An efficient extraction of intervertebral disc (IVD) proteins coupled with the Liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection
A specific discovery proteomic protocol for intervertebral discs relies on an efficient protein extraction using the bead beating homogenizing method with stainless steel grinding balls coupled with a customized lysis buffer, and followed by the protein detection with specific mass spectrometry setting.
Sample Processing Protocol
Sample was homogenized in the Percellys 24 homogenizer, which containing 2.8 mm stainless steel grinding balls and pre-cooled in liquid nitrogen. Speed at 6800 rpm for 20 seconds, repeat the step for 3 times with 20 seconds pulse in between. Then, lysis buffer was added and at 6800 rpm for 20 seconds twice with 20 seconds pulse in between. Then centrifuge the lysate and supernatant was recovered. A Bradford protein assay was performed for quantifying the protein concentration. Protein (50 ug) was reduced with 10 mM Dithiothreitol (DTT) at 37°C for 60 min. and alkylated with 20 mM iodoacetamide (IAA) at 25oC for 45 min. Protein precipitation overnight at -20oC with 80% acetone. Centrifuge for 30 min at 4oC with speed 21380 x g and discard the supernatant. Wash the pellet with 200 uL of cold 80% acetone. Centrifuge for 20 min at 4oC with speed 21380 x g and keep the pellet only. Dissolve the pellet in 8 M urea in 25 mM ammonium bicarbonate (at least 10 uL) and dilute to 1 M urea with 25 mM ammonium bicarbonate. Mix 2 ug modified sequencing grade trypsin with the dissolved sample for 16 h at 37oC. Acidify and quench the protein digestion samples by adding 0.1% Trifluoroacetic acid (TFA) to 1 mL by volume. Peptide was desalted with Waters Oasis HLB 1cc (10 mg) extraction cartridge(s) and dried with Centrivap. Peptide concentration was determined by using the Pierce Quantitative Colorimetric peptide assay. Adjust the peptide concentration to around 0.5 ug/uL for MS analysis.
Data Processing Protocol
Data-dependent acquisition (DDA) were searched Mus musculus Uniprot unreview database (id 86764) and protein identification (ID) were acquired using ProteinPilot 5.0.1 software (Sciex, US) with the following parameters: identification sample type, trypsin digestion, IAA as alkylating agent of cysteine residues, and thorough search effort with inclusion of biological modifications. A combined search of triplicate injections was selected to form a combined search mouse intervertebral disc database Mus musculus Uniprot unreview database (id 86764) in ProteinPilot 5.0 software 7.2 Obtain protein identification at 1% false discovery rate (FDR).
Chuen Lam, The Hong Kong Polytechnic University/School of optometry
Thomas Chuen Lam, Laboratory of Experimental Optometry, Centre for Myopia Research, School of Optometry, The Hong Kong Polytechnic University, Kowloon, Hong Kong ( lab head )