Purpose: This study aims to define an optimized sample processing protocol for synovial fluid proteome analysis using two-dimensional gel electrophoresis (2DE). Methods: 16 synovial fluid (SF) knee samples collected from 16 non-osteoarthritic patients (mean age: 24.65 years old; females 31.5%), undergoing knee arthroscopy for acute meniscal injuries, were analysed. 500μl aliquoted SF samples were treated with two different hyaluronidase concentrations (1 mg/mL vs 1 μg/mL). The 1 μg/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 was performed. Results: SF samples treated with 1 μg/mL hyaluronidase showed a significantly higher protein spots number compared to 1 mg/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-enrichment provided 334 unique mean protein spots in SF proteome, which could not be detected nor in SF samples without depletion, nor samples treated with affinity depletion. Conclusions: This study has validated an optimized sample processing protocol for SF proteome analysis using a gel-based approach. SF samples treatment with hyaluronidase (concentration: 1μg/mL) revealed critical for the correct sample processing. The use of ProteoMinerTM-enrichment is highly recommended in SF proteomic analysis to detect low-abundance proteins.
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;Gesualdo, L;Gnoni, A
2021-01-01
Abstract
Purpose: This study aims to define an optimized sample processing protocol for synovial fluid proteome analysis using two-dimensional gel electrophoresis (2DE). Methods: 16 synovial fluid (SF) knee samples collected from 16 non-osteoarthritic patients (mean age: 24.65 years old; females 31.5%), undergoing knee arthroscopy for acute meniscal injuries, were analysed. 500μl aliquoted SF samples were treated with two different hyaluronidase concentrations (1 mg/mL vs 1 μg/mL). The 1 μg/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 was performed. Results: SF samples treated with 1 μg/mL hyaluronidase showed a significantly higher protein spots number compared to 1 mg/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-enrichment provided 334 unique mean protein spots in SF proteome, which could not be detected nor in SF samples without depletion, nor samples treated with affinity depletion. Conclusions: This study has validated an optimized sample processing protocol for SF proteome analysis using a gel-based approach. SF samples treatment with hyaluronidase (concentration: 1μg/mL) revealed critical for the correct sample processing. The use of ProteoMinerTM-enrichment is highly recommended in SF proteomic analysis to detect low-abundance proteins.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
