In this study we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) interface with a porous tip as the nanospray emitter for use in peptide analysis. A positively charged capillary coating and 0.1% formic acid as background electrolyte were used for separation upstream from mass spectrometry characterization. The influence of the distance between emitter tip and MS inlet, ESI voltage applied, and of the electroosmotic flow (EOF) on electrospray performance and efficiency of the system was investigated in detail. Under optimized conditions, less than 30 amol of a model peptide (angiotensin I) was required for a detection in the base peak electropherogram and positive identification via tandem MS. Three different cationic capillary coatings were investigated for stability, resolution, and EOF and were found to enable reproducible separations by CE-ESI-MS. After optimizing MS settings, the effectiveness of the CE-ESI-MS method developed was compared with a state-of-the-art nano-liquid chromatography (LC)-ESI-MS method by analyzing Arg-C-digested rat testis linker histones with both systems. With comparable amounts of sample applied, the number of identified peptides increased by more than 60% when using CE-ESI-MS. We found that low molecular mass peptides (below 1400 Da) were preferentially identified by CE-ESI-MS, since this group of peptides poorly interacted with the reversed-phase material in the nano-LC system. Finally, total analysis time in LC-ESI-MS for three runs including equilibration was nearly 4 times longer than that of CE-ESI-MS: 246 versus 66 min.

Methylation of the N-terminal region of histones was first described more than 35 years ago, but its biological significance has remained unclear. Proposed functions range from transcriptional regulation to the higher order packing of chromatin in progress of mitotic condensation. Primarily because of the recent discovery of the SET domain-depending H3-specific histone methyltransferases SUV39H1 and Suv39h1, which selectively methylate lysine 9 of the H3 N terminus, this posttranslational modification has regained scientific interest. In the past, investigations concerning the biological significance of histone methylation were largely limited because of a lack of simple and sensitive analytical procedures for detecting this modification. The present work investigated the methylation pattern of histone H4 both in different mammalian organs of various ages and in cell lines by applying mass spectrometric analysis and a newly developed hydrophilic-interaction liquid chromatographic method enabling the simultaneous separation of methylated and acetylated forms, which obviates the need to work with radioactive materials. In rat kidney and liver the dimethylated lysine 20 was found to be the main methylation product, whereas the monomethyl derivative was present in much smaller amounts. In addition, for the first time a trimethylated form of lysine 20 of H4 was found in mammalian tissue. A significant increase in this trimethylated histone H4 was detected in organs of animals older than 30 days, whereas the amounts of mono-and dimethylated forms did not essentially change in organs from young (10 days old) or old animals (30 and 450 days old). Trimethylated H4 was also detected in transformed cells; although it was present in only trace amounts in logarithmically growing cells, we found an increase in trimethylated lysine 20 in cells in the stationary phase.In vivo methylation of the side chains of specific lysines, histidines, and arginines in proteins is a very common phenomenon in nature involving numerous classes of proteins in both prokaryotic and eukaryotic cells (1, 2). During the last several years, studies on the methylation of proteins have yielded many important observations. While these studies were under way, it was generally realized that protein methylation is far more complex and has more ramifications than originally assumed.Methylation is also a well known posttranslational modification reaction of histone proteins on lysine and/or arginine residues with a site selectivity for lysine methylation at specific positions in the N termini of histones H3 and H4. In combination with other posttranslational modifications, i.e. acetylation and phosphorylation, methylation seems to play a significant role in regulating nuclear functions. Thus, it has been suggested that distinct combinations of covalent histone modifications, also referred to as "histone code," provide a specific mark on the hydrophilic histone tails, which, when read by other proteins, cause specific downstream events finally inducing transi...

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr 137 and Thr 154 , the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr 10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serineor threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.The nucleosome core, which consists of 146 bp of DNA wrapped 1.75 times around an octamer of core histones, represents the fundamental subunit of chromatin (for review, see Ref. 1). The H1 or linker histones are associated with the core histone-DNA complex and with the linker DNA between adjacent nucleosomes. Histone H1 is phosphorylated in a cell cycle-dependent manner: levels of H1 phosphorylation are usually lowest in the G 1 phase and rise continuously during S and G 2 . The M phase, where chromatin is highly condensed, shows the maximum number of phosphorylated sites. The individual H1 subtypes, however, differ in their degree of phosphorylation during the cell cycle (2, 3). A number of studies indicate that H1 phosphorylation is more likely involved in chromatin decondensation than in condensation (4). H1 phosphorylation seems to destabilize chromatin structure, thus weakening its binding to DNA. This decondensation of chromatin may give the DNA access to factors involved in transcription and replication in G 1 and S as well as to condensing factors active during mitosis (5). Recent studies demonstrate that H1 phosphorylation regulates specific gene expression in vivo and that it acts by mimicking the partial removal of H1 (6).The H1 histones consist of a globular central region flanked by short N...

