Martin P. Lacey

Dandruff-associated Malassezia genomes reveal convergent and divergent virulence traits shared with plant and human fungal pathogens

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.

Derivatization procedures to facilitate de novo sequencing of lysine‐terminated tryptic peptides using postsource decay matrix‐assisted laser desorption/ionization mass spectrometry

Guanidination of the epsilon-amino group of lysine-terminated tryptic peptides can be accomplished selectively in one step with O-methylisourea hydrogen sulfate. This reaction converts lysine residues into more basic homoarginine residues. It also protects the epsilon-amino groups against unwanted reaction with sulfonation reagents, which can then be used to selectively modify the N-termini of tryptic peptides. The combined reactions convert lysine-terminated tryptic peptides into modified peptides that are suitable for de novo sequencing by postsource decay matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The guanidination reaction is very pH dependent. Product yields and reaction kinetics were studied in aqueous solution using either NaOH or diisopropylethylamine as the base. Methods are reported for derivatizing and sequencing lysine-terminated tryptic peptides at low pmole levels. The postsource decay (PSD) MALDI tandem mass spectra of a model peptide (VGGYGYGAK), the homoarginine analog and the sulfonated homoarginine analog are compared. These spectra show the influence that each chemical modification has on the peptide fragmentation pattern. Finally, we demonstrate that definitive protein identifications can be achieved by PSD MALDI sequencing of derivatized peptides obtained from solution digests of model proteins and from in-gel digests of 2D-gel separated proteins.

Tandem mass spectrometry methods for definitive protein identification in proteomics research.

Optimized procedures have been developed for the addition of sulfonic acid groups to the N‐termini of low‐level peptides. These procedures have been applied to peptides produced by tryptic digestion of proteins that have been separated by two‐dimensional (2‐D) gel electrophoresis. The derivatized peptides were sequenced using matrix‐assisted laser desorption/ionization (MALDI) post‐source decay (PSD) and electrospray ionization‐tandem mass spectrometry methods. Reliable PSD sequencing results have been obtained starting with sub‐picomole quantities of protein. We estimate that the current PSD sequencing limit is about 300 fmol of protein in the gel. The PSD mass spectra of the derivatized peptides usually allow much more specific protein sequence database searches than those obtained without derivatization. We also report initial automated electrospray ionization‐tandem mass spectrometry sequencing of these novel peptide derivatives. Both types of tandem mass spectra provide predictable fragmentation patterns for arginine‐terminated peptides. The spectra are easily interpreted de novo, and they facilitate error‐tolerant identification of proteins whose sequences have been entered into databases.

Sequencing of sulfonic acid derivatized peptides by electrospray mass spectrometry

We report the application of nanoelectrospray ionization tandem mass spectrometry (nES-MS/MS) and capillary LC/microelectrospray MS/MS (cLC/&mgr;ES-MS/MS) for sequencing sulfonic acid derivatized tryptic peptides. These derivatives were specifically prepared to facilitate low-energy charge-site-initiated fragmentation of C-terminal arginine-containing peptides, and to enhance the selective detection of a single series of y-type fragment ions. Both singly and doubly protonated peptides were analyzed by MS/MS and the results were compared with those from their derivatized counterparts. Model peptides and peptides from tryptic digests of gel-isolated proteins were analyzed. Derivatized singly protonated peptides fragment in the same way by nES-MS/MS as they do by post-source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD-MALDI-MS). They produce fragment ion spectra dominated by y-ions, and the simplified spectra are readily interpreted de novo. Doubly protonated peptides fragment in much the same way as their non-derivatized doubly protonated counterparts. The fragmentation of doubly protonated derivatives is especially useful for sequencing peptides that possess a proline residue near the N-terminus of the molecule. The singly protonated forms of these proline-containing derivatives often show enhanced fragmentation on the N-terminal side of the proline and considerably reduced fragmentation on the C-terminal side. In addition, sulfonic acid derivatization increases the in-source fragmentation of arginine-containing peptides. This could be useful for sequence verification and sequence tagging for use in single stage mass spectrometry. Copyright 2000 John Wiley & Sons, Ltd.

Proteomic analysis of simulated occupational jet fuel exposure in the lung.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss‐Webster mice exposed 1 h/day for seven days to aerosolized JP‐8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid‐base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Proteomic analysis of the renal effects of simulated occupational jet fuel exposure

We analyzed protein expression in the cytosolic fraction prepared from whole kidneys in male Swiss‐Webster mice exposed 1 h/day for five days to aerosolized JP‐8 jet fuel at a concentration of 1000 mg/m3, simulating military occupational exposure. Kidney cytosol samples were solubilized and separated via large‐scale, high‐resolution two‐dimensional electrophoresis (2‐DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant changes in soluble kidney proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting and related to ultrastructural abnormalities, altered protein processing, metabolic effects, and paradoxical stress protein/detoxification system responses. These results demonstrate a significant but comparatively moderate JP‐8 effect on protein expression in the kidney and provide novel molecular evidence of JP‐8 nephrotoxicity. Human risk is suggested by these data but conclusive assessment awaits a noninvasive search for biomarkers in JP‐8 exposed humans.

