Multiplatform High-Definition Ion Mobility Separations of the Largest Epimeric Peptides.

Ion mobility spectrometry (IMS) coupled to mass spectrometry (MS) has become a versatile tool to fractionate complex mixtures, distinguish structural isomers, and elucidate molecular geometries. Along with the whole MS field, IMS/MS advances to ever larger species. A topical proteomic problem is the discovery and characterization of d-amino acid-containing peptides (DAACPs) that are critical to neurotransmission and toxicology. Both linear IMS and FAIMS previously disentangled d/l epimers with up to ∼30 residues. In the first study using all three most powerful IMS methodologies─trapped IMS, cyclic IMS, and FAIMS─we demonstrate baseline resolution of the largest known d/l peptides (CHH from Homarus americanus with 72 residues) with a dynamic range up to 100. This expands FAIMS analyses of isomeric modified peptides, especially using hydrogen-rich buffers, to the ∼50-100 residue range of small proteins. The spectra for d and l are unprecedentedly strikingly similar except for a uniform shift of the separation parameter, indicating the conserved epimer-specific structural elements across multiple charge states and conformers. As the interepimer resolution tracks the average for smaller DAACPs, the IMS approaches could help search for yet larger DAACPs. The a priori method to calibrate cyclic (including multipass) IMS developed here may be broadly useful.

Top-down ion mobility/mass spectrometry reveals enzyme specificity: Separation and sequencing of isomeric proteoforms.

Enzymatic catalysis is one of the fundamental processes that drives the dynamic landscape of post-translational modifications (PTMs), expanding the structural and functional diversity of proteins. Here, we assessed enzyme specificity using a top-down ion mobility spectrometry (IMS) and tandem mass spectrometry (MS/MS) workflow. We successfully applied trapped IMS (TIMS) to investigate site-specific N-ε-acetylation of lysine residues of full-length histone H4 catalyzed by histone lysine acetyltransferase KAT8. We demonstrate that KAT8 exhibits a preference for N-ε-acetylation of residue K16, while also adding acetyl groups on residues K5 and K8 as the first degree of acetylation. Achieving TIMS resolving power values of up to 300, we fully separated mono-acetylated regioisomers (H4K5ac, H4K8ac, and H4K16ac). Each of these separated regioisomers produce unique MS/MS fragment ions, enabling estimation of their individual mobility distributions and the exact localization of the N-ε-acetylation sites. This study highlights the potential of top-down TIMS-MS/MS for conducting enzymatic assays at the intact protein level and, more generally, for separation and identification of intact isomeric proteoforms and precise PTM localization.

Full Separation and Sequencing of Isomeric Proteoforms in the Middle-Down Mass Range Using Cyclic Ion Mobility and Electron Capture Dissociation.

Unambiguous identification of distinct proteoforms and their biological functions is a significant analytical challenge due to the many combinations of post-translational modifications (PTM) that generate isomeric proteoforms. Resulting chimeric tandem mass spectra hinder detailed structural characterization of individual proteoforms for mixtures with more than two isomers. Large isomeric peptides and intact isomeric proteins are extremely difficult to distinguish with traditional chromatographic separation methods. Gas-phase ion separation techniques such as ion mobility spectrometry (IMS) methods now offer high resolving power that may enable separation of isomeric biomolecules, such as peptides and proteins. We explored novel high-resolution cyclic ion mobility spectrometry (cIM) combined with an electro-magnetostatic cell for "on-the-fly" electron capture dissociation (ECD) for separation and sequencing of large isomeric peptides. We demonstrate the effectiveness of this approach on ternary mixtures of mono- and trimethylated isomers of histone H3 N-tails (∼5.4 kDa), achieving a complete separation of these isomers with an average resolving power of ∼400 and a resolution of 1.5 and with nearly 100% amino acid sequence coverage. Our results demonstrate the potential of the cIM-MS/MS(ECD) technology to enhance middle-down and top-down proteomics workflows, thereby facilitating the identification of near-identical proteoforms with essential biological functions in complex mixtures.

