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In organometallic chemistry, especially in the catalysis area, accessing the finest tuning of a catalytic reaction pathway requires a detailed knowledge of the steric and electronic influences of the ligands bound to the metal center. Usually, the M–L bond between a ligand and metal is depicted by the Dewar–Chatt–Duncanson model involving two opposite interactions, σ-donor and π-acceptor effects of the ligand. The experimental evaluation of these effects is essential and complementary to in-depth theoretical approaches that are able to provide a detailed description of the M–L bond. In this work, we present a study of LMo(CO)5complexes with L being various tertiary phosphine ligands by means of mass-selected high-resolution photoelectron spectroscopy (PES) performed with synchrotron radiation, DFT, and energy decomposition analyses (EDA) combined with the natural orbitals for chemical valence (NOCV) analysis. These methods enable a separated access of the σ-donor and π-acceptor effects of ligands by probing either the electronic configuration of the complex (PES) or the interaction of the ligand with the metal (EDA). Three series of PR3ligands with various electronic influences are investigated: the strong donating alkyl substituents (PMe3, PEt3, and PiPr3), the intermediate PPhxMe(3–x)(x= 0–3) set, and the PPhxPyrl(3–x)set (x= 0–3 with Pyrl being the strong electron withdrawing pyrrolyl group C4H4N). For each complex, their adiabatic and vertical ionization energies (IEs) could be determined with a 0.03 eV precision. Experiment and theory show an excellent agreement, either for the IE determination or electronic effect analysis. The ability to interpret the spectra is shown to depend on the character of the ligand. “Innocent” ligands provide the spectra that are the most straightforward to analyze, whereas the “non-innocent” ligands (which are ionized prior to the metal center) render the analysis more difficult due to an increased number of molecular orbitals in the energy range considered. A very good linear correlation is finally found between the measured adiabatic ionization energies and the interaction energy term obtained by EDA for each of these two types of ligands, which opens interesting perspective for the prediction of ligand characters.

Rigorous validation of amino acid sequence is fundamental in the characterization of original and biosimilar protein biopharmaceuticals. Widely accepted workflows are based on bottom-up mass spectrometry, and they often require multiple techniques and significant manual work. Here, we demonstrate that optimization of a set of tandem mass spectroscopy (MS/MS) collision energies and automated combination of all available information in the measurements can increase the sequence validated by one technique close to the inherent limits. We created a software (called “Serac”) that consumes results of the Mascot database search engine and identifies the amino acids validated by bottom-up MS/MS experiments using the most rigorous, industrially acceptable definition of sequence coverage (we term this “confirmed sequence coverage”). The software can combine spectra at the level of amino acids or fragment ions to exploit complementarity, provides full transparency to justify validation, and reduces manual effort. With its help, we investigated collision energy dependence of confirmed sequence coverage of individual peptides and full proteins on trypsin-digested monoclonal antibody samples (rituximab and trastuzumab). We found the energy dependence to be modest, but we demonstrated the benefit of using spectra taken at multiple energies. We describe a workflow based on 2–3 LC-MS/MS runs, carefully selected collision energies, and a fragment ion level combination, which yields ∼85% confirmed sequence coverage, 25%–30% above that from a basic proteomics protocol. Further increase can mainly be expected from alternative digestion enzymes or fragmentation techniques, which can be seamlessly integrated to the processing, thereby allowing effortless validation of full sequences.

The active site of the [NiFe]-hydrogenase contains a pentacoordinated iron atom; therefore, a vacant coordination site is available for substrate binding. Nonetheless, most organometallic models of the [NiFe]-hydrogenase failed to reproduce this key feature of the active site. In order to rationalize such behavior, quantum chemical calculations were carried out on a series of [Fe(CN)2(CO)n(dithiolate)]2–n= 1,2 complexes, where dithiolate denotes the ligands (CF3)2C2S22–, (CO2Me)2C2S22–, Ph2C2S22–, C6Cl2H2S22–, C6H4S22–, C2H4S22–, and C3H6S22–. Structural and energetic features are discussed, and a topological analysis based on two scalar fields, the one-electron density and the electron localization function (ELF), has been attempted to describe the nature of the metal–ligand bonds. The present approach contributes to better understand the ability of noninnocent dithiolene to strongly labilize one CO whereas innocent dithiolate cannot. The methodology developed throughout the paper could be useful in the field of the CO-releasing molecules.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/zmass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Collision energy is a key parameter determining the information content of beam-type collision induced dissociation tandem mass spectrometry (MS/MS) spectra, and its optimal choice largely affects successful peptide and protein identification in MS-based proteomics. For an MS/MS spectrum, quality of peptide match based on sequence database search, often characterized in terms of a single score, is a complex function of spectrum characteristics, and its collision energy dependence has remained largely unexplored. We carried out electrospray ionization-quadrupole-time of flight (ESI-Q-TOF)-MS/MS measurements on 2807 peptides from tryptic digests of HeLa and E. coliat 21 different collision energies. Agglomerative clustering of the resulting Mascot score versus energy curves revealed that only few of them display a single, well-defined maximum; rather, they feature either a broad plateau or two clear peaks. Nonlinear least-squares fitting of one or two Gaussian functions allowed the characteristic energies to be determined. We found that the double peaks and the plateaus in Mascot score can be associated with the different energy dependence of b- and y-type fragment ion intensities. We determined that the energies for optimum Mascot scores follow separate linear trends for the unimodal and bimodal cases with rather large residual variance even after differences in proton mobility are taken into account. This leaves room for experiment optimization and points to the possible influence of further factors beyond m/z.

Density functional theoretical methods, including several basis sets and two functionals, were used to collect information on the structure and energetic parameters of poly(ethylene glycol) (PEG), also referred to as poly(ethylene oxide) (PEO), coordinated by alkali metal ions. The oligomer chain is found to form a spiral around the alkali cation, which grows to roughly two helical turns when the oligomer size increases to about the decamer for each alkali ion. Above this size, the additional monomer units do not build the spiral further for Li+and Na+; instead, they form less organized segments outside or next to the initial spiral. The distance of the first layer of coordinating O atoms from the alkali cation is 1.9–2.15 Å for Li+, 2.3–2.5 Å for Na+, 2.75–3.2 Å for K+and 3.5–3.8 Å for Cs+complexes. The number of O atoms in the innermost shell is five, six, seven and eleven for Li+, Na+, K+and Cs+. The collision cross sections with He increase linearly with the oligomer to a very good approximation. No sign of leaning towards the 2/3 power dependence characterizing spherical particles is observed. The binding energy of the cation to the oligomer increases up to polymerization degree of about 10, where it levels off for each alkali-metal ion, indicating that this is approximately the limit of the oligomer size that can be influenced by the alkali cation. The binding energy–degree of polymerization curves are remarkably parallel for the four cations. The limiting binding energy at large polymerization degrees is about 544 kJ mol−1, 460 kJ mol−1, 356 kJ mol−1and 314 kJ mol−1for Li, Na, K and Cs, respectively. The geometrical features are compared with the X-ray and neutron diffraction data on crystalline and amorphous phases of conducting polymers formed by alkali-metal salts and PEG. The implications of the observations concerning collision cross sections and binding energies to ion mobility spectroscopy and mass spectrometry are discussed.

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