Comparative Proteomic Analysis of Osteoblast Lineages

First-dimension analytical gel electrophoresis was performed, followed by second-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% fixed concentration gels, as previously described [47 (link),48 (link)]. The gels were scanned with a Sapphire Biomolecular Imager (Azure Biosystems, Dublin, OH, USA) and digitalized via the image analysis software Sapphire Capture system (Azure Biosystems, Dublin, OH, USA). Spot volumes were log-transformed to generate normally distributed data, and log-normalized volume instead of spot intensities was used in statistical processing to quantify differential expression. All spots were pre-filtered and manually checked before applying the statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥ 1.5). Independent direct comparisons were made between the protein spots related to the OB, OD, and ODM groups, and the fold differences and p-values were calculated using one-way ANOVA. Spots that fulfilled the above-mentioned statistical criteria were submitted for further mass spectrometric (MS) analysis.

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Alshahrani A., Aljada A., Masood A., Mujammami M., Alfadda A.A., Musambil M., Alanazi I.O., Al Dubayee M., Abdel Rahman A.M, & Benabdelkamel H. (2023). Proteomic Profiling Identifies Distinct Regulation of Proteins in Obese Diabetic Patients Treated with Metformin. Pharmaceuticals, 16(10), 1345.

Publication 2023

Sapphire Biomolecular Imager Sapphire Capture system

Anova Azure Electrophoresis Gels Mass spectrometric Protein Sapphire Sds page Spots

Corresponding Organization :

Other organizations : National Guard Health Affairs, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Center, Alfaisal University, King Saud University, King Saud Medical City, King Abdulaziz City for Science and Technology, King Faisal Specialist Hospital & Research Centre

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Variable analysis

independent variables

First-dimension analytical gel electrophoresis
Second-dimension sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) on 12.5% fixed concentration gels

dependent variables

Spot volumes
Differential expression of protein spots related to the OB, OD, and ODM groups

control variables

Experimental procedures previously described in references [47] and [48]
Scanning of gels with Sapphire Biomolecular Imager and digitalization via Sapphire Capture system
Log-transformation of spot volumes to generate normally distributed data
Log-normalized volume used in statistical processing instead of spot intensities
Pre-filtering and manual checking of all spots before applying statistical criteria (ANOVA test, p ≤ 0.05 and fold ≥ 1.5)

controls

No positive or negative controls were explicitly mentioned in the provided information.

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