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

Iron is an essential metal for virtually all organisms. Iron acquisition is well characterized for various organisms, whereas intracellular iron distribution is poorly understood. In contrast to bacteria, plants, and animals, most fungi lack ferritin-mediated iron storage but possess an intracellular siderophore shown to be involved in iron storage. Here we demonstrate that deficiency in the intracellular siderophore ferricrocin causes iron starvation in conidia of Aspergillus fumigatus, demonstrating that ferricrocin is also involved in intra-and transcellular iron distribution. Thus, ferricrocin represents the first intracellular iron transporter identified in any organism.Virtually all organisms require iron as an indispensable cofactor for various metabolic processes, including electron transport and redox reactions. Excess or incorrect storage of iron, however, is toxic, as this metal has the capacity to reinforce the production of reactive oxygen species. Therefore, organisms have evolved precisely regulated iron acquisition systems, which are well characterized in numerous prokaryotes and eukaryotes. In contrast, mechanisms for intracellular distribution of acquired iron are poorly understood. We investigated these mechanisms in Aspergillus fumigatus, a typical saprophytic ascomycete, which has become the most common airborne fungal pathogen of humans, causing life-threatening invasive disease especially in immunocompromised patients (16). A. fumigatus employs four siderophores (low-molecularmass ferric iron chelators) for maintenance of iron homeostasis (5): it excretes two siderophores for solubilization and uptake of iron, and it accumulates two structurally different siderophores, ferricrocin (FC) and its hydroxylated derivative hydroxy-FC, within hyphae and conidia, respectively. Both intracellular siderophores are believed to be involved in intracellular iron storage. The siderophore system became a matter of particular interest as it represents an attractive target for antifungal therapy due to its requirement for virulence of A. fumigatus and its lack in mammalian hosts (12, 13). Recently, extra-and intracellular siderophores have also been implicated in the phytopathogenicity of various ascomycetes (6, 11). Here we demonstrate that intracellular siderophores are also involved in the intracellular long-distance distribution of iron.Extracellular siderophores are utilized by most fungi and bacteria and some plants, whereas intracellular siderophores are found exclusively in fungi, which in contrast to bacteria, plants, and animals lack ferritin-mediated iron storage (5). The function of intracellular siderophores has been studied in the most detail with Aspergillus nidulans and A. fumigatus (5). Several lines of evidence support a role for FC and hydroxy-FC in iron storage in these fungi. (i) FC accumulation increases under conditions of intracellular iron excess (4, 9, 14); (ii) FC deficiency reduces the iron content of conidia by 34% and 76% in A. nidulans and A. fumigatus, respectively (4, 13); ...

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BACKGROUND:The specific forms of pro-B-type natriuretic peptide (proBNP) that occur in human blood are not yet clear. We demonstrated the presence of several proBNP forms in human plasma with a new affinity chromatography method that can be used in combination with nano-liquid chromatography electrospray ionization tandem mass spectrometry (nano-LC-ESI-MS/MS).

Alkylglycerol monooxygenase (glyceryl-ether monooxygenase, EC 1.14.16.5) is the only enzyme known to cleave the O-alkyl bond of ether lipids which are essential components of brain membranes, protect the eye from cataract, interfere or mediate signalling processes, and are required for spermatogenesis. Along with phenylalanine hydroxylase, tyrosine hydroxylase, tryptophan hydroxylase, and nitric oxide synthase, alkylglycerol monooxygenase is one of five known enzymatic reactions which depend on tetrahydrobiopterin. Although first described in 1964, no sequence had been assigned to this enzyme so far since it lost activity upon protein purification attempts. A functional library screen using pools of plasmids of a rat liver expression library transfected to CHO cells was also unsuccessful. We therefore selected human candidate genes by bioinformatic approaches and by proteomic analysis of partially purified enzyme and tested alkylglycerol monooxygenase activity in CHO cells transfected with expression plasmids. Transmembrane protein 195, a predicted membrane protein with unassigned function which occurs in bilateral animals, was found to encode for tetrahydrobiopterin-dependent alkylglycerol monooxygenase. This sequence assignment was confirmed by injection of transmembrane protein 195 cRNA into Xenopus laevis oocytes. Transmembrane protein 195 shows no sequence homology to aromatic amino acid hydroxylases or nitric oxide synthases, but contains the fatty acid hydroxylase motif. This motif is found in enzymes which contain a diiron center and which carry out hydroxylations of lipids at aliphatic carbon atoms like alkylglycerol monooxygenase. This sequence assignment suggests that alkylglycerol monooxygenase forms a distinct third group among tetrahydrobiopterin-dependent enzymes. EC 1.14.16.5 | glyceryl-ether monooxygenase | nitric oxide synthase | phenylalanine hydroxylase | transmembrane protein 195