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
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
复制标题
DOI：
10.1021/jf070337l
复制
发表时间：
2007-07-11
影响因子：
6.1
通讯作者：
Gruppen, Harry
中科院分区：
文献类型：
--
作者：
Kuipers, Bas J. H.;Gruppen, Harry
在存在乙腈和甲酸的情况下，在214 nm处测量20种氨基酸和肽键的摩尔消光系数，以便在用质谱法（RP-HPLC-MS）鉴定后，对从反相高效液相色谱法洗脱的肽进行定量比较。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摩尔消光系数高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摩尔消光系数高6倍。脯氨酸作为单个氨基酸，具有可忽略的摩尔消光系数。然而，当存在于肽链中时（除了在N末端），它的吸收类似于肽键的三倍。甲硫氨酸具有与肽键相似的摩尔消光系数，而所有其他氨基酸具有低得多的摩尔消光系数。使用几种蛋白质和肽，通过氨基酸组成和存在的肽键的数量计算的蛋白质和肽的摩尔消光系数的可预测性进行了验证。大多数的测量和计算的摩尔消光系数是在良好的协议，这表明，它是可能的比较肽分析RP-HPLC-MS定量的方式。该方法能够对水解产物中存在的所有肽进行定量分析，一旦用RP-HPLC-MS鉴定。
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 similar to 30 times higher than that of the peptide bond, whereas the molar extinction coefficients of phenylalanine, tyrosine, and histidine are similar to 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 similar to 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.