Skip to main content

An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples


In order to evaluate the critical components of the process necessary to preserve clinical plasma samples collected at research sites for proteomic analysis, various collection and preservation protocols with controlled experimentation were evaluated. The presence of a protease inhibitor cocktail (PIC) included in the blood draw tube would stabilize the plasma proteins was hypothesized. To test this hypothesis, four plasma samples from each of 14 volunteers were collected. Samples were treated following a standard protocol that included PIC or were subjected to various processing treatments that included time, temperature, different anticoagulants, and the absence of PIC. Large format two dimensional-polyacrylamide gel electrophoresis (2D-PAGE) proteomic analysis and enzyme immunoassay (EIA) were used to detect differences between the treatment groups. A novel 2D-PAGE quality scoring method was developed to determine global differences in the treatment groups, wherein a rating scale questionnaire was used to quantify the quality of each 2D-PAGE gel. The data generated from EIAs, classical 2D-PAGE image analysis and 2D-PAGE quality scoring, each generated similar results. Inclusion of protease inhibitor cocktail in the sample tubes, provided stable and reliable human plasma samples that yielded reproducible results by proteomic analysis. When PIC was included, samples retained stability under less stringent processing, such that refrigeration for several hours before processing or one freeze-thaw cycle had little detrimental effect. We demonstrated that samples without PIC, from either heparin or ethylenediaminetetraacetic acid (EDTA) plasma tubes, gave results that varied significantly from the control samples. Also, even with PIC present in blood tubes, we found it was important to quickly decant the separated plasma from the cellular components found in the blood tubes following centrifugation, as prolonged exposure again yielded different results from the standard procedure.


  1. 1

    Hanash S. Disease proteomics. Nature 2003;422:226–232.

    PubMed  Article  CAS  Google Scholar 

  2. 2

    Hanash SM. Biomedical applications of two-dimensional electrophoresis using immobilized pH gradients: current status. Electrophoresis 2000;21:1202–1209.

    PubMed  Article  CAS  Google Scholar 

  3. 3

    Chambers G, Lawrie L, Cash P, Murray GI. Proteomics: a new approach to the study of disease. J Pathol 2000;192:280–288.

    PubMed  Article  CAS  Google Scholar 

  4. 4

    Seliger B, Kellner R. Design of proteome-based studies in combination with serology for the identification of biomarkers and novel targets. Proteomics 2002;2:1641–1651.

    PubMed  Article  CAS  Google Scholar 

  5. 5

    Rabilloud T. Two-dimensional gel electrophoresis in proteomics: old, old fashioned, but it still climbs up the mountains. Proteomics 2002;2:3–10.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Young DS, Tracy RP. Clinical applications of two-dimensional electrophoresis. J Chromatogr A 1995;698:163–179.

    PubMed  Article  CAS  Google Scholar 

  7. 7

    Le Naour F, Misek DE, Krause MC, et al. Proteomics-based identification of RS/DJ-1 as a novel circulating tumor antigen in breast cancer. Clin Cancer Res 2001;7:3328–3335.

    PubMed  Google Scholar 

  8. 8

    Rui Z, Jian-Guo J, Yuan-Peng T, Hai P, Bing-Gen R. Use of serological proteomic methods to find biomarkers associated with breast cancer. Proteomics 2003;3:433–439.

    PubMed  Article  Google Scholar 

  9. 9

    Zuo X, Hembach P, Echan L, Speicher DW. Enhanced analysis of human breast cancer proteomes using micro-scale solution isoelectrofocusing combined with high resolution 1-D and 2-D gels. J Chromatogr B Analyt Technol Biomed Life Sci 2002;782:253–265.

    PubMed  Article  CAS  Google Scholar 

  10. 10

    Charrier JP, Tournel C, Michel S, et al. Differential diagnosis of prostate cancer and benign prostate hyperplasia using twodimensional electrophoresis. Electrophoresis 2001;22:1861–1866.

    PubMed  Article  CAS  Google Scholar 

  11. 11

    Charrier JP, Tournel C, Michel S, Dalbon P, Jolivet M. Two-dimensional electrophoresis of prostate-specific antigen in sera of men with prostate cancer or benign prostate hyperplasia. Electrophoresis 1999;20:1075–1081.

