Serum/Plasma Proteome An Investigation of Plasma Collection, Stabilization, and Storage Procedures for Proteomic Analysis of Clinical Samples Jeffrey D. Hulmes,* ,1 Deidra Bethea, 1 Keith Ho, 1 Shu-Pang Huang, 2 Deborah L. Ricci, 3 Gregory J. Opiteck 1 Stanley A. Hefta 1 1 Department of Clinical Discovery, Proteomics Unit, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 2, *Department of Clinical Discovery, Biostatistics Unit, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 3, *Department of Clinical Discovery, Pharmacology Unit, Pharmaceutical Research Institute, Bristol-Myers Squibb Company, Princeton, NJ 17 Clinical Proteomics Journal Copyright ©Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/04/01:17–31/$25.00 *Author to whom all correspondence and reprint requests should be addressed: E-mail: jeffrey.hulmes@bms.com Abstract 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 pre- servation protocols with controlled experi- mentation 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 hypoth- esis, four plasma samples from each of 14 vol- unteers were collected. Samples were treated following a standard protocol that included PIC or were subjected to various processing treat- ments 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 ques- tionnaire 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 repro- ducible 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.