Phosphoproteomics and depletion studies in human serum – revealing the differences between healthy state, obesity and type 2 diabetes mellitus

Introduction: Type 2 diabetes mellitus (T2D) is a worldwide known disease with an increasing incidence year by year. Extensive research has proven that besides the numerous risk factors, obesity is the main leading risk factor for the development of T2D. Regrettably, obesity also shows growing tendency because of an unhealthy lifestyle in the modern era.

Aim: The aim of our study was to characterize proteins and phosphoproteins in serum samples from patients with obesity, T2D and healthy controls. To reveal the relevant biological processes between control, obese and T2D groups, network- and gene ontology (GO) analyses were performed.

Materials and methods: 135 serum samples from obese, T2D and age- and sex-matched controls were collected by the Department of Internal Medicine, University of Debrecen. The protein concentration of the samples was determined by BCA assay. After it, the experiment was divided into two parts. Depletion was carried out, the top 14 most abundant proteins were depleted followed by digestion using iST kit (PreOmics) and two fractions were collected from the digested samples. Besides, non-depleted serum samples were used for TMT-labelling. The TMT-labelled peptides were fractionated using a high-pH reverse phase column and subjected to phosphopeptide enrichment. After the preparation of all samples, LC-MS analyses were performed using data-independent acquisition (DIA) method for depleted samples and data-dependent acquisition (DDA) for TMT-labelled samples. Data analyses were performed using DIA-NN and Proteome Discoverer. For network and GO analyses, Cytoscape and clueGO were used and the kinases having a role in the generation of the phosphorylated sites were identified.

Results and conclusion: 232 proteins were found in the depleted samples whereas 268 proteins were identified by TMT-labelling experiments in the three patient groups. Protein clusters and pathways were characteristic to each condition were identified such as cell-cell connections, protein folding and endoplasmic reticulum stress. The complex analysis revealed proteins important in obesity and T2D.

This research was funded by NKFIH FK134605, GINOP-2.3.3-15-2016-00020, KIM NKFIA 2022-2.1.1-NL-2022-00005, European Union's Horizon 2020 research and innovation programme under grant agreement No 739593.