    PubMed  Article  CAS  Google Scholar 

  12. 12

    Poon TC, Johnson PJ. Proteome analysis and its impact on the discovery of serological tumor markers. Clin Chim Acta 2001;313: 231–239.

    PubMed  Article  CAS  Google Scholar 

  13. 13

    Steel LF, Shumpert D, Trotter M, et al. Astrategy for the comparative analysis of serum proteomes for the discovery of biomarkers for hepatocellular carcinoma. Proteomics 2003;3: 601–609.

    PubMed  Article  CAS  Google Scholar 

  14. 14

    Kellner R, Lichtenfels R, Atkins D, et al. Targeting of tumor associated antigens in renal cell carcinoma using proteome-based analysis and their clinical significance. Proteomics 2002;2:1743–1751.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    Sinz A, Bantscheff M, Mikkat S, et al. Mass spectrometric proteome analyses of synovial fluids and plamas from patients suffering from rheumatoid arthritis and comparison to reactive arthritis or osteoarthritis. Electrophoresis 2002;23:3445–3456.

    PubMed  Article  CAS  Google Scholar 

  16. 16

    Smith MA, Bains SK, Betts JC, Choy EH, Zanders ED. Use of two-dimensional gel electrophoresis to measure changes in synovial fluid proteins from patients with rheumatoid arthritis treated with antibody to CD4. Clin Diagn Lab Immunol 2001;8:105–111.

    PubMed  Article  CAS  Google Scholar 

  17. 17

    He QY, Lau GK, Zhou Y, et al. Serum biomarkers of hepatitis B virus infected liver inflammation: A proteomic study. Proteomics 2003;3:666–674.

    PubMed  Article  CAS  Google Scholar 

  18. 18

    Anderson NL, Anderson NG. The human plasma proteome: history, character, and diagnostic prospects. Mol Cell Proteomics 2003; 2:50.

    Article  CAS  Google Scholar 

  19. 19

    Boyanton BL, Jr., Blick KE. Stability studies of twenty-four analytes in human plasma and serum. Clin Chem 2002;48:2242–2247.

    PubMed  CAS  Google Scholar 

  20. 20

    Flower L, Ahuja RH, Humphries SE, Mohamed-Ali V. Effects of sample handling on the stability of interleukin 6, tumour necrosis factor-alpha and leptin. Cytokine 2000;12: 1712–1716.

    PubMed  Article  CAS  Google Scholar 

  21. 21

    Lewis MR, Callas PW, Jenny NS, Tracy RP. Longitudinal stability of coagulation, fibrinolysis, and inflammation factors in stored plasma samples. Thromb Haemost 2001;86: 1495–1500.

    PubMed  CAS  Google Scholar 

  22. 22

    Holodniy M, Rainen L, Herman S, Yen-Lieberman B. Stability of plasma human immunodeficiency virus load in VACUTAINER PPT plasma preparation tubes during overnight shipment. J Clin Microbiol 2000;38:323–326.

    PubMed  CAS  Google Scholar 

  23. 23

    Marshall J, Kupchak P, Zhu W, et al. Processing of Serum Proteins Underlies the Mass Spectral Fingerprinting of Myocardial Infarction. J Proteome Res. 2003;2:361–372.

    PubMed  Article  CAS  Google Scholar 

  24. 24

    Olivieri E, Herbert B, Righetti PG, The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes. Electrophoresis 2001;22:560–565.

    PubMed  Article  CAS  Google Scholar 

  25. 25

    Lollo BA, Harvey S, Liao J, et al. Improved two-dimensional gel electrophoresis representation of serum proteins by using Proto Clear. Electrophoresis 1999;20:854–859.

    PubMed  Article  CAS  Google Scholar 

  26. 26

    Wang YY, Cheng P, Chan DW. A simple affinity spin tube filter method for removing highabundant common proteins or enriching low-abundant biomarkers for serum proteomic analysis. Proteomics 2003;3:243–248.

    PubMed  Article  CAS  Google Scholar 

  27. 27

    Rothemund DL, Locke VL, Liew A, Thomas TM, Wasinger V, Rylatt DB. Depletion of the highly abundant protein albumin from human plasma using the Gradiflow. Proteomics 2003; 3:279–287.

