Analytical challenges and strategies for the characterization of gp96-associated peptides.
This paper describes the methods that were used for the preparation of gp96-associated peptides and the analysis of these peptides using mass spectrometry. A general approach for stripping, enriching, and separating peptides associated with gp96 is presented. Protocols for the demonstration of the diversity of gp96-associated peptides using mass spectrometry and the identification of these peptides using a combination of tandem mass spectrometry and protein database searching are described. Important parameters and factors that affect the outcome of the experiments are discussed.
Liu
Chuanliang
C
Antigenics, Inc., Woburn, MA 01801, USA. cliu@antigenics.com
Ewing
Nigel
N
DeFilippo
Melissa
M
D000818:Animals; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006360:Heat-Shock Proteins / Q000737:chemistry; D007202:Indicators and Reagents; D051379:Mice; D009369:Neoplasms / Q000276:immunology; D010446:Peptide Fragments / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D000951:Antigens, Neoplasm; D006360:Heat-Shock Proteins; D007202:Indicators and Reagents; D010446:Peptide Fragments; C052646:sarcoma glycoprotein gp96 rejection antigens