Ion emission from 1-10 MDa salt clusters: individual charge state resolution with charge detection mass spectrometry.

Salt cluster ions produced by electrospray ionization are used for mass calibration and fundamental investigations into cluster stability and charge separation processes. However, previous studies have been limited to relatively small clusters owing to the heterogeneity associated with large, multiply-charged clusters that leads to unresolved signals in conventional m/z spectra. Here, charge detection mass spectrometry is used to measure both the mass and charge distributions of positively charged clusters of KCl, CaCl2, and LaCl3 with masses between ∼1 and 10 MDa by dynamically measuring the energy per charge, m/z, charge, and mass of simultaneously trapped individual ions throughout a 1 s trapping time. The extent of remaining hydration on the clusters, determined from the change in the frequency of ion motion with time as a result of residual water loss, follows the order KCl < CaCl2 < LaCl3, and is significantly lower than that of a pure water nanodrop, consistent with tighter water binding to the more highly charged cations in these clusters. The number of ion emission events from these clusters also follows this same trend, indicating that water at the cluster surface facilitates charge loss. A new frequency-based method to determine the magnitude of the charge loss resulting from individual ion emission events clearly resolves losses of +1 and +2 ions. Achieving this individual charge state resolution for ion emission events is an important advance in obtaining information about the late stages of bare gaseous ions formation. Future experiments on more hydrated clusters are expected to lead to a better understanding of ion formation in electrospray ionization.

Dynamics of Rayleigh Fission Processes in ∼100 nm Charged Aqueous Nanodrops.

Fission of micron-size charged droplets has been observed using optical methods, but little is known about fission dynamics and breakup of smaller nanosize droplets that are important in a variety of natural and industrial processes. Here, spontaneous fission of individual aqueous nanodrops formed by electrospray is investigated using charge detection mass spectrometry. Fission processes ranging from formation of just two progeny droplets in 2 ms to production of dozens of progeny droplets over 100+ ms are observed for nanodrops that are charged above the Rayleigh limit. These results indicate that Rayleigh fission is a continuum of processes that produce progeny droplets that vary widely in charge, mass, and number.

Dynamic Energy Measurements in Charge Detection Mass Spectrometry Eliminate Adverse Effects of Ion-Ion Interactions.

Ion-ion interactions in charge detection mass spectrometers that use electrostatic traps to measure masses of individual ions have not been reported previously, although ion trajectory simulations have shown that these types of interactions affect ion energies and thereby degrade measurement performance. Here, examples of interactions between simultaneously trapped ions that have masses ranging from ca. 2 to 350 MDa and ca. 100 to 1000 charges are studied in detail using a dynamic measurement method that makes it possible to track the evolution of the mass, charge, and energy of individual ions over their trapping lifetimes. Signals from ions that have similar oscillation frequencies can have overlapping spectral leakage artifacts that result in slightly increased uncertainties in the mass determination, but these effects can be mitigated by the careful choice of parameters used in the short-time Fourier transform analysis. Energy transfers between physically interacting ions are also observed and quantified with individual ion energy measurement resolution as high as ∼950. The mass and charge of interacting ions do not change, and their corresponding measurement uncertainties are equivalent to ions that do not undergo physical interactions. Simultaneous trapping of multiple ions in CDMS can greatly decrease the acquisition time necessary to accumulate a statistically meaningful number of individual ion measurements. These results demonstrate that while ion-ion interactions can occur when multiple ions are trapped, they have negligible effects on mass accuracy when using the dynamic measurement method.

Lighting Up at High Potential: Effects of Voltage and Emitter Size in Nanoelectrospray Ionization.

Effects of electrospray voltage on cluster size and abundance formed from aqueous CsI were investigated with emitter tip diameters between 260 ± 7 nm and 2.45 ± 0.30 µm. Cluster size increases with increasing voltage, increasing solution concentration and increasing emitter diameter consistent with formation of larger initial droplet sizes. For emitters with tip diameters above ∼1 µm, varying the voltage either up or down leads to reproducible voltage-dependent extents of cluster formation. In contrast, higher voltages with submicron diameter emitters can lead to only Cs+ and Cs(H2O)+ and no clusters. This change in ion formation reproducibly occurs at spray potentials >1.3 kV for 260 nm emitters and appears to be induced by a corona discharge and material build-up at the emitter tip. Under conditions where abundant Cs+ is observed and no clusters are formed, ions such as K+ and Cu1+ are also observed but ions with more negative solvation energies, such as Ba2+, are not. Similarly, ions from bradykinin and ubiquitin are observed predischarge but not post discharge. Ions with more positive solvation energies can desorb directly from the air-water interface that is created at the tip of these emitters, whereas ions with more negative solvation energies as well as peptide and protein ions do not. These results indicate that ion desorption directly from solution can occur, and similar experiments with even smaller emitters may lead to new insights into ion formation in electrospray ionization.

