This study explores the potential of a novel electrospray-based method, termed gas-phase electrophoretic mobility molecular analysis (GEMMA), allowing the molecular mass determination of peptides, proteins and noncovalent biocomplexes up to 2 MDa (dimer of immunglobulin M). The macromolecular ions were formed by nano electrospray ionization (ESI) in the 'cone jet' mode. The multiple charged state of the monodisperse droplets/ions generated was reduced by means of bipolar ionized air (generated by an alpha-particle source) to yield exclusively singly charged positive and negative ions as well as neutrals. These ions are separated subsequently at atmospheric pressure using a nano differential mobility analyzer according to their electrophoretic mobility in air. Finally, the ions are detected using a standard condensation particle counter. Data were expressed as electrophoretic mobility diameters by applying the Millikan equation. The measured electrophoretic mobility diameters, or Millikan diameters, of 32 well-defined proteins were plotted against their molecular weights in the range 3.5 to 1920 kDa and exhibited an excellent squared correlation coefficient (r(2) = 0.999). This finding allowed the exact molecular weight determination of large (glyco)proteins and noncovalent biocomplexes by means of this new technique with a mass accuracy of +/-5.6% up to 2 MDa at the femtomole level. From the molecular masses of the weakly bound, large protein complexes thus obtained, the binding stoichiometry of the intact complex and the complex stability as a function of pH, for example, can be derived. Examples of specific protein complexes, such as the avidin or catalase homo-tetramer, are used to illustrate the potential of the technique for characterization of high-mass biospecific complexes. A discussion of current and future applications of charge-reduced nano ESI GEMMA, such as chemical reaction monitoring (reduction process of immunglobulin G) or size determination of an intact virus, a supramolecular complex, and monitoring of partial dissociation of a human rhinoviruses, is provided.

This study represents the first example an environmentally benign, sustainable, and practical synthesis of substituted quinolines and pyrimidines using combinations of 2-aminobenzyl alcohols and alcohols as well as benzamidine and two different alcohols, respectively. These reactions proceed with high atom efficiency via a sequence of dehydrogenation and condensation steps that give rise to selective C-C and C-N bond formations, thereby releasing 2 equiv of hydrogen and water. A hydride Mn(I) PNP pincer complex recently developed in our laboratory catalyzes this process in a very efficient way. A total of 15 different quinolines and 14 different pyrimidines were synthesized in isolated yields of up to 91 and 90%, respectively.

We used a proteomic approach to identify constitutively formed extracellular proteins of Hypocrea atroviridis (Trichoderma atroviride), a known biocontrol agent. The fungus was cultivated on glucose and the secretome was examined by two‐dimensional gel electrophoresis. The two predominant spots were identified by MALDI MS utilizing peptide mass fingerprints and amino acid sequence tags obtained by postsource decay and/or high‐energy collision‐induced dissociation (MS/MS) experiments, and turned out to be the same protein (12 629 Da as determined with MS, pI 5.5–5.7), probably representing the monomer and the dimer. The corresponding gene was subsequently cloned from H. atroviridis and named epl1 (eliciting plant response‐like), because it encodes a protein that exhibits high similarity to the cerato‐platanin family, which comprises proteins such as cerato‐platanin from Ceratocystis fimbriata f. sp. platani and Snodprot1 of Phaeosphaeria nodorum, which have been reported to be involved in plant pathogenesis and elicitation of plant defense responses. Additionally, based on the similarity of the N‐terminus to that of H. atroviridis Epl1, we conclude that a previously identified 18 kDa plant response elicitor isolated from T. virens is an ortholog of epl1. Our results showed that epl1 transcript was present under all growth conditions tested, which included the carbon sources glucose, glycerol, l‐arabinose, d‐xylose, colloidal chitin and cell walls of the plant pathogen Rhizoctonia solani, and also plate confrontation assays with R. solani. Epl1 transcript could even be detected under osmotic stress, and carbon and nitrogen starvation.

We have prepared and structurally characterized a new class of Fe(II) PNP pincer hydride complexes [Fe(PNP-iPr)(H)(CO)(L)]n (L = Br–, CH3CN, pyridine, PMe3, SCN–, CO, BH4–; n = 0, +1) based on the 2,6-diaminopyridine scaffold where the PiPr2 moieties of the PNP ligand are connected to the pyridine ring via NH and/or NMe spacers. Complexes [Fe(PNP-iPr)(H)(CO)(L)]n with labile ligands (L = Br–, CH3CN, BH4–) and NH spacers are efficient catalysts for the hydrogenation of both ketones and aldehydes to alcohols under mild conditions, while those containing inert ligands (L = pyridine, PMe3, SCN–, CO) are catalytically inactive. Interestingly, complex [Fe(PNPMe-iPr)(H)(CO)(Br)], featuring NMe spacers, is an efficient catalyst for the chemoselective hydrogenation of aldehydes. The first type of complexes involves deprotonation of the PNP ligand as well as heterolytic dihydrogen cleavage via metal-alkoxide cooperation, but no reversible aromatization/deprotonation of the PNP ligand. In the case of the N-methylated complex the mechanism remains unclear, but obviously does not allow bifunctional activation of dihydrogen. The experimental results complemented by DFT calculations strongly support an insertion of the C=O bond of the carbonyl compound into the Fe–H bond.

Herein, we describe an efficient coupling of alcohols and amines catalyzed by well-defined isoelectronic hydride Mn(I) and Fe(II) complexes, which are stabilized by a PNP ligand based on the 2,6-diaminopyridine scaffold. This reaction is an environmentally benign process implementing inexpensive, earth-abundant non-precious metal catalysts, and is based on the acceptorless alcohol dehydrogenation concept. A range of alcohols and amines including both aromatic and aliphatic substrates were efficiently converted in good to excellent isolated yields. Although in the case of Mn selectively imines were obtained, with Fe-exclusively monoalkylated amines were formed. These reactions proceed under base-free conditions and required the addition of molecular sieves.

Triacylglycerols were analyzed as cationized species (Li ϩ , Na ϩ , K ϩ ) by high-energy CID at 20 keV collisions utilizing MALDI-TOF/RTOF mass spectrometry. Precursor ions, based on [M ϩ Li] ϩ -adduct ions exhibited incomplete fragmentation in the high and low m/z region whereas [M ϩ K] ϩ -adducts did not show useful fragmentation. Only sodiated precursor ions yielded product ion spectra with structurally diagnostic product ions across the whole m/z range. The high m/z region of the CID spectra is dominated by abundant charge-remote fragmentation of the fatty acid substituents. In favorable cases also positions of double bonds or of hydroxy groups of the fatty acid alkyl chains could be determined. A-type product ions represent the end products of these charge-remote fragmentations. B-and C-type product ions yield the fatty acid composition of individual triacylglycerol species based on loss of either one neutral fatty acid or one sodium carboxylate residue, respectively. Product ions allowing fatty acid substituent positional determination were present in the low m/z range enabling identification of either the sn-1/sn-3 substituents (E-, F-, and G-type ions) or the sn-2 substituent (J-type ion). These findings were demonstrated with synthetic triacylglycerols and plant oils such as cocoa butter, olive oil, and castor bean oil. Typical features of 20 keV CID spectra of sodiated triacylglycerols obtained by MALDI-TOF/RTOF MS were an even distribution of product ions over the entire m/z range and a mass accuracy of Ϯ0.1 to 0.

Multistage mass spectrometry, as implemented using low-energy collision-induced dissociation (CID) analysis in three-dimensional (3D) quadrupole ion traps (QITs), has become a powerful tool for the investigation of protein glycosylation. In addition to the well-known combination of QITs with electrospray ionization (ESI), also a matrix-assisted laser desorption/ionization--quadrupole ion trap--reflectron time-of-flight (MALDI-QIT-rTOF) mass spectrometer has recently become available. This study systematically investigates the differences between these types of instrument, as applied to characterization of glycopeptides from human antithrombin. The glycopeptides were obtained by tryptic digestion followed by lectin-affinity purification. Some significant differences between the ESI-QIT and MALDI-QIT-rTOF approaches appeared, most of them are causally related to the desorption/ionization process. The combination of a vacuum MALDI source with an ion-trap analyzer accentuates some characteristic differences between MALDI and ESI due the longer time frame needed for the trapping process. In contrast to ESI, MALDI generated ions that exhibited considerable metastable fragmentation during trapping. The long time span of the QIT process (ms range) compared with that for conventional rTOF experiments (micros range) significantly magnified the extent of this metastable fragmentation. With the investigated glycopeptides, a complete depletion of the terminal sialic acids of the glycopeptides as well as a variety of other fragment ions was already found in the MS1 spectra from the MALDI-QIT-rTOF instrument. The positive ion low-energy CID spectra (MS2) of the selected glycopeptides obtained using the two different QIT equipped instruments were found to be quite similar. In both approaches, fragmentation of the glycan and peptide structures occurred sequentially, allowing unambiguous sequence determination. In the case of ESI-QIT-MS, fragmentation of the glycan structure occurred at the MS2 stage and fragmentation of the peptide structure was obtained only at the MS3 stage, which indicates the necessity of multistage CID experiments for complete structure elucidation. The MALDI-QIT-rTOF instrument yielded both kinds of fragments at the MS2 stage but without mutual interference.

The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross-linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids. Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L-tyrosine and the subsequent oxidative cross-linking of the modified tyrosine residues (catalyzed by a cytochrome P-450), leading to soluble dityrosine-containing spore wall precursors. We isolated, purified and characterized the dityrosine-containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall. Chemical and spectroscopic analyses showed that this precursor is N,N'-bisformyl dityrosine. In addition, we identified a tyrosine-containing spore wall precursor as N-formyl tyrosine. The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g. by in vitro systems. A dityrosine-containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N-formyl dityrosine. Mature spore walls contain significant amounts of N-formyl dityrosine and N,N'-bisformyl dityrosine. This supports the assumption that the dityrosine-containing macromolecular network on the spore surface has an unusual, non-peptidic structure.