Semi-automated chromatographic procedure for the isolation of acetylated N-terminal fragments from protein digests.
Several published procedures have been combined to develop a general strategy for the specific identification and isolation of the acetylated-N-terminal fragment from all other proteolytic fragments. This ruse can be divided into four steps: (i) succinylation of the substrate to block lysine NH2 groups; (ii) enzymatic digestion of the modified protein; (iii) automated phenylisothiocyanate derivatization of the protease derived fragments to block newly generated "free" N-termini; and (iv) reversed-phase high-performance liquid chromatography with on-line photodiode array spectroscopy. The individual phenylthiocarbamyl-peptide species exhibit an increased reversed-phase retention time and a greater UV (210-297 nm) profile compared to the corresponding control (-phenylisothiocyanate) digest. The N-terminal acetylated fragment shows neither a retention time shift nor an augmented UV profile. To validate each process step, synthetic peptides and acetylated-N-terminal proteins of known sequence were used as test samples. The desired fragment was isolated from three proteins and positively identified by electrospray mass spectrometry and amino acid composition. Proteins with other N-terminal blocking groups should be amenable to this procedure.
Howard Hughes Medical Institute, Core Protein/Peptide Facility, Washington University School of Medicine, St. Louis, MO 63110.
D L
DL
Crimmins
R S
RS
Thoma
D000107:Acetylation; D000595:Amino Acid Sequence; D001322:Autoanalysis / Q000379:methods*; D002147:Calmodulin / Q000737:chemistry / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000378:metabolism; D003574:Cytochrome c Group / Q000737:chemistry / Q000302:isolation & purification; D017879:Isothiocyanates; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000737:chemistry / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification*; D013056:Spectrophotometry, Ultraviolet; D013386:Succinates; D019802:Succinic Acid; D013861:Thiocyanates
D002147:Calmodulin; D003574:Cytochrome c Group; D017879:Isothiocyanates; D010320:Parvalbumins; D010446:Peptide Fragments; D013386:Succinates; D013861:Thiocyanates; C005441:phenylisothiocyanate; D019802:Succinic Acid; D002918:Chymotrypsin