Gel and gel-free proteomics to identify Saccharomyces cerevisiae cell surface proteins.
The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S-S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC-MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. "Bona fide" cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.
Insenser
María Rosa
MR
Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Plaza de Ramón y Cajal s/n, 28040 Madrid, Spain.
Hernáez
María Luisa
ML
Nombela
César
C
Molina
María
M
Molero
Gloria
G
Gil
Concha
C
D019070:Cell Lineage; D002462:Cell Membrane / Q000378:metabolism*; D002473:Cell Wall / Q000378:metabolism; D004229:Dithiothreitol / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005656:Fungal Proteins / Q000378:metabolism; D005800:Genes, Fungal; D013058:Mass Spectrometry / Q000379:methods; D010455:Peptides / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D014774:Virulence
D005656:Fungal Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D004229:Dithiothreitol