Determination of Sialic Acid Isomers from Released N-Glycans Using Ion Mobility Spectrometry.

Complex carbohydrates are ubiquitous in nature and represent one of the major classes of biopolymers. They can exhibit highly diverse structures with multiple branched sites as well as a complex regio- and stereochemistry. A common way to analytically address this complexity is liquid chromatography (LC) in combination with mass spectrometry (MS). However, MS-based detection often does not provide sufficient information to distinguish glycan isomers. Ion mobility-mass spectrometry (IM-MS)─a technique that separates ions based on their size, charge, and shape─has recently shown great potential to solve this problem by identifying characteristic isomeric glycan features such as the sialylation and fucosylation pattern. However, while both LC-MS and IM-MS have clearly proven their individual capabilities for glycan analysis, attempts to combine both methods into a consistent workflow are lacking. Here, we close this gap and combine hydrophilic interaction liquid chromatography (HILIC) with IM-MS to analyze the glycan structures released from human alpha-1-acid glycoprotein (hAGP). HILIC separates the crude mixture of highly sialylated multi-antennary glycans, MS provides information on glycan composition, and IMS is used to distinguish and quantify α2,6- and α2,3-linked sialic acid isomers based on characteristic fragments. Further, the technique can support the assignment of antenna fucosylation. This feature mapping can confidently assign glycan isomers with multiple sialic acids within one LC-IM-MS run and is fully compatible with existing workflows for N-glycan analysis.

Liquid chromatography setup-dependent artefactual methionine oxidation of peptides: The importance of an adapted quality control process.

In both biologics quality control experiments and protein post-translational modification studies, the analytical system used is not supposed to bring any artefactual modifications which could impair the results. In this work, we investigated oxidation of methionine-containing peptides during reversed-phase (RP) chromatographic separation. We first used a synthetic methionine-containing peptide to evaluate this artefactual phenomenon and then considered more complex samples (i.e., plasma and HeLa protein digests). The methionine oxidation levels of the peptides were systematically assessed and compared for the long-term use of the analytical column, the sample trapping time, the gradient length, the sample load and the nature of the stationary phase (HSS T3 from Waters, YMC Triart C18 from YMC Europe GmbH and BEH130 C18 from Waters). In addition to the oxidation of methionine in solution, we observed on the HSS T3 and the BEH130 stationary phases an additional broad peak corresponding to an on-column oxidized species. Considering the HSS T3 phase, our results highlight that the on-column oxidation level significantly increases with the age of the analytical column and the gradient length and reaches 56 % when a 1-year-old column set is used with a 180 min-long LC method. These levels go to 0 % and 18 % for the YMC Triart C18 and the BEH130 C18 phases respectively. Interestingly, the on-column oxidation proportion decreases as the injected sample load increases suggesting the presence of a discrete number of oxidation sites within the stationary phase of the analytical column. Those findings observed in different laboratories using distinct set of columns, albeit to varying degrees, strengthen the need for a standard of methionine-containing peptide that could be used as a quality control to appraise the status of the liquid chromatographic columns.

Radical-Pairing Interactions in a Molecular Switch Evidenced by Ion Mobility Spectrometry and Infrared Ion Spectroscopy.

The digital revolution sets a milestone in the progressive miniaturization of working devices and in the underlying advent of molecular machines. Foldamers involving mechanically entangled components with modular secondary structures are among the most promising designs for molecular switch-based applications. Characterizing the nature and dynamics of their intramolecular network following the application of a stimulus is the key to their performance. Here, we use non-dissociative electron transfer as a reductive stimulus in the gas phase and probe the consecutive co-conformational transitions of a donor-acceptor oligorotaxane foldamer using electrospray mass spectrometry interfaced with ion mobility and infrared ion spectroscopy. A comparison of collision cross section distributions for analogous closed-shell and radical molecular ions sheds light on their respective formation energetics, while variations in their respective infrared absorption bands evidence changes in intramolecular organization as the foldamer becomes more compact. These differences are compatible with the advent of radical-pairing interactions.

Solvent Adducts in Ion Mobility Spectrometry: Toward an Alternative Reaction Probe for Thermometer Ions.

The fragmentation of benzylpyridinium "thermometer" ions is widely used to quantify the energetics of ions studied by mass spectrometry and other hyphenated techniques such as ion mobility. The reaction pathway leads to a benzylium cation with the release of a neutral pyridine. Using trapped ion mobility spectrometry, we noticed that the addition of acetonitrile, present in the electrosprayed solvent mixture, could occur on some electrophilic benzylium cations. This process results in the formation of adducts and in the appearance of a supplementary mobility peak. We here demonstrate that the addition takes place both in the electrospray source and inside the mobility analyzer, thereby evidencing possible outflow of solvent vapors downstream the instrument. By further characterizing the initial kinetics and the resulting equilibrium linked with the addition reaction, we presently discuss these as alternative probes to calibrate ion temperature in the framework of ion mobility.

Effective Temperature and Structural Rearrangement in Trapped Ion Mobility Spectrometry.

Modern ion mobility instrumentation is typically operated above the low field limit, which may activate the ions and cause structural rearrangement or fragmentation during analysis. Here, we quantitatively assessed the internal heating experienced by ions during trapped ion mobility spectrometry (TIMS) experiments. To this end, the fragmentation yields of fragile benzylpyridinium "thermometer" ions were monitored during both the accumulation and analysis steps inside the TIMS tunnel. The corresponding fragmentation rate constants were translated into a vibrational effective temperature Teff,vib. Our results demonstrate significant fragmentation upstream and inside the TIMS tunnel that corresponds to Teff,vib ≈ 510 K during both the accumulation and analysis steps. Broadening our scope to cytochrome c and lysozyme, we showed that although compact "native" folds can be preserved, the collision cross section distributions are highly sensitive to the transmission voltages and the analysis time scale. Our results are discussed with regard to Teff,vib data previously acquired on traveling-wave (TWIMS) ion mobility in the context of native mass spectrometry and conformational landscape exploration.

Kendrick Mass Defect Approach Combined to NORINE Database for Molecular Formula Assignment of Nonribosomal Peptides.

The identification of known (dereplication) or unknown nonribosomal peptides (NRPs) produced by microorganisms is a time consuming, expensive, and challenging task where mass spectrometry and nuclear magnetic resonance play a key role. The first step of the identification process always involves the establishment of a molecular formula. Unfortunately, the number of potential molecular formulae increases significantly with higher molecular masses and the lower precision of their measurements. In the present article, we demonstrate that molecular formula assignment can be achieved by a combined approach using the regular Kendrick mass defect (RKMD) and NORINE, the reference curated database of NRPs. We observed that irrespective of the molecular formula, the addition and subtraction of a given atom or atom group always leads to the same RKMD variation and nominal Kendrick mass (NKM). Graphically, these variations translated into a vector mesh can be used to connect an unknown molecule to a known NRP of the NORINE database and establish its molecular formula. We explain and illustrate this concept through the high-resolution mass spectrometry analysis of a commercially available mixture composed of four surfactins. The Kendrick approach enriched with the NORINE database content is a fast, useful, and easy-to-use tool for molecular mass assignment of known and unknown NRP structures.

Covalent Cross-Linking as an Enabler for Structural Mass Spectrometry.

The number of studies referring to the structural elucidation of intact biomolecular systems using mass spectrometry techniques has gradually increased in the post-2000s literature topics. As part of native mass spectrometry, this domain capitalizes on the kinetic trapping of physiological folds in view of probing solution-like conformational properties of isolated molecules or complexes after their electrospray transfer to the gas phase. Despite its efficiency for a wide array of analytes, this approach is expected to be pushed to its limits when considering highly dynamic systems or when dealing with nonideal operating conditions. To circumvent these limitations, we challenge the adequacy of an original strategy based on cross-linkers to improve the gas-phase stability of isolated proteins and ensure the preservation of folded conformations when measuring with strong transmission voltages, by spraying from denaturing solvents, or trapping for extended periods of time. Tested on cytochrome c, myoglobin, and ß-lactoglobulin cross-linked using BS3, we validated the process as structurally nonintrusive in solution using far-ultraviolet circular dichroism and unraveled the preservation of folded conformations showing better resilience to denaturation on cross-linked species using ion mobility. The resulting collision cross sections were found in agreement with the native fold, and a preservation of the proteins' secondary and tertiary structures was evidenced using molecular dynamics simulations. Our results provide new insights concerning the fate of electro-sprayed cross-linked conformers in the gas phase, while constituting promising evidence for the validation of this technique as part of future structural mass spectrometry workflows.

Two-Parameter Power Formalism for Structural Screening of Ion Mobility Trends: Applied Study on Artificial Molecular Switches.

