Detection of amino acid and peptide phosphate protonation using Raman spectroscopy

Raman spectra of phosphorylated amino acids and peptides undergo pH-dependent changes attributed to protonation of -OPO32− (dibasic) to -OPO3H− (monobasic). Bands at approximately 980 and 1080 cm−1 in solution Raman spectra of phosphoserine and phosphothreonine are assigned to the monobasic and dibasic phosphate groups, respectively. Calibrated Raman peak area ratio measurements, performed as a function of pH, are used to determine the corresponding pKa values of 5.6 (phosphoserine) and 5.9 (phosphothreonine). In peptides, the phosphate Raman bands are difficult to distinguish due to interference from other neighboring bands (particularly those derived from aromatic amino acid residues) as well as the relatively low solubility of peptides. Nevertheless, drop coating deposition Raman (DCDR) spectra obtained from 100-μM peptide solutions reveal pH-dependent second derivative features at approximately 980 and 1080 cm−1, which are indicative of phosphate protonation.