Mass spectrometric quantitation of covalently bound cell wall proteins in Saccharomyces cerevisiae.
The cell wall of yeast consists of an internal skeletal layer and an external layer of glycoproteins covalently linked to the stress-bearing polysaccharides. The cell wall protein (CWP) population consists of over 20 different proteins, and may vary in composition. We present two complementary methods for quantifying CWPs, based on isobaric tagging and tandem MS: (1) absolute quantitation of individual CWPs, allowing estimation of surface densities; and (2) relative quantitation of CWPs, allowing monitoring of the dynamics of the CWP population. For absolute quantitation, we selected a representative group of five proteins (Cwp1p, Crh1p, Scw4p, Gas1p, and Ecm33p), which had 67 x 10(3), 44 x 10(3), 38 x 10(3), 11 x 10(3) and 6.5 x 10(3) of wall-bound copies per cell, respectively. As Cwp1p is predominantly incorporated in the birth scar, this corresponds to a protein density of c. 22 x 10(3) copies microm(-2). For relative quantitation, we compared wild-type cells to gas1Delta cells, in which the cell wall integrity pathway is constitutively activated. The levels of Crh1p, Crh2p, Ecm33p, Gas5p, Pst1p and Pir3p increased about three- to fivefold, whereas the level of Scw4p was significantly decreased. We propose that our methods are widely applicable to other fungi.
Yin
Qing Yuan
QY
Laboratory for Biomacromolecular Mass Spectrometry, Swammerdam Institute for Life Sciences, University of Amsterdam, WV Amsterdam, The Netherlands.
de Groot
Piet W J
PW
de Jong
Luitzen
L
Klis
Frans M
FM
De Koster
Chris G
CG
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000737:chemistry* / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods
D029701:Saccharomyces cerevisiae Proteins