Recent literature provides increasing samples of structural studies relying on ion mobility coupled to mass spectrometry in view of characterizing gas-phase conformation and energetics properties of biomolecular ions. A typical framework consists in experimentally monitoring the collisional cross sections for various experimental conditions and using them as references to select appropriate candidate structures issued from theoretical modeling. Although it has proved successful for structural assignment, this process is resource costly and lengthy, namely due to intricacies in the selection of appropriate input geometries. In the present work, we propose simplified methodologies dedicated to the systematic screening of ion mobility data acquired on systems built from repetitive subunits and detail their application to challenging artificial molecular switch systems. Capitalizing on coarse-grained design, we first demonstrate how the assimilation of subunits into adequately assembled building-blocks can be used for fast assignments of a system topology. Further focusing on topology-specific differential ion mobility trends, we show that the building-block assemblies can be fused into single fully convex solid figure models, i.e., sphere and cylinder, whose projected areas follow a two-parameter power formalism A × nB. We show that the fitting parameters A and B were assigned as structural descriptors respectively associated with the dimensions of each constitutive subunit, i.e., size parameter, and with their assembled tridimensional arrangement, i.e., shape parameter. The present work provides a ready-to-use method for the screening of IM-MS data sets that is expected to facilitate the eventual design of input structures whenever advanced modeling calculations are required.

Multi-Enzymatic Limited Digestion: The Next-Generation Sequencing for Proteomics?

Over the past 40 years, proteomics, generically defined as the field dedicated to the identification and analysis of proteins, has tremendously gained in popularity and potency through advancements in genome sequencing, separative techniques, mass spectrometry, and bioinformatics algorithms. As a consequence, its scope of application has gradually enlarged and diversified to meet specialized topical biomedical subjects. Although the tryptic bottom-up approach is widely regarded as the gold standard for rapid screening of complex samples, its application for precise and confident mapping of protein modifications is often hindered due to partial sequence coverage, poor redundancy in indicative peptides, and lack of method flexibility. We here show how the synergic and time-limited action of a properly diluted mix of multiple enzymes can be exploited in a versatile yet straightforward protocol to alleviate present-day drawbacks. Merging bottom-up and middle-down ideologies, our results highlight broad assemblies of overlapping peptides that enable refined and reliable characterizations of proteins, including variant identification, and their carried modifications, including post-translational modifications, truncations, and cleavages. Beyond this boost in performance, our methodology also offers efficient de novo sequencing capabilities, in view of which we here present a dedicated custom assembly algorithm.

Where Ion Mobility and Molecular Dynamics Meet To Unravel the (Un)Folding Mechanisms of an Oligorotaxane Molecular Switch.

At the interface between foldamers and mechanically interlocked molecules, oligorotaxanes exhibit a spring-like folded secondary structure with remarkable mechanical and physicochemical properties. Among these properties, the ability of oligorotaxanes to act as molecular switches through controlled modulations of their spatial extension over (un)folding dynamics is of particular interest. The present study aims to assess and further characterize this remarkable feature in the gas phase using mass spectrometry tools. In this context, we focused on the [4]5NPR+12 oligorotaxane molecule complexed with PF6- counterion and probed its co-conformational states as a function of the in-source-generated charge states. Data were interpreted in light of electronic secondary structure computations at the PM6 and DFT levels. Our results highlight two major co-conformational groups associated either with folded compact structures, notably stabilized by intramolecular π-π interactions and predominant for low charge states or with fully stretched structures resulting from significant Coulombic repulsions at high charge states. Between, the oligorotaxane adopts intermediate folded co-conformations, suggesting a stepwise unfolding pathway under increasing repulsive Coulombic constraints. The reversibility of this superstructural transition was next interrogated under electron-driven (nondissociative electron transfer) and heat-driven (collision-induced unfolding) activation stimuli. The outcomes support the feasibility to either unfold or (partially) refold the oligorotaxane foldamer on purpose in the gas phase. Our results show that the balance between the stabilizing π-π interactions and the versatile Coulomb interactions dictates the elongation state of the foldamer in the gas phase and emphasizes the adequacy of mass spectrometry tools for the superstructural characterization of desolvated prototypical artificial molecular machines.

Energetics and structural characterization of isomers using ion mobility and gas-phase H/D exchange: Learning from lasso peptides.

State-of-the-art characterization of proteins using MS namely relies on fragmentation methods that allow exploring featured dissociative reaction pathways. These pathways are often initiated by a series of potentially informative mass-constant conformational changes that are nonetheless frequently overlooked by lack of adequate investigation techniques. In the present study, we propose a methodology to readily address both structural and energetic aspects of stereoisomerization reactions using ion mobility coupled with MS. To this end, a commercial spectrometer was used as a reactor comprising an energy resolved collisional activation step intended at promoting controlled conformational changes and a structural assignment step dedicated to the identification of the generated isomers. This identification relies on ion mobility and other on-line coupled techniques, namely an originally designed gas-phase H/D exchange experiment. We here apply this methodology to characterize the isomerization kinetics of capistruin, a 19-residue long lasso-folded peptide. We expect this approach to bring insights into the physical origin of global dissociation thresholds monitored in MS/MS experiments and to set a promising basis for quantitative investigations of the stability of different molecular folds.