Metadata |
datasetIdentifier | PASS00443 |
datasetType | MSMS |
submitter | Weixuan Chen <weixuan.chen@chemistry.gatech.edu> |
submitter_organization | Georgia Institute of Technology |
lab_head_full_name | Ronghu Wu |
lab_head_email | ronghu.wu@chemistry.gatech.edu |
lab_head_organization | Georgia Institute of Technology |
lab_head_country | USA |
datasetTag | BA_for_N_GlyPeptide |
datasetTitle | A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by MS |
publicReleaseDate | 2014-02-25 00:00:00 |
finalizedDate | 2014-03-18 09:40:55 |
summary | These raw files are for comprehensive analysis of protein N-glycosylation in yeast whole cell lysates. |
contributors | Weixuan Chen, Johanna M. Smeekens, and Ronghu Wu |
publication | Weixuan Chen, Johanna M. Smeekens, and Ronghu Wu, A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by MS, molecular & cellular proteomics, submitted |
growth | BY4742 MAT alpha yeast were grown in YPD overnight. Cells were harvested by centrifugation in the exponential growth phase (O.D. was about 1.0 at 600nm). |
treatment | no treatment was applied in our experiments. |
extraction | Cells were resuspended at 4ºC in a buffer containing 50 mM Tris pH 8.2, 8 M urea, 75 mM NaCl, 2% SDS and one protease inhibitor cocktail tablet (complete mini, EDTA-free, Roche) per 10 mL. Cells were lysed using the MiniBeadbeater (Biospec) at maximum speed, four cycles of 40 s each, with 2 min pauses between cycles to minimize protein degradation. After centrifuging at 13,000 rpm, the supernatants were transferred to new tubes. |
separation | The peptide mixture was dissolved in 100 mM ammonium acetate buffer, and incubated for one hour with rotation with magnetic beads bound to boronic acid. After incubation, the beads were washed three times with the binding buffer. Enriched peptides were eluted first by incubation with a 5% formic acid solution at 37 ºC for 30 min. The peptides were further eluted with a solution containing acetonitrile:H2O:trifluoroacetic acid at 50:49:1.
The enriched glycopeptide samples were desalted again using a tC18 Sep-Pak cartridge. Then we performed the separation of glycopeptides using high-pH reversed phase HPLC (pH=10). |
digestion | The purified proteins were digested with trypsin (Promega) at the ratio of ~ 100:1 in 25 mM Tris pH 8.2, 1.5 M urea, at 37ºC for 15 h. |
acquisition | Peptides were separated by reversed-phase chromatography using an UltiMate 3000 binary pump with a 90-min gradient of 4-30% ACN (in 0.125% FA) and detected in a hybrid dual-cell quadrupole linear ion trap – orbitrap mass spectrometer (LTQ Orbitrap Elite, ThermoFisher, with a software of Xcalibur 2.0.7 SPI) using a data-dependent Top20 method. For each cycle, one full MS scan (resolution: 60,000) in the Orbitrap at 1 million AGC target was followed by up to 20 MS/MS in the LTQ for the most intense ions. Selected ions were excluded from further analysis for 90 s. Ions with a single or unassigned charge were not sequenced. Maximum ion accumulation times were 1000 ms for each full MS scan and 50 ms for MS/MS scans. |
informatics | All MS/MS spectra were then searched using the SEQUEST algorithm (version 28). Spectra were matched against a database encompassing sequences of all proteins (6607 protein entries) in the yeast ORFs database (S288C 2010) downloaded from SGD. Each protein sequence was listed in both forward and reversed orientations to estimate false positive rate (FDR) of peptide identification. The following parameters were used for the database search: 20 ppm precursor mass tolerance; 1.0 Da product ion mass tolerance; fully tryptic digestion; up to two missed cleavages; variable modifications: oxidation of methionine (+15.9949) and O18 tag of Asn (+2.9883); fixed modifications: carbamidomethylation of cysteine (+57.0214).
The target-decoy method was employed to evaluate and further control FDRs of glycopeptide identification. Linear discriminant analysis (LDA) was utilized to distinguish correct and incorrect peptide identifications using numerous parameters such as Xcorr, ΔCn, and precursor mass error |
instruments | Thermo Scientific LTQ Orbitrap Elite |
species | Yeast |
massModifications | static: C+57.021464, variable: M+15.99492, N+2.988266 |