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