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Dataset Identifier

Metadata
datasetIdentifierPASS00443
datasetTypeMSMS
submitterWeixuan Chen <weixuan.chen@chemistry.gatech.edu>
submitter_organizationGeorgia Institute of Technology
lab_head_full_nameRonghu Wu
lab_head_emailronghu.wu@chemistry.gatech.edu
lab_head_organizationGeorgia Institute of Technology
lab_head_countryUSA
datasetTagBA_for_N_GlyPeptide
datasetTitleA Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by MS
publicReleaseDate2014-02-25 00:00:00
finalizedDate2014-03-18 09:40:55
summaryThese raw files are for comprehensive analysis of protein N-glycosylation in yeast whole cell lysates.
contributorsWeixuan Chen, Johanna M. Smeekens, and Ronghu Wu
publicationWeixuan Chen, Johanna M. Smeekens, and Ronghu Wu, A Universal Chemical Enrichment Method for Mapping the Yeast N-glycoproteome by MS, molecular & cellular proteomics, submitted
growthBY4742 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).
treatmentno treatment was applied in our experiments.
extractionCells 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.
separationThe 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).
digestionThe 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.
acquisitionPeptides 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.
informaticsAll 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
instrumentsThermo Scientific LTQ Orbitrap Elite
speciesYeast
massModificationsstatic: C+57.021464, variable: M+15.99492, N+2.988266

Official URL for this dataset: http://www.peptideatlas.org/PASS/PASS00443
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Username: PASS00443
Password: TA3755yv

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Listing of files:

   48 Mar 18  2014 Annotated and viewable spectra
  653 Feb 19  2014 PASS00443_DESCRIPTION-2014-01-19_193838.txt
 4.0K Feb 25  2014 PASS00443_DESCRIPTION.txt
 4.0K Feb 19  2014 RawFiles

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