Orthogonal acceleration single-pass time-of-flight mass spectrometry for determination of the exact masses of product ions formed in tandem mass spectrometry experiments

A hybrid magnetic sector/orthogonal acceleration single-pass time-of-flight (oaTOF) mass spectrometer has been used to measure the exact masses of product ions formed in high- and low-collision-energy MSMS experiments. Methods for both external and internal calibration of the mass scale have been evaluated. Mass accuracies of ±25 ppm are routinely achieved for product ions having masses greater than 200 Da. Best case mass accuracies of ±10 ppm have been obtained. Mass accuracy at low mass is limited by the current speed of the time-to-digital converter (TDC). Experiments at reduced accelerating voltages suggest that doubling the TDC acquisition rate will increase low-mass accuracy six-fold. A number of applications of exact mass MSMS are presented, including: characterization of a side-reaction product formed in a combinatorial library synthesis, differentiation of isobaric residues in small peptides and a ‘mimicked’ MS3 measurement to confirm the identity of an unusual product ion previously observed in an ion trap MSMS experiment.

The use of MALDI mass spectrometry to characterize synthetic protein conjugates

Abstract MALDI mass spectrometry has been used to study the conjugation of two small molecules to hen egg white lysozyme and human serum albumin. Hapten densities were determined with both proteins. Synthetic peptide mixtures, designed to eliminate the need for subsequent sequencing experiments, were used to assess the potential reactivities of various amino acid side chains in proteins. Selective hydrolysis reactions were also used to differentiate ester and amide conjugates, which are expected to have quite different in vivo stabilities. Finally, MALDI was used to follow the kinetics of protein conjugation reactions, even for reactions having initial rates differing by as much as three orders of magnitude.

Off-line supercritical fluid chromatography/plasma desorption mass spectrometry for simple mixture characterization

SummarySupercritical fluid chromatography/mass spectrometry (SFC/MS) is a powerful tool for the characterization of less volatile, complex mixtures. Unfortunately, on-line SFC/MS instrumentation is relatively expensive, and instrument time is limited. Here we describe an investigation of off-line SFC/plasma desorption mass spectrometry (PDMS) for the characterization of relatively simple mixtures. As an example of the potential of this approach, PD mass spectra of individual oligomers from a mixture of propoxylated dimethylpyrazoles are presented. The mixture was separated by capillary SFC with ultraviolet (UV) absorbance detection, individual peaks were collected on PDMS foils, and the foils were subjected to PDMS analysis. A variety of PDMS foils were compared, and NaI-pretreated, nitrocellulose-coated, aluminized-mylar foils provided the best results for the mixtures investigated in this work. Thirteen nanograms of an ethoxylated standard provided a signal-to-noise ratio of 3. The reproducibility of the overall method, as implemented here with manual collection, was relatively poor. The relative standard deviation of the PDMS response for an alkyl-phenol ethoxylate standard was 26%. Off-line PDMS characterization of a non-UV-absorbing compound, sucrose octamyristate, is also demonstrated. The retention time of the compound of interest is determined during an SFC separation using flame ionization detection. During a second separation, the detector flame is extinguished and the PDMS foil is held above the detector at the appropriate retention time. While limited in its ability to resolve closely eluting components, the off-line approach does provide greater flexibility and potentially greater mass range than conventional on-line SFC/MS. This approach also has the potential to provide the majority of SFC users, who do not have on-line SFC/MS capabilities, with a relatively straightforward method for obtaining mass spectral information on important peaks in an SFC separation.

ACCURATE DETERMINATION OF MOLECULAR MASS FOR PROTEINS ISOLATED FROM POLYACRYLAMIDE GELS

Publisher Summary This chapter describes accurate determination of molecular mass for proteins isolated from polyacrylamide gels. The molecular mass (MM) of a protein provides important information for identification and purification. SDS-PAGE is commonly used to determine protein MM values because of its simplicity and wide applicability. However, the accuracy of MM values determined from SDS-PAGE is limited because they are a function of relative mobility, which is subject to many experimental variables. This is especially true for small (>10 kD) proteins. This chapter describes a method to remove proteins from SDS gels for analysis by time-of-flight MS. Initial efforts were directed toward sample preparation for PDMS. The sample preparation method was extended to matrix-assisted laser desorption MS (MALDI). PDMS gave accurate MM values for peptides and small proteins, and was reasonably insensitive to gel components such as SDS and dyes. The efforts were then extended to MALDI, which indicated that it was more sensitive and accurate than PDMS for small proteins. However, MALDI analysis of larger proteins often resulted in poor quality spectra.