Kuipers Bas J H, Gruppen Harry
Department of Agrotechnology and Food Sciences, Laboratory of Food Chemistry, Wageningen University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands.
J Agric Food Chem. 2007 Jul 11;55(14):5445-51. doi: 10.1021/jf070337l. Epub 2007 Jun 1.
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.
在乙腈和甲酸存在的条件下，于214 nm处测定了20种氨基酸和肽键的摩尔消光系数，以便在通过质谱鉴定后，对反相高效液相色谱洗脱的肽进行定量比较（反相高效液相色谱-质谱联用，RP-HPLC-MS）。肽键的摩尔消光系数为923 M⁻¹ cm⁻¹。色氨酸的摩尔消光系数比肽键的约高30倍，而苯丙氨酸、酪氨酸和组氨酸的摩尔消光系数比肽键的约高6倍。作为单个氨基酸时，脯氨酸的摩尔消光系数可忽略不计。然而，当它存在于肽链中（N端除外）时，其吸收量约为肽键的3倍。甲硫氨酸的摩尔消光系数与肽键相似，而所有其他氨基酸的摩尔消光系数则低得多。通过氨基酸组成和肽键数量计算得出的蛋白质和肽的摩尔消光系数的可预测性，已使用几种蛋白质和肽进行了验证。大多数测量值和计算值的摩尔消光系数吻合良好，这表明有可能对通过RP-HPLC-MS分析的肽进行定量比较。该方法能够对经RP-HPLC-MS鉴定后的水解产物中存在的所有肽进行定量分析。
