Quantification of peptides and proteins by spectrofluorimetric detection of native fluorescence and mass spectrometry

摘要

We explored the native fluorescence of peptides and proteins as tool that can improve quantification of peptides and proteins by LC-MS. Natively fluorescent amino acids, as well as peptides and proteins were successfully separated by HPLC, and quantified with spectrofluorimetric detection and ESI-MS. Amino acids were separated successfully using HPLC with retention times of 4.2 min, 5.5 min, and 9.8 min for tyrosine, phenylalanine, and tryptophan, respectively. Retention times were 9.6, 11.7, 13.7, 19.8, 22.6, 27.3, and 32.5 min for MRFA, neurotensin fragment, bradykinin, angiotensin II, Glu-fib, leucine enkephalin, and neurotensin, respectively. Cytochrome c, trypsinogen, and myoglobin were separated with retention time of 15.2, 22.5, and 36.7 min, respectively. A good linear relationship was found over the investigated concentration range (580-0.0012 (mu)M for amino acids, 0.51 (mu)M to 230 (mu)M for peptides, and 0.041-4 (mu)M for proteins), as indicated by correlation coefficient of determination (>0.99) for all the calibration curves obtained. Therefore, the detectors connected in series have provided simultaneous measurements of fluorescence intensity and ionization efficiencies, as well as structural characterization of polypeptides. Fluorescence detection provides better sensitivity, linearity, and repeatability than mass spectrometry for most of the small biomolecules (amino acids and peptides) analyzed in this study. Mass spectrometry provides sensitivity and linearity of response for proteins that is comparable to fluorescence detection. For example, for the peptide bradykinin acetate, LOD is 510 nM and 900 nM for fluorescence and MS, respectively. For the protein trypsinogen, the LOD was 41 nM and 41 nM for fluorescence and MS, respectively. Hence, native fluorescence appears to be a better detection method than MS for quantification of peptides. We successfully separated tryptic peptides of BSA and further experiments are underway to characterize tryptic digest of proteins and real biological samples. These experiments will enable quantification of peptide and proteins in complex mixtures.