2D DIGE to compare baseline and post-treatment patient samples
In this study, two-dimensional difference gel electrophoresis (2D DIGE) was used to compare the plasma proteome of 20 patients before and three months after liraglutide treatment. Baseline and post-treatment samples from each patient were labeled with Cy3 or Cy5 using a dye-switching strategy to control for any dye-specific bias. To control for gel-to-gel variation, equal amounts of all samples were pooled and labeled with Cy2 as an internal control. For a given patient, the two labeled samples were combined with the internal control and run on a 2D gel which was scanned for Cy2, Cy3 and Cy5 using the Sapphire Imager. Overlaying the images and quantifying spot intensity identified 128 spots whose intensity was statistically significantly different after liraglutide treatment.
Using the Sapphire to identify proteins affected by liraglutide treatment
To identify the proteins whose expression was affected by liraglutide treatment, a preparative gel was run with a pool of equal amounts of each patient protein sample. The spots of interest were cut from the preparative gel and the protein identified by mass spectrometry. Of the 128 spots, 72 were identified and represented 29 unique proteins. The identities of two spots were further confirmed by chemiluminescent Western blot, also imaged using the Sapphire.
Results from the study
The results indicate that liraglutide affects expression of proteins involved in inflammation, apolipoprotein regulation, and oxidative stress, all processes known to contribute to cardiovascular disease. The authors conclude that effects on these pathways may contribute to the effectiveness of liraglutide treatment and that the results should be further investigated in larger-scale studies involving more patients and longer terms of treatment. If substantiated, the authors suggest the results could lead to the development of biomarkers to assess the cardiovascular benefits of liraglutide.
Using an untargeted proteomic approach
Liraglutide, a glucagon-like peptide-1 receptor agonist, improves glycemic control through action on the pancreas. Liraglutide also reduces cardiovascular risk in patients with diabetes via an unknown mechanism. The authors used untargeted proteomic profiling of plasma from patients treated with liraglutide to identify changes induced by treatment and to see if those changes in the proteome provide insight into liraglutide’s cardiovascular and metabolic benefits.
An advantage of the untargeted proteomic approach used in this research is the potential to discover unexpected things. With an untargeted approach, any protein whose level changes as a result of treatment may be identified, whereas in targeted experiments, only specific candidate proteins are studied to prove or disprove their involvement in a process. Metabolism is complex and involves many interacting pathways and processes, making it an attractive subject for an untargeted approach that can assess many targets simultaneously. In addition, protein levels may better reflect changes in physiology than RNA levels or DNA-level changes.
More published research using the Azure Sapphire:
- Sapphire Biomolecular Imager used in investigation of potential nasal vaccine for SARS-CoV-2
- New tools created for the study of coagulation pathway proteases
- In-cell Westerns used to Demonstrate Potent Anti-inflammatory Activity of Mosquito Saliva Protein
- Sapphire used in Study of Green Tea’s Anti-cancer Effects
- Ekhzaimy AA, Masood A, Benabdelkamel H, et al. Plasma proteomics reveals an improved cardio-metabolic profile in patients with type 2 diabetes post-liraglutide treatment. Diab Vasc Dis Res. 2022;19(3):14791641221094322.