Global stability of plasma proteomes for mass spectrometry-based analyses.
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.
Jim Ayers Institute for Precancer Detection and Diagnosis, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.
Lisa J
LJ
Zimmerman
Ming
M
Li
Wendell G
WG
Yarbrough
Robbert J C
RJ
Slebos
Daniel C
DC
Liebler
D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry*; D015925:Cryopreservation; D005260:Female; D005340:Fibrinogen / Q000737:chemistry; D006461:Hemolysis; D006801:Humans; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D055550:Protein Stability; D020543:Proteome / Q000737:chemistry*; D013048:Specimen Handling; D053719:Tandem Mass Spectrometry; D055815:Young Adult
D001798:Blood Proteins; D010446:Peptide Fragments; D020543:Proteome; D005340:Fibrinogen