Short-chain peptide analysis by high-performance liquid chromatography coupled to electrospray ionization mass spectrometer after derivatization with 9-fluorenylmethyl chloroformate.
Resolution and characterization of short-chain peptides (M(r) = 200-1000) and free amino acids were demonstrated by the use of precolumn derivatization with 9-fluorenylmethyl chloroformate (Fmoc) followed by reverse-phase high-performance liquid chromatography (RP-HPLC) interfaced with an electrospray ionization mass spectrometer (ESI-MS). At pH 10, in addition to derivatization at the N terminus, epsilon-NH(2) and OH groups of lysine and tyrosine residues, respectively, were also derivatized. Fmoc derivatives showed at least 2 orders of magnitude higher ionization potential in the presence of trifluoroacetic acid. The detection levels for both the free amino acid and peptide derivatives were in a few hundred picomoles compared to 10-50 nmol for the underivatized samples. The mass spectra of the peptides before or after derivatization showed the presence of only singly charged ions. However, collision-induced dissociation of the derivatized peptides showed predominance of b-type ions that are relatively less complicated in assigning the peptide sequence.
Gartenmann
K
K
Nestlé Research Center, P.O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland.
Kochhar
S
S
D000596:Amino Acids / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D005449:Fluorenes / Q000737:chemistry*; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry*; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*
D000596:Amino Acids; D005449:Fluorenes; D007202:Indicators and Reagents; D010455:Peptides; C054007:1-(9-fluorenyl)methyl chloroformate