High performance liquid chromatography and time-of-flight secondary ion mass spectrometry: a new dimension in structural analysis of apolipoproteins.
We report the isolation and characterization of an apolipoprotein A-I mutant using a new technique for structural analysis of apolipoproteins based upon the combined techniques of protein isolation by isoelectric focusing in immobilized pH-gradients, reversed-phase HPLC of tryptic peptides, and subsequent molecular weight analysis of isolated peptides by time-of-flight secondary ion mass spectrometry (TOF-SIMS). The particular advantages of the TOF-SIMS procedure in the characterization of proteolytic peptides are the detection limits in the picomole range, the accuracy of molecular weight determination (up to 3000 +/- 1 D), the speed of analysis, and the wide range of applications for involatile biomolecules. The described procedure for the analysis of apolipoproteins requires only 2 ml of serum as starting material. This method can be used to monitor for genetic polymorphisms and posttranslational modifications on a microscale basis. Applying these techniques, we characterized a new apolipoprotein A-I mutant with an amino acid exchange arginine177 by histidine.
D016632:Apolipoprotein A-I; D001053:Apolipoproteins / Q000302:isolation & purification*; D001054:Apolipoproteins A / Q000235:genetics / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D006801:Humans; D007525:Isoelectric Focusing; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D009154:Mutation; D010375:Pedigree; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D016632:Apolipoprotein A-I; D001053:Apolipoproteins; D001054:Apolipoproteins A; D010446:Peptide Fragments; D014357:Trypsin