De novo proteomic sequencing of a monoclonal antibody raised against OX40 ligand.
De novo sequencing of a full-length monoclonal antibody raised against OX40 ligand is described. Using a combination of overlapping complementary proteolytic and chemical digestions, with analysis by mass spectrometry and Edman degradation, both the heavy and light chains were fully sequenced. Particular attention was paid to those modifications that could be susceptible to degradation in the complementarity determining region and Fc region. An overview of the protocol is described, and suggestions for improvements to aid in such sequencing projects in the future are discussed.
Department of Protein Chemistry, Genentech, South San Francisco, CA 94080, USA.
Victoria
V
Pham
William J
WJ
Henzel
David
D
Arnott
Sarah
S
Hymowitz
Wendy N
WN
Sandoval
Bao-Tran
BT
Truong
Henry
H
Lowman
Jennie R
JR
Lill
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000494:pharmacology; D022801:Complementarity Determining Regions / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D053278:OX40 Ligand; D009943:Organophosphorus Compounds; D011145:Polyvinyls / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods*; D000079424:Tumor Necrosis Factor Inhibitors; D048069:Tumor Necrosis Factors / Q000276:immunology*; D005465:Fluorocarbon Polymers
D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D007135:Immunoglobulin Variable Region; D008562:Membrane Glycoproteins; D053278:OX40 Ligand; D009943:Organophosphorus Compounds; D011145:Polyvinyls; D000079424:Tumor Necrosis Factor Inhibitors; D048069:Tumor Necrosis Factors; C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; C089251:TNFSF4 protein, human; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers