Rapid on-membrane proteolytic cleavage for Edman sequencing and mass spectrometric identification of proteins.
A method for the rapid limited enzymatic cleavage of PVDF membrane-immobilized proteins is described. This method allows the fast characterization of PVDF blotted proteins by peptide mass fingerprinting (Henzel, W. J., Billeci, T. M., Stults, J. T., Wong, S. C., Grimley, C., Wantanabe, C., Proc. Natl. Acad. Sci. USA 1993, 90, 5011-5015), LC-MS/MS, or N-terminal sequencing and has been demonstrated on a range of proteins using a full complement of proteolytic enzymes. This technique allows the generation of proteolytic fragments between 5 and 60 min (depending on the enzyme employed), which is significantly faster than previously reported on-membrane digestion methods. To date, this on-membrane rapid digestion protocol has aided in the identification and confirmation of mutation sites in over 200 recombinant proteins.
Pham
Victoria C
VC
Department of Protein Chemistry, Genentech Inc., South San Francisco, CA 94080, USA.
Henzel
William J
WJ
Lill
Jennie R
JR
D000595:Amino Acid Sequence; D016326:Extracellular Matrix Proteins; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D008567:Membranes, Artificial; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry; D010447:Peptide Hydrolases / Q000378:metabolism*; D011145:Polyvinyls; D011506:Proteins / Q000032:analysis*; D018123:Receptors, Interleukin / Q000737:chemistry; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D016212:Transforming Growth Factor beta / Q000276:immunology; D005465:Fluorocarbon Polymers
D016326:Extracellular Matrix Proteins; D007147:Immunoglobulin Light Chains; D008567:Membranes, Artificial; D009363:Neoplasm Proteins; D010447:Peptide Hydrolases; D011145:Polyvinyls; D011506:Proteins; D018123:Receptors, Interleukin; D016212:Transforming Growth Factor beta; C122166:SPON2 protein, human; C492510:interleukin-20 receptor; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers