Quantitation of yeast cell wall proteins respectively) were averaged from the data of 126 wild-type strains in Sa. cerevisiae Morphological Database (SCMD, http://scmd.gi.k.u-tokyo.ac.jp). The surface area S was cal- culated to be c. 60 mm2, which corresponds to an average CWP density in the cell wall of c. 31 103 molecules mm 2. Considering that a spherical protein such as hemoglobin (Mr 64 500), which has a diameter of about 5.5 nm (Lehnin- ger et al., 1993), needs about 24 nm2 to accommodate it on a surface (corresponding to c. 38 103 copies mm 2 in hexago- nal packing; http://mathworld.wolfram.com/CirclePacking. html), these results suggest that CWPs are closely packed on the cell surface, as also suggested by ultrastructural studies (Tokunaga et al., 1986; Baba & Osumi, 1987) and consistent with their being largely present in the form of a monolayer. It has to be noted that CWPs are frequently heavily glycosylated and may carry extended N-chains of up to 200 sugar residues (Orlean, 1997) and thus occupy more space than their protein parts alone. This predicted dense packing of CWPs on the cell surface is also consistent with the observation that the N-chains of CWPs have been shown to limit the permeability of the yeast cell wall for macro- molecules (De Nobel et al., 1990). Absolute quantitation of five covalently linked cell wall proteins In a recent genome-wide study using fusion constructs with a C-terminally attached green ﬂuorescent protein (GFP), the absolute quantity of yeast proteins was determined (Ghaem- maghami et al., 2003). Unfortunately, the analysis of CWPs using GFP fusion constructs did not take into account that the C-terminal hydrophobic part of GPI-modiﬁed proteins is removed in the endoplasmic reticulum by a transamidase complex (Ohishi et al., 2000). Their estimates do not, therefore, reﬂect the protein amount at the cell surface, and are probably too low. For example, a yeast cell was estimated to contain only c. 2000 copies of Cwp1p (Saccharomyces Genome Database; Ghaemmaghami et al., 2003). We there- fore developed an alternative approach for CWP quantita- Table 1. Absolute quantitation of covalently linked cell wall proteins 891 tion based on using deﬁned concentrations of synthetic peptides, isobaric tagging with the iTRAQ reagent, and tandem MS (Ross et al., 2004). To this end, ﬁve CWPs were selected to determine their absolute quantities, including a member from each of three carbohydrate-active enzyme families (the two GPI-dependent CWPs Crh1p and Gas1p, and the ASL-CWP Scw4p) found in the cell wall, Ecm33p, another GPI-dependent CWP of unknown function, which has been postulated to be a carbohydrate-active enzyme as well (Klis et al., 2006), and the structural CWP Cwp1p. To develop a suitable internal standard, one peptide from each target protein was chosen on the basis of its amino acid sequence and the cleaving characteristics of the protease trypsin. The selection criteria we used were as follows: (1) the peptides were observed in previous MS/MS experiments, indicating that they are sensitive to the ionization process; (2) the peptides do not contain methionine or cysteine residues, so that they are stable under oxidative conditions; (3) the peptides contain as few serine and threonine residues as possible, as these may undergo a side reaction with the iTRAQ reagent; and (4) the peptides must be unique for the corresponding protein but not too large to complicate synthesis and analysis. Five peptides (Table 1) were thus order-synthesized, derivatized with an isobaric iTRAQ tag, and evaluated by MALDI-MS/MS. This evaluation process provided qualitative information about peptide ionization efﬁciency in MS collision-induced fragmentation, and about their identity (data not shown). When titrated with a deﬁned amount of the synthetic peptide, an iTRAQ-labeled tryptic peptide from natural CWP is chromatographically indistinguishable from the internal standard peptide and has an identical mass. There- fore, in a typical MALDI-MS spectrum of CWPs (Fig. 2a), the subsequent MALDI-MS/MS analysis provides a highly speciﬁc and sensitive measurement of both the internal standard and the analyte, directly from a mixture of hundreds of cell wall peptides. In this MS spectrum, peaks of interest (i.e. synthetic peptides and their native counter- parts from target CWPs) could be easily recognized on the Protein GPI protein Crh1p Cwp1p Ecm33p Gas1p ASL protein Scw4p Synthetic peptide Residue numbers Peptide mass Observed mass Copies cell 1 10 3 WFTDLK SSSGFYAIK VIDGFNK TAEFK WLLEQIQR 68–73 106–114 285–291 248–252 301–308 808.41 958.48 791.42 594.30 1084.60 1097.63 1247.70 1080.64 883.52 1229.71 44 67 6.5 11 38 Observed masses ([M1H]1) of iTRAQ-labeled peptides in the MS spectrum (Fig. 2a). The iTRAQ reagent labels the N-termini and the e-amino group from lysine residues of peptides. Peptide identities were conﬁrmed by MS/MS before and after the labeling. GPI, glycosylphosphatidylinositol; ASL, alkali-sensitive linkage. FEMS Yeast Res 7 (2007) 887–896 c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.