888 pressure. In response to cell wall stress, the cell wall integrity pathway is activated, resulting in increased expression of a speciﬁc set of CWPs, massive deposition of chitin in the lateral walls, and much higher levels of the CWP–polysac- charide complex CWP ! b-1,6-glucan’chitin (Kapteyn et al., 1997; Jung & Levin, 1999; Lagorce et al., 2003; Levin, 2005). A representative cell wall mutant is gas1D, in which a gene encoding a b-1,3-glucan remodeling enzyme is deleted (Popolo & Vai, 1999). Quantitation of CWPs by gel-based methods is notor- iously difﬁcult, because CWPs are heavily glycosylated and often also phosphorylated. They occur as mixtures of multi- ple glycoforms and phosphoforms, resulting in multiple fuzzy spots per protein (Weig et al., 2004). In this study, we used isobaric tagging and tandem MS (MS/MS) for absolute and relative quantitation of CWPs. Our method is gel- independent and does not require a speciﬁc antiserum or a tagged form of the target proteins. In addition, as the number of fully sequenced fungal genomes is rapidly grow- ing, and because many fungi, such as Candida spp., Sc. pombe, Aspergillus niger, Fusarium oxysporum and Ustilago maydis, and also the oomycete Phytophthora ramorum, possess covalently bound CWPs, our methods will be widely applicable (Ruiz-Herrera et al., 1996; Schoffelmeer et al., 1996, 2001; Brul et al., 1997; Weig et al., 2004; Damveld et al., 2005; Meijer et al., 2006; De Groot et al., 2007). Materials and methods Yeast strains and media Cells from the wild-type strain FY833 (MATa his3D200 ura3- 52 leu2D1 lys2D202 trp1D63), and the mutant strain AR104 (FY833 gas1::LEU2), were grown to exponential phase at 30 1C in YPD medium (1% yeast extract, 2% Bacto peptone and 2% glucose). At harvest, the OD600 nm was 1.2; OD600 nm = 1.0 corresponds to 1.66 0.05 107 cells mL 1 (mean SD, n = 3) as measured with a Multisizer Coulter Counter (Beckman Coulter, Fullerton, CA). Cell wall isolation and protein content determination Cell walls were isolated as described by De Groot et al. (2004). The cell wall protein content was determined with the BCA protein assay (Perbio Science, Erembodegem, Belgium) as described by Kapteyn et al. (1997). Cell wall digestion Sodium dodecyl sulfate-extracted cell walls were reduced, alkylated, and freeze-dried overnight, as previously de- scribed (Yin et al., 2005). Freeze-dried cell walls (4 0.1 mg; mean SD, n = 3) were resuspended in 50 mL c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. Q.Y. Yin et al. of 0.5 M triethylammonium bicarbonate, pH 8.5, and in- cubated overnight at 37 1C with sequencing-grade trypsin (Roche Applied Science, Mannheim, Germany), using a protein/enzyme ratio of 50 : 1. This procedure has been immunologically shown to result in a complete loss of the signal in the case of Cwp1p, Pir2p, and Ccw14p, indicating that tryptic digestion is exhaustive (Yin et al., 2005). Digested samples were centrifuged, and the supernatants containing the solubilized peptides were completely dried in a vacuum concentrator. Preparation of defined standard solutions of labeled peptides The synthetic peptides WFTDLK(68–73) (for Crh1p), SSSGFYIK(106–114) (for Cwp1p), VIDGFNK(285–291) (for Ecm33p), TAEFK(248–252) (for Gas1p) and WLLE- QIQR(301–308) (for Scw4p) were purchased from KJ Ross- Petersen ApS (Klampenborg, Denmark). The sequence identity of the peptides was veriﬁed by amino acid analysis and conﬁrmed by MS. Synthetic peptides, 0.4–0.7 mg, were dissolved in 10 mL of 0.5 M triethylammonium bicarbonate, pH 8.5. Five microliters of iTRAQ114 reagent (Applied Biosystem, Foster City, CA) was added, and the mixture was incubated at room temperature for 1 h. The iTRAQ114- labeled synthetic peptides were diluted 100- to 1000-fold, and were used as stock solutions. The concentrations of the standard peptides in stock solutions were determined spec- trophotometrically on the basis of the molar extinction coefﬁcients of phenylalanine, tryptophan or tyrosine resi- dues (195, 5579, and 1405 M 1 cm 1, respectively) (Fasman, 1976). The UV absorbances at 257.5 nm (for peptides TAEFK and VIDGFNK), 280 nm (for peptides WFTDLK and WLLEQIQR) and 274 nm (for peptide SSSGFYIK) were used to calculate peptide concentrations. Stock solutions of the ﬁve peptides were mixed to obtain a working standard solution, in which the concentration of each peptide was normalized to 0.32–0.54 mM. Isobaric labeling of cell wall peptides and synthetic peptides Absolute and relative amounts of CWPs were determined using the iTRAQ methodology (Ross et al., 2004). Figure 1a presents an overview of the essential steps of the quantita- tion experiments. In brief, peptide samples were incubated with one or more of the four available iTRAQ reagents (Applied Biosystem) to derivatize the N-termini and the lysine residues of the peptides. Each iTRAQ reagent mole- cule consists of a reporter group (based on N-methylpiper- azine), a mass balance group (the carbonyl group), and a peptide-reactive group (N-hydroxylsuccinimide ester) (Fig. 1b). The overall added mass of reporter and balance components of the molecule is kept constant so that FEMS Yeast Res 7 (2007) 887–896