Abstract
BackgroundNormal 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.MethodsProtein 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.ResultsFully 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.ConclusionThe 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.
Highlights
Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments
A second key contribution of this paper is to show that the peptides from proteins expressed in tissues and cells may be identified and quantified by micro electrospray with LC–ESI–MS/MS of endogenous peptides with a simple linear ion trap [36] that shows a low type I error rate by comparison to a null model of random MS/MS spectra, or noise spectra, and computing protein p-values using X!TANDEM that shows a low False Discovery Rate using the method of Benjamini and Hochberg [37]
EDTA plasma collected directly onto ice were analyzed by C18 preparative chromatography followed by micro electrospray LC–ESI–MS/MS of samples incubated on ice or ice plus protease inhibitors
Summary
Normal human EDTA plasma samples were collected on ice, processed ice cold, and stored in a freezer at – 80 °C prior to experiments. There is considerable variation in the peptides observed, and even the trends reported, in the study of degradation of blood proteins that may differ between groups, likely from the large variation that occurs immediately after sample collection [2, 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. One key contribution of this study is the collection of EDTA plasma directly onto ice for cold processing to establish a reliable baseline compared to plasma peptides at room temperature
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