890 water10.1% formic acid, and mobile phase B was 80% acetonitrile10.1% formic acid. For the separation of pep- tides, a linear gradient of 5–95% B over 30 min was employed. Eluting peptides were directly electrosprayed into a Micromass quadrupole time-of-ﬂight mass spectrometer (Waters, Milford, MA). The most abundant ions from the survey spectrum, ranging from m/z 350 to 2000, were automatically selected for collision-induced fragmentation using MASSLYNX software. Fragmentation was conducted with argon as collision gas at a pressure of 4 10 5 bars as measured on the quadrupole pressure gauge. Resulting MS/ MS spectra were processed with the MAXENT3 algorithm embedded in MASSLYNX software to generate peak lists. Data analysis Mass spectra were transformed and submitted to MASCOT (version 2.05, Matrix Science, London, UK) by the script provided in the data acquisition and analysis software ANALYST QS (Applied Biosystems), or by the peak list (pkl) file generated from the software MASSLYNX (Waters). Each MS/MS spectrum was searched against an in-house database (Yin et al., 2005), resulting in a set of tryptic peptide matches with confidence values. These peptide identifica- tions were then combined using the MASCOT search engine to yield a set of cell wall protein identifications with confidence values. The MASCOT searches were run using the following parameters: fixed carbamidomethyl modifica- tion of cysteine residues; fixed iTRAQ modification of free amine in the N-terminus and lysine; and variable methio- nine oxidation, and glutamine and asparagine deamidation. Incomplete tryptic digestion with one missed cleavage was allowed; precursor error tolerance was set at 0.8 Da; MS/ MS fragment tolerance was set at 0.4 Da; and charge was set at11. Furthermore, only peptides matching the identi- fied protein better than any other protein in the database (first rank) with P o 0.05 for random occurrence were retained for further analysis. For each MS/MS spectrum acquired, signature ion peak areas at m/z 114.1, 115.1, 116.1 and 117.1 were extracted using ANALYST QS or MASSLYNX software. For absolute quantitation, in the three mixing experi- ments, MS/MS spectra that showed the closest signal-to- noise ratios at the signature ion region were selected for quantitation. For relative quantitation, the ratios of the raw peak heights were normalized against the amount of dried cell walls used. Because the accuracy of ion intensity measurement may be affected by its signal-to-noise ratio in the time-of-ﬂight type of mass spectrometer used in this study, spectra with the intensity of reporter ion peaks below a predeﬁned threshold were considered as noise and were ﬁltered using MASSLYNX software. The remaining spectra were used to calculate the mean peptide ratios and SDs. We took c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. Q.Y. Yin et al. into consideration that each protein can potentially be identified by a number of peptides and every peptide can be measured multiple times. Finally, protein ratios from the duplicate labeling experiments (iTRAQ114 vs. iTRAQ116, and iTRAQ117 vs. iTRAQ114) were averaged. For proteins quantified with only two peptides, the raw data were manually inspected, and a protein ratio was calculated only if the two peptide ratios differed by no more than 25%. Results and discussion Cellular quantitation of covalently linked cell wall proteins Previously, we identiﬁed 19 covalently bound CWPs of Sa. cerevisiae, seven of which (Crh1p, Crh2p, Gas1p, Gas3p, Gas5p, Scw4p, and Scw10p) were classiﬁed as glycoside hydrolases (Henrissat & Davies, 1997). Phenotypic analysis of deletion mutants lacking the corresponding encoding genes indicated that most of them have altered cell wall properties, which reinforces the importance of the identiﬁed proteins for proper cell wall formation. These studies, however, did not provide quantitative information on the CWPs identiﬁed. To address this question, we determined as a ﬁrst step the total number of CWP molecules in a log- phase haploid cell. Yeast cells, 5 108 (harvested from 25 mL of medium), were freeze-dried, yielding a dry weight of 9.3 0.2 mg (mean SD, n = 3), which corresponds to 18.6 0.4 pg (mean SD, n = 3) biomass per cell. In paral- lel, the same number of cells was used to isolate cell walls as previously described (De Groot et al., 2004), showing that the cell wall dry weight accounted for c. 21% of the total biomass (3.9 0.3 pg cell 1; mean SD, n = 3). This is in agreement with a previous report showing that in rich medium the cell wall accounts for c. 20% of the dry biomass per cell (Aguilar-Uscanga & Franc¸ois, 2003). The peptide parts of the covalently bound CWPs were determined using the BCA protein assay to be 4% of the wall biomass, which corresponds to 0.16 pg cell 1, consistent with a previous report (Kapteyn et al., 2001). Assuming a mean molecular mass of CWPs of 50 kDa, a haploid cell contains 1.9 106 covalently attached CWP molecules. As CWPs seem to be uniformly distributed over the cell surface (Tokunaga et al., 1986; Baba & Osumi, 1987), it is possible to estimate their surface density by calculating the cell surface of an average yeast cell. Yeast cells were assumed to approximate two prolate spheroids (the mother cell and the bud cell) in conjunction with each other. The surface area S was calculated from the semi-major (a) and semi- minor (b) axes and the equation S = 2p(b21abœ/sinœ), where œ = arccos(a/b). The values for the major and minor axes of the adult cell (4.57 and 3.82 mm, respectively) and for the major and minor axes of the bud (1.85 and 1.70 mm, FEMS Yeast Res 7 (2007) 887–896