HUMAN PROTEINPEDIA 


Experimental Details - HuPA_00047
Experiment type Mass spectrometry
Short description Glycosylation analysis
Experimental description Saliva was collected from healthy individuals. Proteins from saliva were precipitated using ethanol. The proteins were then either prefractionated by pI using Zoom-IEF fractionation or used as such without any prefractionation. Proteins from Zoom fractions were precipitated using acetone. The proteins obtained with or without prefractionation were resuspended in coupling buffer. Sodium periodate was added to oxidize sugars on the glycoproteins. Glycoproteins were then immobilized on an agarose-Hz resin via the sugars. The glycoproteins were digested in situ using trypsin. Non glycopeptides were washed with 1.5 M NaCl, water, methanol, 80% ACN/ 0.1% TFA and 100mM Ammonium Bicarbonate solution. The beads were then resuspended in 100mM ammonium bicarbonate. PNGase F was added to cleave formerly glycosylated N-glycopeptides from the sugars. The peptides were dried and resuspended in 0.1% formic acid and analyzed by LC-MS/MS.
Principal Investigator's NameDr. Joseph Loo
TitleProfessor
E-mailjloo@chem.ucla.edu
AddressUniversity of California, Los Angeles (UCLA)
402 Paul Boyer Hall (MBI)
Los Angeles, CA 90095
CountryUSA
Lab URLhttp://loolab.chem.ucla.edu/
Data submitted byPrasanna Ramachandran
TitlePh.D. student
E-mailpramacha@chem.ucla.edu
Published/Unpublished Published
Journal name
PubMed ID 16740002
Sample source
Tissue: Saliva [eVOC:EV:0100515]
Cell line:
Source organism Homo sapiens [Taxonomy:9606]
Labeling technique None
Protease used Trypsin
Is the sample from in gel No
Reduction and Alkylation Reduction of samples was done with TCEP and alkylation with iodoacetamide
Mass spectrometer
Instrument: QSTAR [PSI:1000190]
Vendor: ABI/SCIEX [PSI:1000121]
Ionization method ESI [PSI:1000073]
Fragmentation method CID [PSI:1000133]
Mass tolerance used for database searching (MS) 0.3 Da
Mass tolerance used for database searching (MS/MS) 0.3 Da
Database used for searching IPI
Search engine used Mascot
Download MS raw dataset  
Download processed files  



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