    PubMed  Article  CAS  Google Scholar 

  28. 28

    Corthals GL, Wasinger VC, Hochstrasser DF, Sanchez JC. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000;21:1104–1115.

    PubMed  Article  CAS  Google Scholar 

  29. 29

    Herbert B, Righetti PG. A turning point in proteome analysis: sample prefractionation via multicompartment electrolyzers with isoelectric membranes. Electrophoresis 2000;21: 3639–3648

    PubMed  Article  CAS  Google Scholar 

  30. 30

    Zuo X, Echan L, Hembach P, et al. Towards global analysis of mammalian proteomes using sample prefractionation prior to narrow pH range two-dimensional gels and using one-dimensional gels for insoluble and large proteins. Electrophoresis 2001;22:1603–1615.

    PubMed  Article  CAS  Google Scholar 

  31. 31.

    Collins PJ, Juhl C, Lognonne JL. Image analysis of 2D gels: considerations and insights. Cell Mol Biol (Noisy-le-grand) 1994;40:77–83.

    CAS  Google Scholar 

  32. 32

    Nishihara JC, Champion KM. Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis 2002;23:2203–2215.

    PubMed  Article  CAS  Google Scholar 

  33. 33

    Hughes GJ, Frutiger S, Paquet N, et al. Plasma protein map: an update by microsequencing. Electrophoresis 1992;13:707–714.

    PubMed  Article  CAS  Google Scholar 

  34. 34

    Sanchez JC, Appel RD, Golaz O, et al. Inside SWISS-2DPAGE database. Electrophoresis 1995;16:1131–1151.

    PubMed  Article  CAS  Google Scholar 

  35. 35

    Hoogland C, Sanchez JC, Walther D, et al. Two-dimensional electrophoresis resources available from ExPASy. Electrophoresis 1999; 20:3568–3571.

    PubMed  Article  CAS  Google Scholar 

  36. 36

    Hoving S, Gerrits B, Voshol H, Muller D, Roberts RC, van Oostrum J. Preparative twodimensional gel electrophoresis at alkaline pH using narrow range immobilized pH gradients. Proteomics 2002;2:127–134.

    PubMed  Article  CAS  Google Scholar 

  37. 37

    Streiner DL. A checklist for evaluating the usefulness of rating scales. Can J Psychiatry 1993; 38:140–148.

    PubMed  CAS  Google Scholar 

  38. 38.

    Agresti A. Analysis of Ordinal Categorical Data. New York: John Wiley & Sons, Inc., 1984.

    Google Scholar 

  39. 39

    Rhodes AE, Lin E, Streiner DL. Confronting the confounders: the meaning, detection, and treatment of confounders in research. Can J Psychiatry 1999;44:175–179.

    PubMed  CAS  Google Scholar 

  40. 40

    Han AC, Edelson MI, Peralta Soler A, et al. Cadherin expression in glandular tumors of the cervix. Cancer 2000;89:2053–2058.

    PubMed  Article  CAS  Google Scholar 

  41. 41

    Muller N, Reinacher-Schick A, Baldus S, et al. Smad4 induces the tumor suppressor E-cadherin and P-cadherin in colon carcinoma cells. Oncogene 2002;21:6049–6058.

    PubMed  Article  CAS  Google Scholar 

  42. 42

    Blankenberg S, Rupprecht HJ, Bickel C, et al. Circulating cell adhesion molecules and death in patients with coronary artery disease. Circulation 2001;104:1336–1342.

    PubMed  CAS  Google Scholar 

  43. 43.

    Cybulsky MI, Liyama K, Li H, et al. A major role for VCAM-1, but not ICAM-1, in early atherosclerosis. J Clin Invest 2001;107:1255–1262.

    PubMed  CAS  Article  Google Scholar 

Download references

Author information



Corresponding author

Correspondence to Jeffrey D. Hulmes.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hulmes, J.D., Bethea, D., Ho, K. et al. An investigation of plasma collection, stabilization, and storage procedures for proteomic analysis of clinical samples. Clin Proteom 1, 17–31 (2004).

Download citation

  • Issue Date:

  • DOI:

Key Words

  • Proteomics
  • plasma collection
  • twodimensional gel electrophoresis
  • sample stability
  • protease inhibitors
  • 2D-PAGE evaluation