Sequential Fractionation and Two-dimensional Gel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome
2002; Elsevier BV; Volume: 1; Issue: 12 Linguagem: Inglês
10.1074/mcp.m200062-mcp200
ISSN1535-9484
AutoresAída Pitarch, Miguel Sánchez, César Nombela, Concha Gil,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoThe cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome. The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome. In the last 2 decades, the opportunistic fungus Candida albicans has been the center of considerable medical interest because it remains the cause of substantial morbidity and mortality in immunocompromised hosts, such as cancer patients, human immunodeficiency virus-infected individuals, or transplant recipients, among others (1.Vincent J.L. Anaissie E. Bruining H. Demajo W. el Ebiary M. Haber J. Hiramatsu Y. Nitenberg G. Nystrom P.O. Pittet D. Rogers T. Sandven P. Sganga G. Schaller M.D. Solomkin J. 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Nevertheless, hyphal growth may be more critical for the pathogenesis of Candida since hyphae adhere more strongly to mammalian cells, promote tissue penetration, and provide a mechanism to escape the attack by macrophages. Its ability to switch between the budding and mycelial forms could be one of several factors involved in its virulence in systemic infections (6.Cutler J.E. Putative virulence factors of Candida albicans..Annu. Rev. Microbiol. 1991; 37: 115-121Google Scholar, 7.van Burik J.A. Magee P.T. Aspects of fungal pathogenesis in humans.Annu. Rev. Microbiol. 2001; 55: 743-772Google Scholar). This yeast-to-hypha transition is favored by several environmental conditions such as growth at 37 °C and near neutral pH or exposure to an inductor (serum, N-acetylglucosamine, or proline) (8.Ernst J.F. Transcription factors in Candida albicans—environmental control of morphogenesis.Microbiology. 2000; 146: 1763-1774Google Scholar). Since the cell wall, the outermost cellular structure, determines the shape of fungal cell, the morphogenetic conversion from the yeast to the filamentous form requires cell wall remodeling, which involves alterations in its composition and organization. This means that the cell wall is a plastic and dynamic structure that is constantly changing in response to environmental signals and the different stages of the fungal cell cycle (9.Valentin E. Mormeneo S. Sentandreu R. The cell surface of Candida albicans during morphogenesis.Contrib. Microbiol. 2000; 5: 138-150Google Scholar, 10.Chaffin W.L. Lopez-Ribot J.L. Casanova M. Gozalbo D. Martinez J.P. Cell wall and secreted proteins of Candida albicans: identification, function, and expression.Microbiol. Mol. Biol. Rev. 1998; 62: 130-180Google Scholar). Because of its privileged location within the cell, the cell wall is also the initial point of contact between the cell and its environment and thus contributes to host-fungus interactions (e.g. recognition, adhesion, etc.) (11.Calderone R.A. Recognition between Candida albicans and host cells.Trends Microbiol. 1993; 1: 55-58Google Scholar). In addition, given that mammalian cells lack a cell wall, this cellular compartment could be a promising molecular target site to search for new specific antifungal drugs (12.Groll A.H. De Lucca A.J. Walsh T.J. Emerging targets for the development of novel antifungal therapeutics.Trends Microbiol. 1998; 6: 117-124Google Scholar). In light of this, a better knowledge of C. albicans cell wall structure and composition may therefore contribute to the understanding of its involvement in fungal morphogenesis and pathogenesis as well as to the discovery of novel antifungal therapies. The C. albicans cell wall is mainly composed of three components interconnected by covalent bonds: β-1,3- and β-1,6-glucans (50–60%), mannoproteins (30–40%), and chitin (0.6–9%) (10.Chaffin W.L. Lopez-Ribot J.L. Casanova M. Gozalbo D. Martinez J.P. Cell wall and secreted proteins of Candida albicans: identification, function, and expression.Microbiol. Mol. Biol. Rev. 1998; 62: 130-180Google Scholar). Cell wall structure has been studied most extensively in Saccharomyces cerevisiae (13.Klis F. Mol P. Hellingwerf K. Brul S. Dynamics of cell wall structure in Saccharomyces cerevisiae..FEMS Microbiol. Rev. 2002; 26: 239Google Scholar, 14.Molina M. Gil C. Pla J. Arroyo J. Nombela C. Protein localisation approaches for understanding yeast cell wall biogenesis.Microsc. Res. Tech. 2000; 51: 601-612Google Scholar, 15.Kapteyn J.C. Van Den E.H. Klis F.M. The contribution of cell wall proteins to the organization of the yeast cell wall.Biochim. Biophys. 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Covalent association of β-1,3-glucan with β-1,6-glucosylated mannoproteins in cell walls of Candida albicans..J. Bacteriol. 1995; 177: 3788-3792Google Scholar, 20.Sanjuan R. Zueco J. Stock R. Font de Mora J. Sentandreu R. Identification of glucan-mannoprotein complexes in the cell wall of Candida albicans using a monoclonal antibody that reacts with a (1,6)-β-glucan epitope.Microbiology. 1995; 141: 1545-1551Google Scholar, 21.Kapteyn J.C. Hoyer L.L. Hecht J.E. Muller W.H. Andel A. Verkleij A.J. Makarow M. Van Den E.H. Klis F.M. The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants.Mol. Microbiol. 2000; 35: 601-611Google Scholar) concerning the cell wall organization of C. albicans have demonstrated that a similar model is also valid for this pathogenic fungus (10.Chaffin W.L. Lopez-Ribot J.L. Casanova M. Gozalbo D. Martinez J.P. Cell wall and secreted proteins of Candida albicans: identification, function, and expression.Microbiol. Mol. Biol. Rev. 1998; 62: 130-180Google Scholar, 22.Klis F.M. de Groot P. Hellingwerf K. Molecular organization of the cell wall of Candida albicans..Med. Mycol. 2001; 39: 1-8Google Scholar, 23.Chauhan N. Li D. Singh P. Calderone R. Kruppa M. Calderone R.A. The Cell Wall of Candida spp. Candida and Candidiasis. ASM Press, Washington, D. C.2002: 159-175Google Scholar). The yeast cell wall, located outside the plasma membrane, seems to be a layered structure whose electron-dense outer layer consists of mannoproteins, whereas the electron-transparent inner layer is composed of β-1,3-glucan and chitin. Consistent with this, β-1,3-glucan and chitin form a microfibrillar network, providing rigidity to the cell wall, in which mannoproteins are embedded and determine the porosity of the cell wall (10.Chaffin W.L. Lopez-Ribot J.L. Casanova M. Gozalbo D. Martinez J.P. Cell wall and secreted proteins of Candida albicans: identification, function, and expression.Microbiol. Mol. Biol. Rev. 1998; 62: 130-180Google Scholar). Chitin can be glycosidically linked to the non-reducing ends of β-1,3-glucan and/or β-1,6-glucan (24.Kapteyn J.C. Ram A.F. Groos E.M. Kollar R. Montijn R.C. Van Den E.H. Llobell A. Cabib E. Klis F.M. Altered extent of cross-linking of beta1, 6-glucosylated mannoproteins to chitin in Saccharomyces cerevisiae mutants with reduced cell wall β1,3-glucan content.J. Bacteriol. 1997; 179: 6279-6284Google Scholar). In turn, cell wall proteins (CWPs) 1The abbreviations used are: CWP, cell wall protein; 2-DE, two-dimensional electrophoresis; ACN, acetonitrile; AmBic, ammonium bicarbonate; DTT, dithiothreitol; GPI, glycosylphosphatidylinositol; Pir, protein with internal repeats; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; TOF/TOF, tandem TOF; MS, mass spectrometry; MS/MS, tandem MS1The abbreviations used are: CWP, cell wall protein; 2-DE, two-dimensional electrophoresis; ACN, acetonitrile; AmBic, ammonium bicarbonate; DTT, dithiothreitol; GPI, glycosylphosphatidylinositol; Pir, protein with internal repeats; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; TOF/TOF, tandem TOF; MS, mass spectrometry; MS/MS, tandem MS can be coupled to cell wall components in different ways (22.Klis F.M. de Groot P. Hellingwerf K. Molecular organization of the cell wall of Candida albicans..Med. Mycol. 