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

Abstract

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.

Truncated Amyloid-β(11–40/42) from Alzheimer Disease Binds Cu2+ with a Femtomolar Affinity and Influences Fiber Assembly

“…A␤ and A␤ lack a tyrosine residue so the peptide absorbance at 214-nm ⑀ 214 ϭ 923 cm Ϫ1 M Ϫ1 per amide was used (49). All lyophilized peptides typically contained 10 -20% moisture by weight.…”

Cooperative induction of ordered peptide and fatty acid aggregates

“…The crude peptide was purified by reverse-phase HPLC on a Kinetex C18 5 μm 100 Å 250x10 mm column (Phenomenex) in a gradient of acetonitrile in 0.1% TFA. The purified peptide was quantitated by its calculated extinction coefficient at 214 nm of 6797 M -1 cm -1 (19). Pure peptide was stored in lyophilized aliquots and dissolved in water just before use.…”

Antibody Recognition of a Human Chorionic Gonadotropin Epitope (hCGβ66–80) Depends on Local Structure Retained in the Free Peptide

“…Sample Preparation-The peptides were dissolved directly in 10 mM phosphate buffer solution (pH 6.8), with a final concentration of 0.11 mM (0.177 mg ml Ϫ1 ). The peptides stock concentration was confirmed by UV-visible spectroscopy using a calculated extinction coefficient of ⑀ 214 ϭ 21,807 M Ϫ1 cm Ϫ1 (20). For the samples with the fluorinated alcohol HFIP, water and 0.2 M buffer solutions were used to ensure the overall buffer concentration remained 10 mM.…”