Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa. Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta, a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans, which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.fungal genomics ͉ fungal proteomics ͉ seborrheic dermatitis ͉ skin ͉ fungal mating

A method has been developed for de novo peptide sequencing using matrix-assisted laser desorption ionization mass spectrometry. This method will facilitate biological studies that require rapid determination of peptide or protein sequences, e.g., determination of posttranslational modifications, identification of active compounds isolated from combinatorial peptide libraries, and the selective identification of proteins as part of proteome studies. The method involves fast, one-step addition of a sulfonic acid group to the N terminus of tryptic peptides followed by acquisition of postsource decay (PSD) fragment ion spectra. The derivatives are designed to promote efficient charge site-initiated fragmentation of the backbone amide bonds and to selectively enhance the detection of a single fragment ion series that contains the C terminus of the molecule (y-ions). The overall method has been applied to pmol quantities of peptides. The resulting PSD fragment ion spectra often exhibit uninterrupted sequences of 20 or more amino acid residues. However, fragmentation efficiency decreases considerably at amide bonds on the C-terminal side of Pro. The spectra are simple enough that de novo sequence tagging is routine. The technique has been successfully applied to peptide mixtures, to high-mass peptides (up to 3,600 Da) and to the unambiguous identification of proteins isolated from two-dimensional gel electrophoresis. The PSD spectra of these derivatized peptides often allow far more selective protein sequence database searches than those obtained from the spectra of native peptides.Knowledge of protein sequences is fundamentally important for understanding many physiological processes at the molecular level (1). Tandem mass spectrometry has become an increasingly essential tool for protein and peptide sequencing because of its speed, sensitivity, and applicability to complex mixtures (2). Recently, postsource decay (PSD) matrix-assisted laser desorption ionization (MALDI) mass spectrometry was developed for high-sensitivity peptide sequencing applications (3-8). Subpicomole limits of analysis were reported as a result of the high yield of fragment ions and the high ion transmission inherent with time-of-flight mass spectrometry (5). Kaufmann et al. (5) also noted several problems associated with PSD MALDI sequencing of peptides, including the complexity of the resulting fragmentation patterns and the lack of computer algorithms capable of interpreting the complex spectra. The recent incorporation of delayed extraction (DE) (9, 10), a technique designed to improve precursor-ion mass resolution and mass-measurement accuracy, reduced the rate of PSD fragmentation by at least an order of magnitude. As a result, many low-intensity precursor ions obtained by DE MALDI do not produce enough PSD fragmentation to allow derivation of even short sequence tags (7).Positively charged derivatives have been used in desorption mass spectrometry for many years to improve sensitivity by enhancing ionization efficiencies (11, 12...

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A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20-50 mg of 95-99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4-8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.

A new fast, simple, and water-compatible derivatization strategy improves protein identification.

Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology.

Solid-phase sulfonation of tryptic peptides adsorbed to C 18 mZipTips 2 has been carried out to facilitate de novo sequencing with mass spectrometry. Peptides are reacted with the sulfonation reagent while they are still adsorbed to the solid phase. Excess reagent passes through the ZipTip 2 to waste. Washing the products before subsequent elution from the mini-column also affords sample cleanup prior to analysis. Near quantitative N-terminal sulfonation can be achieved reliably at room temperature in only a few seconds. The method has been applied successfully to model peptides and to solution or in-gel digests of proteins. Current sequencing limits are about 100 fmol of protein.Multiplexed sample sulfonation reactions have been carried out with a manual 8-position micropipettor or using centrifugal force to reliably pass reagents and wash solutions over sampleloaded ZipTips 2 . With multiplexing, overall preparation times have been reduced to about 1 min per sample. The solid-phase format facilitates efficient use of precious digest samples by enabling them to be recovered from the matrix-assisted laser desorption/ionization (MALDI) sample stage after mass fingerprinting, derivatized and re-analyzed by MALDI postsource decay mass spectrometry. Copyright # 2002 John Wiley & Sons, Ltd.Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is widely used for the identification of proteins that have been separated by 1D-or 2D-gel electrophoresis. Typically, gel-separated proteins are digested with trypsin, 1 and the resulting mixtures of tryptic peptides are analyzed by MALDI-MS. The measured masses of the tryptic peptides are compared to theoretical peptide masses calculated for each protein in various sequence databases. The resulting lists of candidate proteins that most closely match the input data are rank ordered using various scoring algorithms. 2±6 In favorable cases definitive protein identifications are possible using this simple`peptide mass fingerprinting' approach. 7 Unfortunately, peptide mass fingerprinting can also lead to ambiguous protein identifications. This may result because of insufficient protein sequence coverage. Limited sequence coverage is often observed at low analyte levels because of poor recovery of tryptic peptides from the gels and because of variable peptide response under MALDI conditions. It is also known that Lysterminated tryptic peptides are less responsive than Arg-terminated tryptic peptides. 8 Furthermore, co-migrating proteins may produce complex mixtures of tryptic peptides that are difficult to de-convolute and interpret. Finally, the sequences of some analyte proteins are novel, so their experimentally observed tryptic mass fingerprints cannot be matched with confidence to any entries in the databases.Complementary tandem mass spectrometry methods are often used to sequence tryptic peptides to resolve ambiguous protein identifications resulting from MALDI mass fingerprinting. The discriminating power of peptide sequence data is extremely high. Defin...