2001; 39: 1-8Google Scholar). Nevertheless, the total number and functions of CWPs are still poorly known. Several chemical and/or enzymatic strategies for their isolation, both from intact cells (25.Casanova M. Lopez-Ribot J.L. Martinez J.P. Sentandreu R. Characterization of cell wall proteins from yeast and mycelial cells of Candida albicans by labelling with biotin: comparison with other techniques.Infect. Immun. 1992; 60: 4898-4906Google Scholar, 26.Lopez-Ribot J.L. Alloush H.M. Masten B.J. Chaffin W.L. Evidence for presence in the cell wall of Candida albicans of a protein related to the hsp70 family.Infect. Immun. 1996; 64: 3333-3340Google Scholar) or from isolated cell walls after cell breakage (18.Kapteyn J.C. Montijn R.C. Dijkgraaf G.J. Klis F.M. Identification of β-1,6-glucosylated cell wall proteins in yeast and hyphal forms of Candida albicans..Eur. J. Cell Biol. 1994; 65: 402-407Google Scholar, 19.Kapteyn J.C. Montijn R.C. Dijkgraaf G.J. Van Den E.H. Klis F.M. Covalent association of β-1,3-glucan with β-1,6-glucosylated mannoproteins in cell walls of Candida albicans..J. Bacteriol. 1995; 177: 3788-3792Google Scholar, 27.Elorza M.V. Murgui A. Sentandreu R. Dimorphism in Candida albicans: contribution of mannoproteins to the architecture of yeast and mycelial cell walls.J. Gen. Microbiol. 1985; 131: 2209-2216Google Scholar, 28.Mormeneo S. Rico H. Iranzo M. Aguado C. Sentandreu R. Study of supramolecular structures released from the cell wall of Candida albicans by ethylenediamine treatment.Arch. Microbiol. 1996; 166: 327-335Google Scholar, 29.Ruiz-Herrera J. Mormeneo S. Vanaclocha P. Font-de-Mora J. Iranzo M. Puertes I. Sentandreu R. Structural organization of the components of the cell wall from Candida albicans..Microbiology. 1994; 140: 1513-1523Google Scholar), have been described. Alternatively, another approach for studying CWPs involves the analysis of proteins secreted into the medium when protoplasts are regenerating their cell walls (30.Elorza M.V. Rico H. Gozalbo D. Sentandreu R. Cell wall composition and protoplast regeneration in Candida albicans..Antonie Leeuwenhoek. 1983; 49: 457-469Google Scholar, 31.Kapteyn J.C. Dijkgraaf G.J. Montijn R.C. Klis F.M. Glucosylation of cell wall proteins in regenerating spheroplasts of Candida albicans..FEMS Microbiol. Lett. 1995; 128: 271-277Google Scholar, 32.Pitarch A. Pardo M. Jimenez A. Pla J. Gil C. Sanchez M. Nombela C. Two-dimensional gel electrophoresis as analytical tool for identifying Candida albicans immunogenic proteins.Electrophoresis. 1999; 20: 1001-1010Google Scholar). In this study, in an attempt to obtain an overall view of different mannoproteins that make up the C. albicans wall structure, cell wall fractionation was carried out. We evaluated the protein profiles of different enriched cell wall fractions from yeast and hyphal forms using two-dimensional PAGE, allowing the establishment of reference 2-DE maps of C. albicans cell wall. Several cell envelope-associated proteins were analyzed by mass spectrometry. We report the identification of some non-classical CWPs previously described at the cell surface from other organisms, suggesting the presence of alternative secretory pathways hitherto undiscovered. We also highlight the usefulness of cell wall fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against bona fide CWPs in the characterization of their interactions with other structural cell wall components. Cells of C. albicans strain SC5314 (33.Gillum A.M. Tsay E.Y. Kirsch D.R. Isolation of the Candida albicans gene for orotidine-5`-phosphate decarboxylase by complementation of S. cerevisiae ura3 and E. coli pyrF mutations.Mol. Gen. Genet. 1984; 198: 179-182Google Scholar) were grown in YPD medium (1% Difco yeast extract, 2% peptone, 2% glucose, and 2% agar) at 28 °C up to an A600 nm of 0.5–1 and washed with water. Cells were then resuspended at up to 105 cells/ml in Lee medium (34.Lee K.L. Buckley H.R. Campbell C.C. An amino acid liquid synthetic medium for the development of mycelial and yeast forms of Candida albicans..Sabouraudia. 1975; 13: 148-153Google Scholar) at pH 4.3 or 6.7 and incubated for 6 h at 37 °C to obtain yeast or hyphal forms, respectively. Both assays were assessed by phase-contrast microscopy. Cell wall fractionation was performed basically as described previously (35.Mrsa V. Seidl T. Gentzsch M. Tanner W. Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae..Yeast. 1997; 13: 1145-1154Google Scholar, 36.Kapteyn J.C. Montijn R.C. Vink E. de la Cruz J. Llobell A. Douwes J.E. Shimoi H. Lipke P.N. Klis F.M. Retention of Saccharomyces cerevisiae cell wall proteins through a phosphodiester-linked β-1,3-β-1,6-glucan heteropolymer.Glycobiology. 1996; 6: 337-345Google Scholar) with some modifications. Yeast cells and hyphae were collected by centrifugation and filtration, respectively, and washed five times with lysis buffer (10 mm Tris-HCl, pH 7.4, 1 mm phenylmethylsulfonyl fluoride). Subsequently cells were resuspended in ice-cold lysis buffer and lysed mechanically with an equal volume of glass beads in a cell homogenizer (Braun, MSK). This procedure was carried out until complete cell breakage, verified beforehand by phase-contrast microscopic examinations and a posteriori by the failure of cells to grow on YPD-chloramphenicol plates. Lysed cells were separated by centrifugation at 3000 × g for 10 min in a cell wall fraction (pellet) and a soluble cytoplasmic fraction (supernatant). Following this, the cell wall fraction was washed five times with ice-cold water and rinsed another five times with each of the following ice-cold solutions: 5% NaCl, 2% NaCl, 1% NaCl, and 1 mm phenylmethylsulfonyl fluoride. Isolated cell walls were extracted by boiling with SDS extraction buffer (50 mm Tris-HCl, pH 8.0, 0.1 m EDTA, 2% SDS, 10 mm DTT) for 10 min each time. This treatment was carried out once again, discarding the last extract. SDS-resistant walls were washed five times with ice-cold water and then a further 10 times with ice-cold 0.1 m NaAc, pH 5.5, 1 mm phenylmethylsulfonyl fluoride. The remaining pellet was divided into two fractions. One fraction was extracted with 30 mm NaOH overnight at 4 °C. The other fraction was digested at 37 °C for 17 h with Quantazyme ylg (Quantum Biotechnologies Inc., Montreal, Canada), a recombinant β-1,3-glucanase, in 50 mm Tris-HCl, pH 7.5, 10 mm DTT. Then Quantazyme-resistant cell walls were digested at 37 °C for 18 h with exochitinase (Sigma), isolated from Serratia marcescens, in 50 mm potassium phosphate buffer, pH 6.3. A flow chart of the experimental procedure is illustrated in Fig. 1. CWPs were precipitated with trichloroacetic acid/acetone. Protein concentration was measured with the Bradford assay (Bio-Rad). Samples containing 500 μg (analytical gels) or 5–10 mg (preparative gels) of protein were solubilized in a lysis buffer (7 m urea, 2 m thiourea, 2% CHAPS, 65 mm dithioerythritol, 2% Pharmalyte pH 3–10 (Amersham Biosciences), bromphenol blue) (37.Rabilloud T. Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis.Electrophoresis. 1998; 19: 758-760Google Scholar) and were then applied onto Immobiline pH 3–10 non-linear DryStrips (18 cm long, Amersham Biosciences). Isoelectric focusing was performed on an IPGphor system (Amersham Biosciences) at 15 °C using the following program: (i) for analytical gels: passive rehydration for 16 h, 500 V for 1 h, 500–2000 V for 1 h, and 8000 V for 5.5 h, or alternatively (ii) for preparative gels: 30 V (active rehydration) for 13 h, 500 V for 1 h, 1000 V for 1 h, 2000 V for 1 h, 2000–5000 V for 3 h, and 8000 V for 11 h. After this, immobilized pH gradient strips were reduced (2% dithioerythritol) and then alkylated (2.5% iodoacetamide) in equilibration buffer (6 m urea, 50 mm Tris-HCl, pH 6.8, 30% glycerol, 2% SDS) (38.Bjellqvist B. Sanchez J.C. Pasquali C. Ravier F. Paquet N. Frutiger S. Hughes G.J. Hochstrasser D. Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins.Electrophoresis. 1993; 14: 1375-1378Google Scholar). The second dimension run was carried out on homogeneous 10% T, 1.6% C (piperazine diacrylamide) polyacrylamide gels (1.5 mm thick) at 40 mA per gel for 6 h using a Protean II gel tank (Bio-Rad). Mr and pI values were estimated using internal two-dimensional SDS-PAGE and external SDS-PAGE standards (Bio-Rad). Preparative gels were silver-stained as described by Shevchenko et al. (39.Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Google Scholar). Alternatively, analytical gels were fixed first in 40% methanol and 10% acetic acid for 1 h and then in 5% ethanol and 5% acetic acid overnight. The gels were rinsed with 7.5% acetic acid and incubated in 10% glutaraldehyde for 30 min. After this the gels were extensively washed with water and stained with an ammoniacal silver nitrate solution for 30 min. The gels were washed and then developed in 0.01% citric acid and 0.1% formaldehyde. Staining was halted with 5% Tris and 2% acetic acid. Preparative gels were stained as reported elsewhere (40.Pardo M. Ward M. Pitarch A. Sanchez M. Nombela C. Blackstock W. Gil C. Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry.Electrophoresis. 2000; 21: 2651-2659Google Scholar). Staining with the periodic acid-Schiff reagent was performed according to Zacharius et al. (41.Zacharius R.M. Zell T.E. Morrison J.H. Woodlock J.J. Glycoprotein staining following electrophoresis on acrylamide gels.Anal. Biochem. 1969; 30: 148-152Google Scholar). Briefly, analytical gels were fixed in 12% trichloroacetic acid and washed with water. They were incubated in 1% periodic acid and 3% acetic acid for 50 min and rinsed with water for 18 h. Gels were incubated in Schiff's reagent (Merck) in the dark for 50 min and washed first with 0.5% sodium bisulfite and then with water. Two-dimensional images were captured by scanning the silver-stained gels using a GS-690 imaging densitometer (Bio-Rad) and digitalized with Multi-Analyst software (Bio-Rad). Different two-dimensional images were processed, including detection, volumetric quantification, matching, statistical analysis (Student's t test), and editing of molecular masses and pI of spots, using Melanie 3.0 software (Bio-Rad). Analytical two-dimensional PAGE and SDS-PAGE gels were electroblotted onto nitrocellulose membranes in Towbin buffer at 50 mA overnight (42.Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Google Scholar). Blots were processed following standard protocols (43.Gallager S. Winston S.E. Fuller S.A. Hurrell J.G.R. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Immunoblotting and Immunodetection. Current Protocols in Molecular Biology. Greene Publishing Associates and Wiley Interscience, New York1997: 10.8.1-10.8.21Google Scholar). Monoclonal antibody against C. albicans Enop (dilution 1:6000) and polyclonal antibodies against C. albicans Pgkp (dilution 1:5000) and S. cerevisiae Gapp (dilution 1:10000), Bgl2p (dilution 1:5000), Hsp150p/Pir2p (dilution 1:5000), Gas1p (dilution 1:3000), and Sec14p (dilution 1:3000) were used for immunodetection. Immunoreactive spots were detected using horseradish peroxidase-labeled anti-mouse or anti-rabbit IgGs (Amersham Biosciences), depending on the first antibody, and an enhanced chemiluminescence detection system (ECL, Amersham Biosciences). Spots of interest were manually excised from preparative silver- or Coomassie-stained 2-DE gels, depending on their intensity and relative volume. Silver-stained gel pieces were destained as described by Gharahdaghi et al. (44.Gharahdaghi F. Weinberg C.R. Meagher D.A. Imai B.S. Mische S.M. Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity.Electrophoresis. 