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Updated: Feb 17, 2026
Sample Preparation for Endopeptidomic Analysis in Human Cerebrospinal Fluid
Published on: December 4, 2017
Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography
Jaimie Dufresne1, Angelique Florentinus-Mefailoski1, Juliet Ajambo1
1Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3 Canada.
Background:
Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at - 80 °C prior to experiments. Plasma test samples from the - 80 °C freezer were thawed on ice or intentionally warmed to room temperature.
Methods:
Protein content was measured by CBBR binding and the release of alcohol soluble amines by the Cd ninhydrin assay. Plasma peptides released over time were collected over C18 for random and independent sampling by liquid chromatography micro electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) and correlated with X!TANDEM.
Results:
Fully tryptic peptides by X!TANDEM returned a similar set of proteins, but was more computationally efficient, than "no enzyme" correlations. Plasma samples maintained on ice, or ice with a cocktail of protease inhibitors, showed lower background amounts of plasma peptides compared to samples incubated at room temperature. Regression analysis indicated that warming plasma to room temperature, versus ice cold, resulted in a ~ twofold increase in the frequency of peptide identification over hours-days of incubation at room temperature. The type I error rate of the protein identification from the X!TANDEM algorithm combined was estimated to be low compared to a null model of computer generated random MS/MS spectra.
Conclusion:
The peptides of human plasma were identified and quantified with low error rates by random and independent sampling that revealed 1000s of peptides from hundreds of human plasma proteins from endogenous tryptic peptides.
