Quantitation of yeast cell wall proteins 889 Fig. 1. (a) Experimental steps of absolute and relative quantitation of cell wall proteins using iTRAQ. For absolute quantitation, wild-type cell wall peptides were labeled with iTRAQ115 reagent and then mixed with iTRAQ114-labeled synthetic peptides. The peptide mixture a was subse- quently subjected to MALDI-MS/MS. For relative quantitation, equal amounts of dry cell walls from wild-type and gas1D cells were digested with trypsin. The resulting peptides were labeled with iTRAQ114 and iTRAQ116, respectively. The experiment was repeated on samples from a replicate culture with iTRAQ117 for the wild-type sample and iTRAQ114 for the gas1D sample (see ‘Materials and methods’). The peptide mixture b was subsequently subjected to LC-MS/MS. (b) A diagram of the iTRAQ reaction. Each iTRAQ reagent molecule consists of a reporter group (based on N-methylpiperazine), a mass balance group (the carbonyl group), and a peptide-reactive group (N-hydroxylsuccinimide ester). The overall mass of reporter and balance components of the molecule is kept constant using differential isotopic enrichment with 13C, 15N and 18O atoms. Peptides from differently labeled samples are therefore chroma- tographically indistinguishable and isobaric, but yield speciﬁc reporter ions at m/z 114, 115, 116 or 117 following MS/MS. FEMS Yeast Res 7 (2007) 887–896 derivatized peptides from different samples are isobaric (i.e. of the same mass) and chromatographically indistinguish- able, but yield speciﬁc reporter ions at m/z 114, 115, 116 or 117 following collision-induced dissociation in a mass spectrometer. The relative concentrations of the peptides are thus deduced from the relative intensities of the corre- sponding reporter ions. For absolute quantitation, dried peptides from 2 mg of cell walls were resuspended in 20 mL of acetonitrile and 10 mL of 0.5 M triethylammonium bicarbonate, pH 8.0. Ten mi- croliters of iTRAQ115 reagent was added, and the reaction mixture was incubated at room temperature for 1 h. The labeled peptides were dried to remove organic solvent, and resuspended in 10 mL of H2O. Subsequently, the labeled cell wall peptides were mixed with the working standard solu- tion of the synthetic peptides at ratios of 1 : 5, 1 : 1 and 5 : 1 (v:v). For relative quantitation, equal amounts of dried cell walls from wild-type and gas1Dcells were digested with the endoprotease trypsin. The resulting peptides were labeled with iTRAQ reagents (iTRAQ114 on the wild-type sample and iTRAQ116 on the gas1D sample). The experiment was repeated on samples from a replicate culture with a different combination of iTRAQ tags, i.e. iTRAQ117 for the wild-type sample, and iTRAQ114 for the gas1D sample. Matrix- assisted laser- desorption ionization (MALDI)-MS/MS analysis For absolute quantiﬁcation, peptides were collected on Ziptip mC18 pipette tips (Millipore, Bedford, MA), washed with 0.1% formic acid, and eluted with 60% acetonitrile/0.1% formic acid. Subsequently, 0.5 mL of the eluate was mixed with 0.5 mL of a-hydroxy cinnaminic acid (10 mg mL 1 in acetonitrile/ethanol 1 : 1, v:v). The mixture was spotted on a MALDI target plate and allowed to dry. MS was performed using a QSTAR XL Hybrid LC/MS/MS system (Applied Biosystems, Framingham, MA) with its oMALDI (orthogonal MALDI) ion source. To obtain precursor masses of target peptides, the mass spectrometer was set to perform data acquisition in the positive ion mode, with a selected mass range of m/z 800–2000. Peptides were selected for MS/MS, and the time of summation of MS/MS events was set at 3 s. Liquid chromatography (LC)-MS/MS analysis For relative quantiﬁcation, six LC-MS/MS runs were per- formed for each sample of mixed peptides from wild-type and gas1D cells. Proteolytic digests derived from 30 mg of freeze-dried cell walls were fractionated on a 150 mm 75 mm (length inner diameter) reversed-phase capillary column (PepMap C18, Dionex). Sample introduction and mobile phase delivery at 300 nL min 1 were performed using an Ultimate nano-LC system (Dionex, Sunnyvale, CA) equipped with a 10-mL injection loop. Mobile phase A was c 2007 The Authors Journal compilation c 2007 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd.