Direct atmospheric pressure coupling of polyacrylamide gel electrophoresis to mass spectrometry for rapid protein sequence analysis.
Using laser desorption-atmospheric pressure chemical ionization we describe a novel approach for coupling mass spectrometry to polyacrylamide gel electrophoresis. In contrast to other approaches, the method allows for the direct sampling of a polyacrylamide gel-embedded protein without the addition of any exogenous matrixes and is performed at atmospheric pressure. After electrophoresis and enzymatic digestion, the gel is analyzed at AP by photons that desorb neutral peptide molecules, followed by corona discharge ionization in the gas-phase, and subsequent mass analysis. Our experimental results demonstrate the method to (1) rapidly identify electrophoresed proteins via "peptide fingerprinting" using protein databases, (2) detect single-amino acid polymorphisms, and (3) has potential for sub-picomole sensitivity while still maintaining in situ gel desorption-ionization at ambient conditions.
Coon
Joshua J
JJ
Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
Steele
Heather A
HA
Laipis
Philip J
PJ
Harrison
W W
WW
D001274:Atmospheric Pressure; D002256:Carbonic Anhydrases / Q000032:analysis / Q000235:genetics; D030562:Databases, Protein; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D009211:Myoglobin / Q000032:analysis; D017354:Point Mutation; D020539:Sequence Analysis, Protein / Q000295:instrumentation / Q000379:methods*
D009211:Myoglobin; D002256:Carbonic Anhydrases