The enantioselectivity of amine-catalyzed reactions of aldehydes with electrophiles is often explained by simple steric arguments emphasizing the role of the bulky group of the catalyst that prevents the approach of the electrophile from the more hindered side. This standard steric shielding model has recently been challenged by the discovery of stable downstream intermediates, which appear to be involved in the rate-determining step of the catalytic cycle. The alternative model, referred to as the Curtin-Hammett scenario of stereocontrol, assumes that the enantioselectivity is related to the stability and reactivity of downstream intermediates. In our present computational study, we examine the two key processes of the catalytic Michael reaction between propanal and β-nitrostyrene that are relevant to the proposed stereoselectivity models, namely the C-C bond formation and the protonation steps. The free energy profiles obtained for the pathways leading to the enantiomeric products suggest that the rate- and stereodetermining steps are not identical as implied by the previous models. The stereoselectivity can be primarily controlled by C-C bond formation even though the reaction rate is dictated by the protonation step. This kinetic scheme is consistent with all observations of experimental mechanistic studies including those of mass spectrometric back reaction screening experiments, which reveal a mismatch between the stereoselectivity of the back and the forward reactions.
The enzyme CTP:phosphocholine cytidylyltransferase (CCT) is essential in the lipid biosynthesis of Plasmodia (Haemosporida), presenting a promising antimalarial target. Here, we identified two independent gene duplication events of CCT within Apicomplexa and characterized a truncated construct of Plasmodium falciparum CCT that forms a dimer resembling the molecular architecture of CCT enzymes from other sources. Based on biophysical and enzyme kinetics methods, our data show that the CDPcholine product of the CCT enzymatic reaction binds to the enzyme considerably stronger than either substrate (CTP or choline phosphate). Interestingly, in the presence of Mg 2+ , considered to be a cofactor of the enzyme, the binding of the CTP substrate is attenuated by a factor of 5. The weaker binding of CTP:Mg 2+ , similarly to the related enzyme family of aminoacyl tRNA synthetases, suggests that, with lack of Mg 2+ , positively charged side chain(s) of CCT may contribute to CTP accommodation. Thermodynamic investigations by isothermal titration calorimetry and fluorescent spectroscopy studies indicate that accommodation of the choline phosphate moiety in the CCT active site is different when it appears on its own as one of the substrates or when it is linked to the CDP-choline product. A tryptophan residue within the active site is identified as a useful internal fluorescence sensor of enzyme-ligand binding. Results indicate that the catalytic mechanism of Plasmodium falciparum CCT may involve conformational changes affecting the choline subsite of the enzyme. Structured digital abstract• PfCCT MDK and PfCCT MDK bind by mass spectrometry studies of complexes (View interaction)• PfCCT MDK and PfCCT MDK bind by comigration in gel electrophoresis (View interaction)• PfCCT MDK and PfCCT MDK bind by molecular sieving (View interaction)
Mass spectrometric analysis of glycopeptides is an emerging strategy for analysis of glycosylation patterns. Here we present an approach using energy resolved collision induced decomposition (CID) spectra to determine structural features of glycopeptides. Fragmentation of multiply protonated glycopeptides proceeds by a series of competing charge separation processes by cleavage of a glycosidic bond, each producing two charged products: a singly charged, "B" type sugar (oxonium) ion, and a complementary high mass fragment. Energy requirements (activation energies) of these processes are similar to each other, and are far less, than that required for peptide fragmentation. At higher collision energies these first generation products fragment further, yielding a complex fragmentation pattern. Analysis of low energy spectra (those corresponding to ca. 50% survival yield) are straightforward; the ions observed correspond to structural features present in the oligosaccharide, and are not complicated by consecutive reactions. This makes it feasible to identify and distinguish antenna-and core-fucosylated isomers; antenna fucosylation usually suggests presence of the Lewis-X antigen. In general, analysis of the triply protonated molecules are most advantageous, where neutral losses and monosaccharide oxonium ion formation are less abundant.
Quadrupole time‐of‐flight (QTof) collision‐induced dissociation (CID) and Orbitrap higher‐energy collisional dissociation (HCD) are the most commonly used fragmentation techniques in mass spectrometry‐based proteomics workflows. The information content of the MS/MS spectra is first and foremost determined by the applied collision energy. How can we set up the two instrument types to achieve maximum transferability? To answer this question, we compared MS/MS spectra obtained on a Bruker QTof CID and a Thermo Q‐Exactive Focus Orbitrap HCD instrument as a function of collision energy using the similarity index. Results show that with a few eV lower collision energy setting on HCD (Orbitrap‐specific CID) than on QTof CID, nearly identical MS/MS spectra can be obtained for leucine enkephalin pentapeptide standard, for selected +2 and +3 enolase tryptic peptides and for a large number of peptides in a HeLa protein digest. The Bruker QTof was able to produce colder ions, which may be significant to study inherently labile compounds. Further, we examined energy dependence of peptide identification confidence, as characterized by Mascot scores, on the HeLa peptides. In line with earlier QTof results, this dependence shows one or two maxima (unimodal or bimodal behavior) on Orbitrap. The fraction of bimodal peptides is lower on Orbitrap. Optimal energies as a function of m/z show a similar linear trend on both instruments, which suggests that with appropriate collision energy adjustment, matching conditions for proteomics can be achieved. Data have been deposited in the MassIVE repository (MSV000086434).
Mass‐spectrometry coupled to liquid chromatography is an indispensable tool in the field of proteomics. In the last decades, more and more complex and diverse biochemical and biomedical questions have arisen. Problems to be solved involve protein identification, quantitative analysis, screening of low abundance modifications, handling matrix effect, and concentrations differing by orders of magnitude. This led the development of more tailored protocols and problem centered proteomics workflows, including advanced choice of experimental parameters. In the most widespread bottom‐up approach, the choice of collision energy in tandem mass spectrometric experiments has outstanding role. This review presents the collision energy optimization strategies in the field of proteomics which can help fully exploit the potential of MS based proteomics techniques. A systematic collection of use case studies is then presented to serve as a starting point for related further scientific work. Finally, this article discusses the issue of comparing results from different studies or obtained on different instruments, and it gives some hints on methodology transfer between laboratories based on measurement of reference species.
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 concept of using disulfides as an oxidant for Cu(i) is introduced as part of a Cu-catalyzed process leading to the formation of benzothiazole from an iminodisulfide under an inert atmosphere.
Electrospray ionization of dilute aqueous solutions of copper(II) chloride-containing traces of pyridine (py) as well as ammonia permits the generation of the gaseous ions (py)(2) Cu(+) and (py)(2) CuCl(+) , of which the latter is a formal copper(II) compound, whereas the former contains copper(I). Collision-induced dissociation of the mass-selected ions in an ion-trap mass spectrometer (IT-MS) leads to a loss of pyridine from (py)(2) Cu(+) , whereas an expulsion of atomic chlorine largely prevails for (py)(2) CuCl(+) . Theoretical studies using density functional theory predict a bond dissociation energy (BDE) of BDE[(py)(2) Cu(+) -Cl] = 125 kJ mol(-1) , whereas the pyridine ligand is bound significantly stronger, i.e. BDE[(py)CuCl(+) -py] = 194 kJ mol(-1) and BDE[(py)Cu(+) -py] = 242 kJ mol(-1) . The results are discussed with regard to the influence of the solvation on the stability of the Cu(I) /Cu(II) redox couple.
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