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

Abstract

BackgroundHigh 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.ResultsThe 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.ConclusionsThe 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.

Quantitative assessment of human serum transferrin receptor in breast cancer patients pre- and post-chemotherapy using peptide immunoaffinity enrichment coupled with targeted proteomics

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The conserved metalloprotease invadolysin is present in invertebrate haemolymph and vertebrate blood

“…4F,G). This band was less readily detected with the G6456 antibody which may be due to the proximity of albumin and immunoglobulin heavy chain, accounting for 70–80% of the total protein content of plasma (Liu et al, 2011; Steel et al, 2003). To improve the detection of invadolysin, abundant plasma proteins were removed using a commercially-available abundant plasma protein removal kit (see Materials and Methods).…”

Detection of tyrosine-phosphorylated proteins in hepatocellular carcinoma tissues using a combination of GST-Nck1-SH2 pull-down and two-dimensional electrophoresis

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