Abstract
Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins.
Highlights
The human plasma proteome contains proteins from all tissues and its composition is hypothesized to reflect dynamic
Using shotgun proteomics and targeted analyses by multiple reaction monitoring (MRM), we examined the stability of blood proteomes at the peptide level under variable storage and handling conditions commonly encountered in a clinic setting, where blood is routinely drawn for purposes other than proteomic analyses
Quantitative plasma protein measurements may be affected by proteolysis or oxidation because of uncontrolled preanalytical variables
Summary
The human plasma proteome contains proteins from all tissues and its composition is hypothesized to reflect dynamic. These recommendations were based primarily on data from matrix-assisted laser desorption/ionization (MALDI) and surface-enhanced laser desorption/ionization (SELDI) analyses, which detect a relatively small number of abundant, low molecular weight proteins and peptides in undigested plasma or serum. Our experiments characterized changes at the peptide level in plasma proteins as a function of [1] time from blood collection to plasma isolation at 4 °C or room temperature, [2] number of freeze-thaw cycles performed on plasma samples, and [3] hemolysis prior to plasma preparation.
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