The state of the N-terminus of recombinant proteins: determination of N-terminal methionine (formylated, acetylated, or free).
The removal of N-terminal methionine from proteins produced by recombinant DNA techniques is often far from quantitative. Furthermore, a proportion of the methionylated product may be N alpha-blocked and thus not easily accessible to conventional (Edman) techniques of protein characterization. In this paper, a method for overcoming the resulting analytical problems is described. The technique is based on perdeuteroacetylation (performed only if unblocked methionine is to be determined), cleavage with cyanogen bromide, extraction of any acylhomoserine lactone into ethyl acetate, formation of a chemical derivative, and analysis by combined gas-liquid chromatography/mass spectrometry (GC/MS). The remaining cyanogen bromide fragments, insoluble in ethyl acetate, are available for further analysis by mass spectrometric or other methods if required. Using an acylhomoserine lactone labeled with a stable isotope as internal standard, the method is semiquantitative. It should be possible to develop a quantitative method if appropriate polypeptide standards are prepared. N-Terminal processing of eight recombinant-derived proteins is discussed.
Département de Biochimie Médicale, CMU, Geneva, Switzerland.
K
K
Rose
L A
LA
Savoy
M G
MG
Simona
R E
RE
Offord
P T
PT
Wingfield
R J
RJ
Mattaliano
D R
DR
Thatcher
D000107:Acetylation; D005561:Formates / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry; D008715:Methionine / Q000032:analysis*; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000032:analysis; D011994:Recombinant Proteins / Q000032:analysis*
D005561:Formates; D011994:Recombinant Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D008715:Methionine