Quantitation of yeast cell wall proteins 1997; Hamada et al., 1999). The ASL-CWP Scw4p seems to be moderately expressed in the cell wall. Relative quantitation of the cell wall proteome To examine the changes in the CWP population as a result of cell wall stress, we performed a quantitative proteomics study comparing wild-type and gas1D cells. In total, 367 peptides were identiﬁed, of which 113 were suitable for quantitation (Table 2). Fourteen cell wall proteins were unambiguously identiﬁed, and their abundance ratios (gas1D cells vs. wild-type cells) were quantiﬁed. Although most of the abundance ratios of covalently bound proteins were derived from multiple observations, this was not the case for proteins from Pir family. The members of this protein family are highly homologous, and all contain a variable number of glutamine-containing internal repeats and a C-terminal domain containing four cysteine residues with ﬁxed spacing (Fig. 3). The internal repeat domain is highly mannosylated, and is therefore not identiﬁed by our approach. Only the C-terminal regions of these proteins provide suitable tryptic peptides for identiﬁcation and quantitation. However, as this region is highly conserved, the sequences of four frequently identiﬁed peptides (Fig. 3a–d) are (partially) shared between the members of the Pir family. Therefore, unambiguous identiﬁcation and quanti- tation of individual Pir proteins can only be based on those peptides that are unique to each individual protein (Fig. 3, bold and underlined). This resulted in a relatively low 893 frequency of observations of unique peptides from Pir1p and Pir3p. It has to be noted that none of the identiﬁed peptides in this study is unique to the ﬁfth member of this family (Pir5/YJL160C). Therefore, its presence cannot be veriﬁed. Finally, in a previous study, we identiﬁed six other covalently linked CWPs in exponentially growing yeast cells (Yin et al., 2005); two of them could not be identiﬁed by trypsin digestion, but were instead identiﬁed using the endoprotease Glu-C and are thus missed here. The remain- ing four CWPs are relatively rare, and might have been missed because of the lower sensitivity inherent in the approach taken in the current study. The protein level of isolated walls of gas1D cells is about twice as high as in wild-type cell walls, consistent with the known constitutive activation of the cell wall integrity path- way in these cells (7.7% vs. 4.0%, own observation; Kapteyn et al., 1997). This implies that, without any changes in the relative protein composition in the wall, the abundance ratios should average about two. The abundance ratios in Table 2 indicate that activation of the cell wall integrity pathway as a result of the cell wall defects in gas1D cells triggers a considerable increase in the absolute cell wall levels of ﬁve putative carbohydrate-active enzymes (Crh1p, Crh2p, Gas5p, and also Ecm33p and Pst1p), and four predicted structural CWPs (Cwp1p, Pir2p, Pir3p, and Pir4p), whereas the level of Scw4p is strongly decreased. For comparison, we have included the corresponding mRNA ratios obtained by microarray analysis of gas1D and wild-type cells (Lagorce et al., 2003). Interestingly, the ratios of the wall-bound forms Table 2. Relative quantitation of covalently linked cell wall proteins in WT and gas1D cells Number of quantiﬁed peptides Protein ratio SD mRNA ratiow Function and propertiesz Protein GPI proteinz Crh1p Crh2p Cwp1p Ecm33p Gas1p‰ Gas3p Gas5p Pst1p Ssr1p ASL proteinz Pir1p Pir2p Pir3p Pir4p Scw4p 15 6 24 6 4 6 2 17 7 1 8 2 7 8 FEMS Yeast Res 7 (2007) 887–896 Protein ratios (gas1D/wild type) are means of peptide ratios from two duplicated labeling experiments. wmRNA data were obtained from www.dkfz.de/funct_genome/yeast-data.html. zGPI, glycosylphosphatidylinositol; ASL, alkali-sensitive linkage; GH, glycoside hydrolases, classiﬁcation according to Henrissat & Davies (1997); CFEM, common in fungal extracellular membrane/wall proteins. ‰Gas1p data in gas1D cells represent the background noise of MS experiments. 4.65 3.58 2.95 4.76 0.18 0.99 3.00 5.33 1.42 1.09 2.98 3.17 2.09 0.50 0.65 0.59 0.79 1.39 0.05 0.26 0.61 1.74 0.25 0.80 0.34 0.27 0.08 3.84 0.86 1.10 1.15 0.04 0.81 1.42 3.87 0.83 3.26 2.30 6.64 3.42 0.78 GH16, transglycosylase crosslinking chitin to b-1,6-glucan GH16, transglycosylase crosslinking chitin to b-1,6-glucan Contains a Pir-like repeat Related to cell wall biogenesis GH72, transglucosidase extending b-1,3-glucan GH72 GH72, transglucosidase Related to cell wall biogenesis CFEM domain Conserved 4-C domain Conserved 4-C domain Conserved 4-C domain Conserved 4-C domain GH17, b-1,3-glucanase c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.