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Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

ABSTRACT: BACKGROUND:High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical cord serum/plasma will be variable. Therefore, the primary purposes of the present study are to investigate the efficiencies of several commonly-used commercial kits during high abundance protein depletion from umbilical cord serum and to determine which kit yields the most effective and reproducible results for further proteomics research on umbilical cord serum. RESULTS:The immunoaffinity based kits (PROTIA-Sigma and 5185-Agilent) displayed higher depletion efficiency than the immobilized dye based kit (PROTBA-Sigma) in umbilical cord serum samples. Both the PROTIA-Sigma and 5185-Agilent kit maintained high depletion efficiency when used three consecutive times. Depletion by the PROTIA-Sigma Kit improved 2DE gel quality by reducing smeared bands produced by the presence of high abundance proteins and increasing the intensity of other protein spots. During image analysis using the identical detection parameters, 411 ± 18 spots were detected in crude serum gels, while 757 ± 43 spots were detected in depleted serum gels. Eight spots unique to depleted serum gels were identified by MALDI- TOF/TOF MS, seven of which were low abundance proteins. CONCLUSIONS:The immunoaffinity based kits exceeded the immobilized dye based kit in high abundance protein depletion of umbilical cord serum samples and dramatically improved 2DE gel quality for detection of trace biomarkers.

SUBMITTER: Liu B

PROVIDER: S-EPMC3105942 | biostudies-literature | 2011 May

REPOSITORIES: biostudies-literature

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Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

Liu Bin B Qiu Fang-Hua FH Voss Courtney C Xu Yun Y Zhao Ming-Zhe MZ Wu Yan-Xin YX Nie Jing J Wang Zi-Lian ZL

Proteome science 20110509 1

<h4>Background</h4>High abundance protein depletion is a major challenge in the study of serum/plasma proteomics. Prior to this study, most commercially available kits for depletion of highly abundant proteins had only been tested and evaluated in adult serum/plasma, while the depletion efficiency on umbilical cord serum/plasma had not been clarified. Structural differences between some adult and fetal proteins (such as albumin) make it likely that depletion approaches for adult and umbilical co ...[more]

PMID: 21554704

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Project description:BackgroundHuman autologous serum (AS) and umbilical cord serum (UCS) both contain growth and neurotrophic factors that promote corneal healing.AimOur objectives were to compare equine AS and UCS cytokine and growth factor profiles and to assess the safety and clinical feasibility of the therapeutic use of UCS eye drops in cases of spontaneous complex ulcers.Study designProspective clinical trial.MethodsVitamin A insulin growth factor, platelet-derived growth factor-BB, transforming growth factor (TGF)-β1 (enzyme-linked immunosorbent assay), interleukin (IL)-1β, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were determined in 10 AS collected from different horses and 10 UCS sampled at delivery. Six client-owned horses presenting with complex non-healing corneal defects of >5 mm2 were included in a clinical trial and treated with conventional therapy and conditioned UCS drops for 8-15 days. Ulcer surface and time to complete epithelialization were recorded.ResultsMedian concentrations of vitamin A, insulin growth factor, and platelet-derived growth factor-BB were not significantly different in AS compared with UCS (respectively, 14.5 vs. 12.05 μg/ml; 107.8 vs. 107.3 pg/ml; and 369.1 vs. 924.2 pg/ml). TGF-β1 median concentration in UCS was significantly higher than in AS (3,245 vs. 2571pg/ml) (p = 0.04). IL-1β, IL-6, interferon-γ, and monocyte chemoattractant protein 1 concentrations were variable in AS and undetectable in UCS. The corneal median ulcerative area was 37.2 mm2 (6.28-57.14 mm2) and had a duration of 4-186 days (median 19 days). All lesions healed within 13-42 days (median 17 days). No adverse effects nor recurrences within 1 month were noticed.LimitationsThe sample size was small. Spontaneous corneal epithelial defects presented with variable clinical characteristics. There were no age-matched control horses to assess corneal healing time and rate.Conclusion and clinical significanceEquine UCS may be beneficial, as it contains no pro-inflammatory cytokines and a greater concentration of TGF-β1 compared with AS. Topical UCS appears safe and may potentially be used as adjunctive therapy for equine complex non-healing ulcers.

Dataset Information

Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

Publications

Evaluation of three high abundance protein depletion kits for umbilical cord serum proteomics.

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