892 Fig. 2. MS and MS/MS spectra of peptides from a tryptic cell wall protein digest titrated with synthetic peptides. (a) Overview of a MALDI- MS spectrum showing the total cell wall protein digest mixed with labeled synthetic peptides (peptide mixture a in Fig. 1). The base peak labeled with an asterisk is from a shared peptide of Pir proteins (peptide b in Fig. 3). The ﬁve indicated precursor ions were fragmented to obtain quantitative information. (b) The MS/MS spectrum of the precursor ion 1080.6 [M1H]1 corresponding to the peptide VIDGFN K from Ecm33p ( denotes the labeled amino groups). The sequence was derived from both b-ion and y-ion series, conﬁrming the identity of the peptide. (c) Expanded view of the signature ion region of three MS/MS spectra of Ecm33p peptide VIDGFNK from 5 108 cells (peaks at 115.1) titrated with (from top to bottom) 1.08, 5.4 and 27 pmol of synthetic peptide (peaks at 114.1), allowing an accurate estimation of the absolute quantity of the peptide studied. basis of their m/z value (Fig. 2a, mass-labeled). Taking the peptide VIDGFN K of Ecm33p ( denotes the labeled amino groups) as an example, this particular peptide has two labeling sites, resulting in an observed precursor m/z of 1080.6. The precursor ion could be sent to fragmentation for sequence veriﬁcation and for quantiﬁcation. Figure 2b c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. Q.Y. Yin et al. shows a representative MS/MS spectrum. The series of both y and b product ions from MS/MS and the precursor ion m/z have been used to verify the identity of this peptide. The isobaric tags from the iTRAQ label produced abundant MS/ MS signature ions of m/z 114.1 and 115.1, representing the ion of the N-methylpiperazinylated N-terminus. The stabi- lity of the resulting alkylated immonium ion is universal for essentially all modiﬁed tryptic peptides, as it is independent of the amino acid at the N-terminus of the peptide. The relative intensities of the signature ions correspond to the proportions of the synthetic and natural peptides. Figure 2c zooms in on the reporter region of the MS/MS spectra of the peptide VIDGFNK derived from 5 108 yeast cells that were titrated with 1.1, 5.4 and 26.9 pmol of standard peptides (from top to bottom, respectively). The peaks in the middle spectrum showed the closest signal-to-noise ratio, and the ratio of the peak areas at m/z 114.1 and 115.1 was then used to determine the amount (5.4 pmol) of this peptide in the original cell wall material, resulting in an estimated 6.5 103 wall-bound Ecm33p molecules per cell (Table 1). The absolute quantitation of all ﬁve CWPs is summarized in Table 1. Our experimental results are in reasonable agreement with the notion that dividing haploid cells contain on average 1.9 106 covalently attached CWPs per cell (see above), considering that there are over 20 CWPs incorpo- rated in the wall of a log-phase-grown cell. Cwp1p is the most abundant cell wall protein observed in this study; the amount of 67 103 per cell is indeed much higher than previously reported by Ghaemmaghami et al. (2003). As- suming that Cwp1p is preferentially incorporated in the birth scar area (Smits et al., 2006), and using an estimated birth scar area of c. 3 mm2 (measured from Fig. 2, Smits et al., 2006), this would correspond to a local density of Cwp1p of c. 22 103 copies mm 2. As the average CWP density of the cell wall is c. 31 103 molecules mm 2, this suggests that Cwp1p is the major CWP found in this area. Interestingly, Cwp1 can function as a crosslinking protein, because it is one of the few GPI-CWPs that also possesses a Pir repeat, which may be used to couple it directly to b-1,3-glucan in addition to its linkage to b-1,6-glucan (Kapteyn et al., 2001). This suggests that it may protect the birth scar against cell wall damage during cytokinesis. Wall-bound Crh1p is pre- sent at 44 103 copies per cell. As its incorporation in the wall is cell cycle-dependent, and as it is only found at the emerging bud site and, later in the cell cycle, in the neck region, its local density is considerable, consistent with its reported function in interconnecting chitin to b-glucan (Rodrı´quez-Pen˜a et al., 2000; Cabib et al., 2007). The low copy numbers per cell of the wall-bound forms of Gas1p and Ecm33p are consistent with the empirical and theoretical evidence that Gas1p and Ecm33p are mainly targeted to the plasma membrane (Conzelmann et al., 1988; Caro et al., FEMS Yeast Res 7 (2007) 887–896