Application of a fluorescent peptide assay to the optimization of peptide generation from patient-derived breast cancer xenografts

摘要

Knowing the peptide concentration of proteolytic digestion is important, as it allows for sample evaluation and normalization before injection during the mass spectrometry analysis. The standard methods to measure proteins, such as UV absorption or protein assays, work poorly with peptides or consume too much peptides. Insufficient characterization and normalilzation leads to difficulties in standardization and reproducibility of mass spectrometry analysis. Recently, ThermoScientific developed a new product, Pierce Quantitative Fluorometric Peptide Assay, to address the needs. We β-tested the product and found it to be sensitive, accurate and reproducible. The assay is based on the reaction between the labelling reagent and the N-terminal primary amine of the peptides. During the testing, a variety of peptides were serially diluted and the limit of quantification (LOQ) was determined to be 15 ng/ul. The quantification curve is liner in the range of 3 ng/ul to 1000 ng/ul. The concentrations obtained by the fluorescent assay for the tested peptides correlate well with the concentrations obtained from AAA analysis. Using this method we were able to evaluate the peptide recovery of tryptic digestions of different complex proteins with different digestion methods, such as FASP, Barocyler digest and Thermomixer digest. We could detect digested peptides from as low as 100 ng of protein input. Second, we used this quantitation method to optimize methods for detergent tolerance removal by PASP. Third, using this method we are able to normalize the samples before injection into the LC-MS for relative quantitation of differences between multiple samples. The N-terminal primary amine labeling of the fluorometric peptide assay also makes it possible to normalize the peptides for TMT labelling as well as the monitoring the completion of the labelling.