The proteins cleaved by endogenous tryptic proteases in normal EDTA plasma by C18 collection of peptides for liquid chromatography micro electrospray ionization and tandem mass spectrometry.

The tryptic peptides from ice cold versus room temperature plasma were identified by C18 liquid chromatography and micro electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Samples collected on ice showed low levels of endogenous tryptic peptides compared to the same samples incubated at room temperature. Plasma on ice contained peptides from albumin, complement, and apolipoproteins and others that were observed by the X!TANDEM and SEQUEST algorithms. In contrast to ice cold samples, after incubation at room temperature, greater numbers of tryptic peptides from well characterized plasma proteins, and from cellular proteins were observed. A total of 583,927 precursor ions and MS/MS spectra were correlated to 94,669 best fit peptides that reduced to 22,287 correlations to the best accession within a gene symbol and to 7174 correlations to at least 510 gene symbols with ≥ 5 independent MS/MS correlations (peptide counts) that showed FDR q-values ranging from E-9 (i.e. FDR = 0.000000001) to E-227. A set of 528 gene symbols identified by X!TANDEM and SEQUEST including C4B showed ≥ fivefold variation between ice cold versus room temperature incubation. STRING analysis of the protein gene symbols observed from endogenous peptides in normal plasma revealed an extensive protein-interaction network of cellular factors associated with cell signalling and regulation, the formation of membrane bound organelles, cellular exosomes and exocytosis network proteins. Taken together the results indicated that a pool of cellular proteins, or protein complexes, in plasma are apparently not stable and degrade soon after incubation at room temperature.

Random and independent sampling of endogenous tryptic peptides from normal human EDTA plasma by liquid chromatography micro electrospray ionization and tandem mass spectrometry.

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.

Freeze-dried plasma proteins are stable at room temperature for at least 1 year.

Thirty human EDTA plasma samples from male and female subjects ranging in age from 24 to 74 years were collected on ice, processed ice cold and stored frozen at -80 °C, in liquid nitrogen (LN2), or freeze dried and stored at room temperature in a desiccator (FDRT) or freeze dried and stored at -20 °C for 1 year (FD-20). In a separate experiment, EDTA plasma samples were collected onto ice, processed ice cold and maintained on ice ± protease inhibitors versus incubated at room temperature for up to 96 h. Random and independent sampling by liquid chromatography and tandem mass spectrometry (LC-ESI-MS/MS), as correlated by the MASCOT, OMSSA, X!TANDEM and SEQUEST algorithms, showed that tryptic peptides from complement component 4B (C4B) were rapidly released in plasma at room temperature. Random sampling by LC-ESI-MS/MS showed that peptides from C4B were undetectable on ice, but peptides were cleaved from the mature C4B protein including NGFKSHALQLNNR within as little as 1 h at room temperature. The frequency and intensity of precursors within ± 3 m/z of the C4B peptide NGFKSHALQLNNR was confirmed by automated targeted analysis where the precursors from MS/MS spectra that correlated to the target sequence were analyzed in SQL/R. The C4B preproprotein was processed at the N terminus to release the mature chain that was cleaved on the carboxyl side of the isoprene C2 domain within a polar C terminal sequence of the mature C4B protein, to reveal the thioester reaction site, consistent with LC-ESI-MS/MS and Western blot. Random sampling showed that proteolytic peptides from complement component C4B were rarely observed with long term storage at - 80 °C in a freezer or in liquid nitrogen (LN2), freeze drying with storage at - 20 °C (FD-20 °C) or freeze drying and storage at room temperature (FDRT). Plasma samples maintained at room temperature (RT) showed at least 10-fold to 100-fold greater frequency of peptide correlation to C4B and measured peptide intensity compared to samples on ice for up to 72 h or stored at - 80 °C, LN2, FDRT or FD-20 °C for up to a year.