Leo J. de Koning

A structure for the yeast prohibitin complex: Structure prediction and evidence from chemical crosslinking and mass spectrometry

The mitochondrial prohibitin complex consists of two subunits (PHB1 of 32 kD and PHB2 of 34 kD), assembled into a membrane‐associated supercomplex of approximately 1 MD. A chaperone‐like function in holding and assembling newly synthesized mitochondrial polypeptide chains has been proposed. To further elucidate the function of this complex, structural information is necessary. In this study we use chemical crosslinking, connecting lysine side chains, which are well scattered along the sequence. Crosslinked peptides from protease digested prohibitin complexes were identified with mass spectrometry. From these results, spatial restraints for possible protein conformation were obtained. Many interaction sites between PHB1 and PHB2 were found, whereas no homodimeric interactions were observed. Secondary and tertiary structural predictions were made using several algorithms and the models best fitting the spatial restraints were selected for further evaluation. From the structure predictions and the crosslink data we derived a structural building block of one PHB1 and one PHB2 subunit, strongly intertwined along most of their length. The size of the complex implies that approximately 14 of these building blocks are present. Each unit contains a putative transmembrane helix in PHB2. Taken together with the unit building block we postulate a circular palisade‐like arrangement of the building blocks projecting into the intermembrane space.

Selective Enrichment of Azide-Containing Peptides from Complex Mixtures

A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.

Effects of fluconazole on the secretome, the wall proteome, and wall integrity of the clinical fungus Candida albicans

ABSTRACT Fluconazole is a commonly used antifungal drug that inhibits Erg11, a protein responsible for 14α-demethylation during ergosterol synthesis. Consequently, ergosterol is depleted from cellular membranes and replaced by toxic 14α-methylated sterols, which causes increased membrane fluidity and drug permeability. Surface-grown and planktonic cultures of Candida albicans responded similarly to fluconazole at 0.5 mg/liter, showing reduced biomass formation, severely reduced ergosterol levels, and almost complete inhibition of hyphal growth. There was no evidence of cell leakage. Mass spectrometric analysis of the secretome showed that its composition was strongly affected and included 17 fluconazole-specific secretory proteins. Relative quantification of 14N-labeled query walls relative to a reference standard mixture of 15N-labeled yeast and hyphal walls in combination with immunological analysis revealed considerable fluconazole-induced changes in the wall proteome as well. They were, however, similar for both surface-grown and planktonic cultures. Two major trends emerged: (i) decreased incorporation of hypha-associated wall proteins (Als3, Hwp1, and Plb5), consistent with inhibition of hyphal growth, and (ii) increased incorporation of putative wall repair-related proteins (Crh11, Pga4, Phr1, Phr2, Pir1, and Sap9). As exposure to the wall-perturbing drug Congo red led to a similar response, these observations suggested that fluconazole affects the wall. In keeping with this, the resistance of fluconazole-treated cells to wall-perturbing compounds decreased. We propose that fluconazole affects the integrity of both the cellular membranes and the fungal wall and discuss its potential consequences for antifungal therapy. We also present candidate proteins from the secretome for clinical marker development.

Hyphal induction in the human fungal pathogen Candida albicans reveals a characteristic wall protein profile

The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host-pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscoves modified Dulbeccos medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a (15)N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.

High front- and back-end resolution MS/MS in Fourier transform ion cyclotron resonance mass spectrometry☆

Abstract It is demonstrated that each of the molecular ions of cyclopropylbenzene and di- n -propyl sulphide, having the same nominal mass m/z 118 and generated from a nearly 1:1 mixture by 70 eV electron impact in a Fourier transform ion cyclotron resonance mass spectrometer, can be selectively isolated in the cell by ejection of all other ions from the cell. This corresponds to a so-called front-end resolution of primary ion selection for MS/MS experiments in excess of 35 000 at m/z 118. Subsequent collisionally induced dissociation (CID) experiments on each of the mass-selected molecular ions have been performed using helium, introduced via a pulsed valve, as collision gas. A so-called back-end resolution of 205 000–475 000 has been obtained for the CID product ions over the mass range m/z 61–117, while the accuracy of the mass measurements on the corresponding ions is shown to be better than 1 ppm.

Surface stress induces a conserved cell wall stress response in the pathogenic fungus Candida albicans.

ABSTRACT The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using 15N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.