Top-Down Ion Mobility Separations of Isomeric Proteoforms.

Continuing advances in proteomics highlight the ubiquity and biological importance of proteoforms─proteins with varied sequence, splicing, or distribution of post-translational modifications (PTMs). The preeminent example is histones, where the PTM pattern encodes the combinatorial language controlling the DNA transcription central to life. While the proteoforms with distinct PTM compositions are distinguishable by mass, the isomers with permuted PTMs commonly coexisting in cells generally require separation before mass-spectrometric (MS) analyses. That was accomplished on the bottom-up and middle-down levels using chromatography or ion mobility spectrometry (IMS), but proteolytic digestion obliterates the crucial PTM connectivity information. Here, we demonstrate baseline IMS resolution of intact isomeric proteoforms, specifically the acetylated H4 histones (11.3 kDa). The proteoforms with a single acetyl moiety on five alternative lysine residues (K5, K8, K12, K16, K20) known for distinct functionalities in vivo were constructed by two-step native chemical ligation and separated using trapped IMS at the resolving power up to 350 on the Bruker TIMS/ToF platform. Full resolution for several pairs was confirmed using binary mixtures and by unique fragments in tandem MS employing collision-induced dissociation. This novel capability for top-down proteoform characterization is poised to open major new avenues in proteomics and epigenetics.

Multianalytical Approach for Deciphering the Specific MS/MS Transition and Overcoming the Challenge of the Separation of a Transient Intermediate, Quinonoid Dihydrobiopterin.

Despite recent technological developments in analytical chemistry, separation and direct characterization of transient intermediates remain an analytical challenge. Among these, separation and direct characterization of quinonoid dihydrobiopterin (qH2Bip), a transient intermediate of tetrahydrobiopterin (H4Bip)-dependent hydroxylation reactions, essential in living organisms, with important and varied human pathophysiological impacts, are a clear illustration. H4Bip regeneration may be impaired by competitive nonenzymatic autoxidation reactions, such as isomerization of qH2Bip into a more stable 7,8-H2Bip (H2Bip) isomer, and subsequent nonenzymatic oxidation reactions. The quinonoid qH2Bip intermediate thus plays a key role in H4Bip-dependent hydroxylation reactions. However, only a few experimental results have indirectly confirmed this finding while revealing the difficulty of isolating qH2Bip from H4Bip-containing solutions. As a result, no current H4Bip assay method allows this isomer to be quantified even by liquid chromatography-tandem mass spectrometry (MS/MS). Here, we report isolation, structural characterization, and abundance of qH2Bip formed upon H4Bip autoxidation using three methods integrated into MS/MS. First, we characterized the structure of the two observed H2B isomers using IR photodissociation spectroscopy in conjunction with quantum chemical calculations. Then, we used differential ion mobility spectrometry to fully separate all oxidized forms of H4Bip including qH2Bip. These data are consistent and show that qH2Bip can also be unambiguously identified thanks to its specific MS/MS transition. This finding paves the way for the quantification of qH2Bip with MS/MS methods. Most importantly, the half-life value of this intermediate is nearly equivalent to that of H4Bip (tens of minutes), suggesting that an accurate method of H4Bip analysis should include the quantification of qH2Bip.

Disentangling Lipid Isomers by High-Resolution Differential Ion Mobility Spectrometry/Ozone-Induced Dissociation of Metalated Species.

The preponderance and functional importance of isomeric biomolecules have become topical in biochemistry. Therefore, one must distinguish and identify all such forms across compound classes, over a wide dynamic range as minor species often have critical activities. With all the power of modern mass spectrometry for compositional assignments by accurate mass, the identical precursor and often fragment ion masses render this task a steep challenge. This is recognized in proteomics and epigenetics, where proteoforms are disentangled and characterized employing novel separations and non-ergodic dissociation mechanisms. This issue is equally pertinent to lipidomics, where the lack of isomeric depth has thwarted the deciphering of functional networks. Here we introduce a new platform, where the isomeric lipids separated by high-resolution differential ion mobility spectrometry (FAIMS) are identified using ozone-induced dissociation (OzID). Cationization by metals (here K+, Ag+, and especially Cu+) broadly improves the FAIMS resolution of isomers with alternative C═C double bond (DB) positions or stereochemistry, presumably via metal attaching to the DB and reshaping the ion around it. However, the OzID yield diminishes for Ag+ and vanishes for Cu+ adducts. Argentination still strikes the best compromise between efficient separation and diagnostic fragmentation for optimal FAIMS/OzID performance.

Differential Ion Mobility Separations of d/l Peptide Epimers.

Life was originally assumed to utilize the l-amino acids only. Since 1980s, the d-amino acid-containing peptides (DAACPs) were detected in animals, often at extremely low levels with tremendous functional specificity. As the unguided proteomic algorithms based on peptide masses are oblivious to DAACPs, many more are believed to be hidden in organisms and novel methods to tackle DAACPs are sought. Linear ion mobility spectrometry (IMS) can distinguish and characterize the d/l-epimers but is restricted by poor orthogonality to MS as in other contexts. We now bring to this area the newer technique of differential IMS (FAIMS). The orthogonality of MS to high-resolution FAIMS exceeded that to linear IMS by 6×, the greatest factor found for biomolecules so far. Hence, FAIMS has achieved the 2.5× resolution of trapped IMS on average despite a lower resolving power, fully separating all 18 pairs of representative epimer species with masses of ∼400-5,000 Da and charge states of 1-6. A constant isomer resolution over these ranges allows projecting success for yet larger DAACPs.

Identification and quantification of amino acids and related compounds based on Differential Mobility Spectrometry.

Amino acids and related compounds constitute a class of biomarkers which is analyzed for early diagnosis of metabolic diseases (MDs). Protocols based on liquid chromatography hyphenated to tandem mass spectrometry (LC-MS/MS) are routinely used for MD diagnosis. Our ultimate objective is to evaluate the analytical performance of differential mobility spectrometry (DMS) hyphenated to MS/MS, in the perspective of using DMS-MS/MS as an alternative or complementary method for the topics of emergency in metabolic diagnosis and newborn rapid screening. The aim of the present study is to evaluate the robustness of a DMS-MS/MS protocol for the separation, identification, and quantification of amino acids and related compounds. Performance in terms of peak capacity and separation of isobaric and isomeric species is compared to those using drift tube type ion mobility spectrometry instruments. High reproducibility of the measurement of the DMS compensation voltage (CV) of metabolites shows that this CV parameter, or the corresponding electric field, could be used for application in metabolite identification. Multiple measurements show that the CV value of each AA or related compound is stable over a large period of time (6 months). Potential effects of matrix or concentration of the analytes on the DMS identifier are found to be negligible. Quantification of a selected set of metabolites in human plasmas has been carried out. The method linearity, intra-assay and inter-assay precision, detection limit, quantification limit and trueness analysis were assessed as adequate for both physiological and pathological conditions. Concentration levels of metabolites derived with our DMS-MS protocols were found to be in good agreement with those obtained with routine LC-MS/MS protocols used for the diagnosis of MDs at the Hospital Robert Debré (Paris).

Resolution and Assignment of Differential Ion Mobility Spectra of Sarcosine and Isomers.

Due to their central role in biochemical processes, fast separation and identification of amino acids (AA) is of importance in many areas of the biomedical field including the diagnosis and monitoring of inborn errors of metabolism and biomarker discovery. Due to the large number of AA together with their isomers and isobars, common methods of AA analysis are tedious and time-consuming because they include a chromatographic separation step requiring pre- or post-column derivatization. Here, we propose a rapid method of separation and identification of sarcosine, a biomarker candidate of prostate cancer, from isomers using differential ion mobility spectrometry (DIMS) interfaced with a tandem mass spectrometer (MS/MS) instrument. Baseline separation of protonated sarcosine from α- and ß-alanine isomers can be easily achieved. Identification of DIMS peak is performed using an isomer-specific activation mode where DIMS- and mass-selected ions are irradiated at selected wavenumbers allowing for the specific fragmentation via an infrared multiple photon dissociation (IRMPD) process. Two orthogonal methods to MS/MS are thus added, where the MS/MS(IRMPD) is nothing but an isomer-specific multiple reaction monitoring (MRM) method. The identification relies on the comparison of DIMS-MS/MS(IRMPD) chromatograms recorded at different wavenumbers. Based on the comparison of IR spectra of the three isomers, it is shown that specific depletion of the two protonated α- and ß-alanine can be achieved, thus allowing for clear identification of the sarcosine peak. It is also demonstrated that DIMS-MS/MS(IRMPD) spectra in the carboxylic C=O stretching region allow for the resolution of overlapping DIMS peaks. Graphical Abstract ᅟ.

Ligand-induced decarbonylation in diphosphine-ligated palladium acetates [CH3CO2Pd((PR2)2CH2)]+ (R = Me and Ph).

A new decarbonylation reaction is observed for [(K2-acetate)Pd(K2-diphosphine)]+ complexes. Gas-phase IR experiments identify the product as [CH3Pd(OP(Ph2)CH2PPh2)]+. DFT calculations uncovered a plausible mechanism involving O atom abstraction by the diphosphine ligand within the coordination sphere to yield the acetyl complex, [CH3COPd(OP(Ph2)CH2PPh2)]+, which then undergoes decarbonylation.

Fingerprints of Both Watson-Crick and Hoogsteen Isomers of the Isolated (Cytosine-Guanine)H+ Pair.

 Gas phase protonated guanine-cytosine (CGH+) pair was generated using an electrospray ionization source from solutions at two different pH (5.8 and 3.2). Consistent evidence from MS/MS fragmentation patterns and differential ion mobility spectra (DIMS) point toward the presence of two isomers of the CGH+ pair, whose relative populations depend strongly on the pH of the solution. Gas phase infrared multiphoton dissociation (IRMPD) spectroscopy in the 900-1900 cm-1 spectral range further confirms that the Watson-Crick isomer is preferentially produced (91%) at pH = 5.8, while the Hoogsteen isomer predominates (66%) at pH = 3.2). These fingerprint signatures are expected to be useful for the development of new analytical methodologies and to trigger isomer selective photochemical studies of protonated DNA base pairs.

Velocity of a Molecule Evaporated from a Water Nanodroplet: Maxwell-Boltzmann Statistics versus Non-Ergodic Events.

The velocity of a molecule evaporated from a mass-selected protonated water nanodroplet is measured by velocity map imaging in combination with a recently developed mass spectrometry technique. The measured velocity distributions allow probing statistical energy redistribution in ultimately small water nanodroplets after ultrafast electronic excitation. As the droplet size increases, the velocity distribution rapidly approaches the behavior expected for macroscopic droplets. However, a distinct high-velocity contribution provides evidence of molecular evaporation before complete energy redistribution, corresponding to non-ergodic events.

Proton Migration in Clusters Consisting of Protonated Pyridine Solvated by Water Molecules.

Proton transfer (PT) from protonated pyridine to water molecules is observed after excitation of microhydrated protonated pyridine (Py) clusters PyH(+) (H2 O)n (n=0-5) is induced by a single collision with an Ar atom at high incident velocity (95×10(3)  m s(-1) ). Besides the fragmentation channel associated with the evaporation of water molecules, the charged-fragment mass spectrum shows competition between the production of the PyH(+) ion (or its corresponding charged fragments) and the production of H(+) (H2 O) or H(+) (H2 O)2 ions. The increase in the production of protonated water fragments as a function of the number of H2 O molecules in the parent cluster ion as well sd the observation of a stable H(+) (H2 O)2 fragment, even in the case of the dissociation of PyH(+) (H2 O)2 , are evidence of the crucial role of PT in the relaxation process, even for a small number of solvating water molecules.