1999; 20: 601-605Google Scholar). Briefly, gel spots were incubated in 100 mm sodium thiosulfate and 30 mm potassium ferricyanide, rinsed twice in 25 mm ammonium bicarbonate (AmBic) and once in water, shrunk with 100% acetonitrile (ACN) for 15 min, and dried in a Savant SpeedVac for 20–30 min. Alternatively, Coomassie Blue-stained gel pieces were destained with ACN, washed twice with 50% ACN in 25 mm AmBic, and vacuum-dried. Both types of spots were reduced with 10 mm dithioerythritol in 25 mm AmBic for 30 min at 56 °C and subsequently alkylated with 55 mm iodoacetamide in 25 mm AmBic for 20 min in the dark. Gel pieces were alternately washed with 25 mm AmBic and ACN, and dried under vacuum. Following this, certain excised spots were also in-gel deglycosylated by treatment with 200 units/ml PNGase F (N-glycosidase F, Roche Molecular Biochemicals) in 25 mm AmBic overnight at 37 °C. Glycans were removed from the gel pieces using six changes of 25 mm AmBic with sonication for 30 min. Gel spots were subsequently shrunk with ACN and vacuum-dried. All gel pieces were incubated with 12.5 ng/μl sequencing grade trypsin (Roche Molecular Biochemicals) in 25 mm AmBic overnight at 37 °C. After digestion, the supernatants (crude extracts) were separated. Peptides were extracted from the gel pieces first into 50% ACN, 1% trifluoroacetic acid and then into 100% ACN. All extracts were pooled, and the volume was reduced by SpeedVac. One microliter of each sample (both crude extracts and extracted peptides) and then 0.4 μl of 3 mg/ml α-cyano-4-hydroxycinnamic acid matrix (Sigma) in 50% ACN, 0.01% trifluoroacetic acid were spotted onto a MALDI target. Samples for MS/MS sequencing were mixed 1:1 with α-cyano matrix (5 mg/ml in 50% ACN, 0.3% trifluoroacetic acid) and spotted on the stained steel plate. MALDI-TOF MS analyses were performed on a Voyager-DE STR instrument (PerSeptive Biosystems, Framingham, MA). Peptides were selected in the mass range of 700–4500 Da. All mass spectra were externally calibrated with the Sequazyme peptide mass standards kit (PerSeptive Biosystems) and internally with trypsin autolysis peaks. MS/MS sequencing analyses were carried out using the MALDI-tandem time-of-flight mass spectrometer 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA). The peptide mass fingerprinting and peptide fragment-ion data obtained from MALDI-TOF and MS/MS analyses, respectively, were used to search for protein candidates in two sequence databases (i.e. SWISS-PROT/TrEMBL non-redundant protein database (www.expasy.ch/sprot) and a nearly complete C. albicans genomic database, namely CandidaDB (genolist.pasteur.fr/CandidaDB)) using MS-Fit and MS-Tag (prospector.ucsf.edu), ProFound (prowl.rockefeller.edu), and/or Mascot (www.matrixscience.com) software programs. Initial search parameters were as follows: Cys as S-carbamidomethyl derivative and Met in oxidized form, one missed cleavage site, peptide mass tolerance of 50 ppm, and MS/MS tolerance of ±0.5 Da. Nucleotide sequence data for C. albicans were obtained from the Stanford Genome Technology Center website at www.sequence.stanford.edu/group/candida. Sequencing of C. albicans was accomplished with the support of the NIDCR, National Institutes of Health and the Burroughs Wellcome Fund. Information about coding sequences and proteins were obtained from CandidaDB available at www.pasteur.fr/Galar_Fungail/CandidaDB/, which has been developed by the Galar Fungail European Consortium (QLK2-2000-00795). Cell walls were obtained by mechanical disruption of C. albicans yeast and hyphal forms, verifying complete cell breakage by microscopic examinations and by lack of growth on YPD-agar plates. This confirmation was carried out to avoid cell lysis of putative intact cells by subsequent enzymatic extractions and thus contamination by intracellular material. In addition, cell walls were extensively washed with solutions of decreasing concentrations of NaCl to remove any extracellular or cytosolic protein contaminants that might be adher
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