Computer‐assisted mass spectrometric analysis of naturally occurring and artificially introduced cross‐links in proteins and protein complexes

A versatile software tool, virtualmslab, is presented that can perform advanced complex virtual proteomic experiments with mass spectrometric analyses to assist in the characterization of proteins. The virtual experimental results allow rapid, flexible and convenient exploration of sample preparation strategies and are used to generate MS reference databases that can be matched with the real MS data obtained from the equivalent real experiments. Matches between virtual and acquired data reveal the identity and nature of reaction products that may lead to characterization of post‐translational modification patterns, disulfide bond structures, and cross‐linking in proteins or protein complexes. The most important unique feature of this program is the ability to perform multistage experiments in any user‐defined order, thus allowing the researcher to vary experimental approaches that can be conducted in the laboratory. Several features of virtualmslab are demonstrated by mapping both disulfide bonds and artificially introduced protein cross‐links. It is shown that chemical cleavage at aspartate residues in the protease resistant RNase A, followed by tryptic digestion can be optimized so that the rigid protein breaks up into MALDI‐MS detectable fragments, leaving the disulfide bonds intact. We also show the mapping of a number of chemically introduced cross‐links in the NK1 domain of hepatocyte growth factor/scatter factor. The virtualmslab program was used to explore the limitation and potential of mass spectrometry for cross‐link studies of more complex biological assemblies, showing the value of high performance instruments such as a Fourier transform mass spectrometer. The program is freely available upon request.

Segmented fourier transform and its application to fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry: ion abundances and mass measurements

Abstract A method has been developed to analyze quantitatively the frequency components of digitized time-domain signals. This method, named segmented Fourier transform (SEFT), has been applied to Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry where it enables accurate ion abundance and mass measurements to be made, thereby minimizing errors stemming from the sampled nature of the signal, the non-uniformity of the frequency distribution of the excitation power, the spectral peak overlap, and the non-uniformity of the spectral line shapes due to non-uniform relaxation of the individual frequency components. The results satisfy the theoretical expectations and show that from very modest data sets ion abundances with accuracies of about 0.1% and mass measurements with errors in the low parts per million range can be gained. Even for broad-band mass spectra recorded under conditions appropriate to ion/molecule reaction studies at relatively high cell pressures of up to 2×10 −6 torr, the above accuracies can be maintained. In this respect the SEFT method is superior to known alternative data handling methods, which under the same conditions allow errors of up to 300% and hundreds of parts per million for ion abundance and mass determinations, respectively.

Comprehensive Two-Dimensional Liquid Chromatography with Stationary-Phase-Assisted Modulation Coupled to High-Resolution Mass Spectrometry Applied to Proteome Analysis of Saccharomyces cerevisiae

Stationary-phase-assisted modulation is used to overcome one of the limitations of contemporary comprehensive two-dimensional liquid chromatography, which arises from the combination of a first-dimension column that is typically narrow and long and a second-dimension column that is wide and short. Shallow gradients at low flow rates are applied in the first dimension, whereas fast analyses (at high flow rates) are required in the second dimension. Limitations of this approach include a low sample capacity of the first-dimension column and a high dilution of the sample in the complete system. Moreover, the relatively high flow rates used for the second dimension make direct (splitless) hyphenation to mass spectrometry difficult. In the present study we demonstrate that stationary-phase-assisted modulation can be implemented in an online comprehensive two-dimensional LC (LC × LC) setup to shift this paradigm. The proposed active modulation makes it possible to choose virtually any combination of first- and second-dimension column diameters without loss in system performance. In the current setup, a 0.30 mm internal diameter first-dimension column with a relatively high loadability is coupled to a 0.075 mm internal diameter second-dimension column. This actively modulated system is coupled to a nanoelectrospray high-resolution mass spectrometer and applied for the separation of the tryptic peptides of a six-protein mixture and for the proteome-wide analyses of yeast from Saccharomyces cerevisiae. In the latter application, about 20000 MS/MS spectra are generated within 24 h analysis time, resulting in the identification of 701 proteins.

An Aptly Positioned Azido Group in the Spacer of a Protein Cross‐Linker for Facile Mapping of Lysines in Close Proximity

Cross‐links between amino acid residues in close proximity can provide distance constraints for the validation of models of the 3D structure proteins. The mapping of cross‐links by the identification of linked peptides in proteolytic digests is facilitated by cleavable cross‐linkers that enable isolation of the cleavage products while preserving information about the linkage. We present an amine‐specific cross‐linker, bis(succinimidyl)‐3‐azidomethyl glutarate (BAMG), that fulfils these requirements. Two parallel reaction pathways are induced by tris(carboxyethyl)phosphine (TCEP) in cross‐linked peptides from BAMG‐treated cytochrome c. One pathway leads to cleavage of the cross‐linked species, while in the other the azido group of BAMG is reduced to an amino group without cleavage. Cross‐linked peptides and peptides modified by partially hydrolysed BAMG yield distinct sets of TCEP‐induced reaction products. These can be isolated by reversed‐phase diagonal chromatography and identified by mass spectrometry to reveal the identity of the parent compounds. The ease with which cross‐link‐derived reaction products can be isolated and identified indicates that the mapping of cross‐links in complex biological assemblies and mixtures of protein complexes might become feasible in the near future.