Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling

Abstract

Blood is an ideal source for biomarker discovery. However, little has been done to address the effects of sampling, handling and storage procedures on serum/plasma proteomes. We used magnetic bead-based MALDI-TOF MS to systematically evaluate the influence of each procedure on low-molecular-weight serum/plasma proteome profiling on the basis of the whole spectra. We found that sampling procedures, including the selection of blood collection tubes and anticoagulants, variations in clotting time or time lag before centrifugation, and hemolysis, displayed significant effects on the proteomes. Moreover, serum and plasma were mutually incompatible for proteome comparison. By contrast, overnight fasting, handling procedures, including centrifugation speeds (1500 x g vs. 3000 x g) or time (15 min vs. 30 min), and storage conditions, such as at 4 degrees C or 25 degrees C for up to 24 h or at -80 degrees C for up to 3 months, and repeated freeze/thaw of up to ten cycles, had relatively minor effects on the proteomes based upon our analysis of about 100 peaks. We concluded that low-molecular-weight serum/plasma proteomes were diversely affected by sampling, handling and storage with most change from variations of sampling procedures. We therefore suggest the necessity of standardizing sampling procedure for proteome comparison and biomarker discovery.

Evaluation of Anticoagulants for Serologic Assays of Cholera Vaccination

“…In contrast, some proteins, such as beta-2 microglobulin and histidine-rich protein 2, were detected at higher levels in sera than in plasma (28,29), whereas a close relationship between serum and plasma levels was shown for IgG and IgA levels to mycobacterial antigens or 3-nitrotyrosine (30,31). Although platelets are relatively stable in heparin and ACD anticoagulants, the protein profiles of plasma extracted from EDTA-treated blood are very different from those of plasma prepared with ACD or heparin because EDTA causes platelet clumping and aggregation, resulting in changes in plasma protein content (32,33).…”

Proteomics Analysis of Porcine Serum Proteins by LC-MS/MS after Foot-and-Mouth Disease Virus (FMDV) Infection

“…Serum samples after infection with FMDV were used as the treatment group, and were taken within 6 days of infection from six pigs with outward clinical symptoms of FMDV infection which characterized with vesicles on the lips and feet [4]. Six replicates of each serum sample were isolated and stored at -80°C until use [11].…”

Strategies for Protein Separation

“…During proteomics analysis there are many potential contributors to variability that can compromise the approach repeatability and reproducibility. The variations can begin in sample collection, specimen processing techniques, storage, and instrument performance (Hsieh at al, 2006;Banks et al, 2005;Pilny et al, 2006). All steps in the proteome analysis can offer a source of variations.…”