Abstract
Purpose: This study aims to define, for the first time, a standard sample processing protocol for synovial fluid proteome analysis for a gel based-approach by two-dimensional gel electrophoresis (2DE). Methods: 29 synovial fluid knee samples collected from 29 patients (mean age: 68.19 years old; female: 55.17%; male: 44.83%) undergoing total knee arthroplasty (TKA) were analysed All the patients underwent a clinical, radiological and laboratory evaluation according to the current clinical practice. Five-hundred μl aliquoted SF was sample treated with two different hyaluronidase concentrations (1μg/ml vs 1mg/ml). The 1μg/ml hyaluronidase treated samples underwent two different high-abundance protein depletion techniques: affinity columns (ProteoPrep®, Sigma-Aldrich) or protein-enrichment kit (ProteoMinerTM, Bio-Rad Laboratories). Standardization of SF two-dimensional gel electrophoresis (2DE) map wa performed. Results: SF treated with 1μg/mL hyaluronidase showed a significantly higher protein spots number compared to 1mg/mL hyaluronidase treated samples (p=0.001). A significantly higher protein spots number was obtained in SF treated with ProteoMinerTM, compared to SF albumin and IgG-depleted (p=0.001). Moreover, ProteoMinerTM-enrichement provided 334 unique mean protein spots in SF proteome, which could not be detected nor in SF samples without depletion, nor in samples treated with affinity depletion.
Beyond pre-analytical and analytical concerns in the study of synovial fluid proteome: description of an optimized Gel-based protocol
Bizzoca, D;Rocchetti, M T;Scacco, S;Taurino, F;Vicenti, G;Ranieri, E;Gesualdo, L;Moretti, B;Gnoni, A
2021-01-01
Abstract
Purpose: This study aims to define, for the first time, a standard sample processing protocol for synovial fluid proteome analysis for a gel based-approach by two-dimensional gel electrophoresis (2DE). Methods: 29 synovial fluid knee samples collected from 29 patients (mean age: 68.19 years old; female: 55.17%; male: 44.83%) undergoing total knee arthroplasty (TKA) were analysed All the patients underwent a clinical, radiological and laboratory evaluation according to the current clinical practice. Five-hundred μl aliquoted SF was sample treated with two different hyaluronidase concentrations (1μg/ml vs 1mg/ml). The 1μg/ml hyaluronidase treated samples underwent two different high-abundance protein depletion techniques: affinity columns (ProteoPrep®, Sigma-Aldrich) or protein-enrichment kit (ProteoMinerTM, Bio-Rad Laboratories). Standardization of SF two-dimensional gel electrophoresis (2DE) map wa performed. Results: SF treated with 1μg/mL hyaluronidase showed a significantly higher protein spots number compared to 1mg/mL hyaluronidase treated samples (p=0.001). A significantly higher protein spots number was obtained in SF treated with ProteoMinerTM, compared to SF albumin and IgG-depleted (p=0.001). Moreover, ProteoMinerTM-enrichement provided 334 unique mean protein spots in SF proteome, which could not be detected nor in SF samples without depletion, nor in samples treated with affinity depletion.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
