Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis.
The molar extinction coefficients of 20 amino acids and the peptide bond were measured at 214 nm in the presence of acetonitrile and formic acid to enable quantitative comparison of peptides eluting from reversed-phase high-performance liquid chromatography, once identified with mass spectrometry (RP-HPLC-MS). The peptide bond has a molar extinction coefficient of 923 M(-1) cm(-1). Tryptophan has a molar extinction coefficient that is approximately 30 times higher than that of the peptide bond, whereas the molar extinction coefficients of phenylalanine, tyrosine, and histidine are approximately six times higher than that of the peptide bond. Proline, as an individual amino acid, has a negligible molar extinction coefficient. However, when present in the peptide chain (except at the N terminus), it absorbs approximately three times more than a peptide bond. Methionine has a similar molar extinction coefficient as the peptide bond, while all other amino acids have much lower molar extinction coefficients. The predictability of the molar extinction coefficients of proteins and peptides, calculated by the amino acid composition and the number of peptide bonds present, was validated using several proteins and peptides. Most of the measured and calculated molar extinction coefficients were in good agreement, which shows that it is possible to compare peptides analyzed by RP-HPLC-MS in a quantitative way. This method enables a quantitative analysis of all peptides present in hydrolysates once identified with RP-HPLC-MS.
Kuipers
Bas J H
BJ
Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
Gruppen
Harry
H
D000596:Amino Acids / Q000737:chemistry*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D010455:Peptides / Q000737:chemistry*; D011392:Proline / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*
D000596:Amino Acids; D010455:Peptides; D011506:Proteins; D011392:Proline