS has received particular interest in life science research. In addition to describing the fundamental aspects of ExD radical ion chemistry, this tutorial provides practical guidelines for peptide de novo sequencing with ExD MS, as well as reviews some of the current capabilities and limitations of these techniques. The merits of ExD MS are discussed primarily within the context of life science research.

Heterolytic N-Cα bond cleavage in electron capture and transfer dissociation of peptide cations.

Mass spectrometry techniques employing electron capture and electron transfer dissociation represent powerful approaches for the analysis of biological samples. Despite routine employment in analytical fields, the underlying physical processes dictating peptide fragmentation remain less understood. Among the most accepted mechanisms, the Cornell proposal of McLafferty postulates that the homolytic cleavage of N-C(α) bonds located in the peptide backbone occurs on the right (C-terminal) side of a hydrogen acceptor carbonyl group. Here, we illustrate that an alternative "enol" mechanism, based on a heterolytic N-C(α) bond cleavage located on the left (N-terminal) side of an acceptor carbonyl group, not only is thermodynamically viable but also often represents the energetically preferred cleavage route.

Borophosphonate cages: easily accessible and constitutionally dynamic heterocubane scaffolds.

A versatile and experimentally facile procedure for the synthesis of borophosphonate cages of the general formula [tBuPO(3)BR'](4) is described. The method involves heating of tert-butylphosphonic acid with a boronic acid in toluene to give borophosphonates in [4+4] condensation reactions. The products display a heterocubane structure with bent P-O-B bridges as evidenced by crystallographic analyses. Scrambling experiments show that the borophosphonate cages are constitutionally dynamic scaffolds with exchange reactions occurring on the hour time scale at room temperature. The cages can be decorated with reactive groups such as aldehydes. Our results demonstrate that borophosphonate cages are interesting building blocks for dynamic covalent chemistry.