title: Evaluation of two high-abundance protein depletion kits and optimization of downstream isoelectric focusing.
abstract: Disease biomarkers for diagnostic and prognostic purposes are most likely within an extremely low concentration range and are thus masked by the presence of high‑abundance proteins. Therefore, removing high‑abundance proteins is the main challenge for identifying disease biomarkers. In addition, the solution obtained from high‑abundance protein depletion kits contains a rich array of compounds, which interfere with isoelectric focusing (IEF). In the present study, the effect of two commercial kits was evaluated and the downstream IEF protocol was optimized. High‑resolution results could be obtained according to the following conditions: The ProteoPrep Blue Albumin and IgG Depletion kit depleted albumin and IgG; immobilized pH gradient strips (typically 18 cm) were rehydrated with sample buffer containing 250 µg serum proteins at 30 v for 6 h, 60 v for 6 h, 200 v for 2 h, 500 v for 2 h, 1,000 v for 2 h, 5,000 v for 2 h, 10,000 v for 2 h and then focusing at 10,000 v up to 110 k vhs. In addition, the protein spots identified by matrix‑assisted laser desorption ionization time‑of‑flight mass spectrometry demonstrated that all proteins had a low abundance. The present study not only provides a definite and effective method for removing high‑abundance proteins, but also provides a proper protocol (protocol C) for downstream IEF. The present study includes a comprehensive investigation of serum proteomics, which paves the way for serum protein research.
authors.affiliation: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Fanghua
authors.initials: F
authors.lastname: Qiu
authors.affiliation: Department of Laboratory Medicine, Guangdong Academy of Medicine Science and Guangdong General Hospital, Guangzhou, Guangdong 510000, P.R. China.
authors.forename: Tieying
authors.initials: T
authors.lastname: Hou
authors.affiliation: Department of Neurology, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Dehong
authors.initials: D
authors.lastname: Huang
authors.affiliation: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Xue
authors.affiliation: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Dongyan
authors.initials: D
authors.lastname: Liang
authors.affiliation: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Qiuming
authors.initials: Q
authors.lastname: Li
authors.affiliation: Department of Clinical Laboratory, Guangzhou Hospital of Traditional Chinese Medicine, Guangzhou, Guangdong 510130, P.R. China.
authors.forename: Weimiao
authors.initials: W
authors.lastname: Lin
mesh_terms: D000328:Adult; D001798:Blood Proteins / Q000032:analysis*; D003110:Colonic Neoplasms / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification*; D007525:Isoelectric Focusing / Q000379:methods*; D008297:Male; D008875:Middle Aged; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D001798:Blood Proteins; D007074:Immunoglobulin G; D012709:Serum Albumin