Current Proteomics - Volume 20, Issue 2, 2023

Dry Blood Spots (DBS) have been used in combination with liquid chromatography-mass spectrometry for targeted proteomics to identify sensitive and specific novel biomarkers. DBS presents several advantages over other traditional blood sampling methods. This review discusses the past, present and future of the technology, focusing on studies with clinical and population relevance. Arguments for and against DBS are presented by discussing technological advances, particularly those related to Mass Spectrometry (MS) and Multiple Reaction Monitoring (MRM), sample preparation issues, disease biomarkers, pharmacokinetics, and pharmacodynamics. There will be a focus on proteomic studies that rely on DBS as a sampling method. In this context, numerous studies on the diagnosis and treatment of several diseases. To date, proteomic reports of studies using DBS have shown that DBS can facilitate diagnosis and prognosis. DBS offers several advantages that make it a viable option for many fields. Moreover, some of its disadvantages can be easily overcome through automation to increase reproducibility and reduce protocol variability and standardization of parameters such as the volume of sample used. Within this context, here we propose to review the advantages and disadvantages of using DBS for blood proteomics and provide an understanding of how current DBS-based protocols are being conducted for future standardization and protocol optimization.

Background: Chlamydiasis is a widespread bacterial infection in the world. Serological tests are expensive, and in addition, intrinsic antigens can cause cross-reactions and make the diagnosis process difficult. Multi-epitope protein antigens are novel and potential diagnostic markers that have the capability of more accurate and cheaper diagnosis. Therefore, in this study, the main goal is to design a new protein vaccine, including multiple epitopes of B cells with dominant immunity from three proteins named MOMP, ompA and Pgp3D from C. trachomatis.Methods: The amino acid sequences were obtained from the UniProt database. The areas with the highest antigenicity were identified using the EMBOSS server. Linear B cell epitopes were determined using BCPRED, ABCpred, and Bepipred servers. Epitopes with the highest antigenicity were connected using the EAAAK linker.Results: Two epitopes from MOMP, two from ompA, and one from Pgp3D were selected. These epitopes were connected to each other with the EAAAK linker. Three residues (0.592), 16 residues (0.76), 36 residues (0.578), and 37 residues (0.734) were obtained from the prediction of the spatial structure of the B cell multiple epitopes designed with ElliPro. Model 1 of RaptorX was selected as the best structure. In this model, the ERRAT quality, ProSA-web z-score, and Verify3D were 83.1169, - 5.17 and 84.62% with PASS score, respectively. Moreover, the Ramachandran plot showed that 86.093% of the amino acid residues were located in the favored region. To achieve the highest level of protein expression, the designed multi-epitope reverse-translated with the Genscript server and was cloned in E. coli. The highest level of expression was achieved, and a CAI score of 0.91 was reported. The gene GC content was 51.98%, and the contribution of low-frequency codons was 0%.Conclusion: The results confirmed that the designed construct could identify C. trachomatis with high sensitivity and specificity in serum samples of patients with chlamydiasis. However, further experimental studies are needed for final confirmation.

Background: Cold exposure can induce inflammation-related injury in lung tissue, but the exact mechanism is still unclear.Objective: The study aimed to clarify the proteomic characteristics of lung tissue under cold exposure.Methods: Forty mice were randomly equally divided into a control group and a model group. The model group was exposed to - 20°C for two weeks (4 hours per day), while the control group was maintained at 22 ±2°C. H&E staining and ELISA were used to verify the injury of lung tissue. Furthermore, a quantitative analysis of the overall proteome in the lung of mice exposed to cold stress was conducted by using LC-MS/MS. 15 differentially expressed proteins were selected for PRM validation.Results: According to our results, cold exposure induced lung injury, and the expressions of 151 proteins were upregulated and those of 95 proteins were downregulated. Bioinformatics analysis showed that differentially expressed proteins were associated with tricarboxylic acid cycle, fat metabolism, glycolysis, and oxidative phosphorylation. The expression of gabra2, Klkb1, and complement- related proteins was significantly upregulated. The results of PRM validation were consistent with those of proteomics.Conclusion: We found changes in glycolysis, gabra2, Klkb1, and the complement system in the lung tissue of cold-stressed mice, which may play an important role in cold stress-induced lung injury.

Background: Ricin is the most toxic protein known. It is part of the ribosome-inactivating proteins, RIPs, type 2, which has generated importance in his research; it is possible to detoxify this protein with phenolic compounds; however, it is essential to understand how this detoxification occurs. To analyze using electrophoresis, UV-visible spectroscopy, and high-performance liquid chromatography (HPLC) the protein ricin with the flavonol quercetin, understanding the detoxification process.Methods: The UV-visible analysis was performed on both the supernatant and the precipitate of the samples; these results were analyzed using one-factor analysis of variance (ANOVA) and a Tukey test with a significance level of 0.05.Results: 34.9 μg/mL of total protein and 4.2 μg / mL of ricin were obtained in the extraction method. Eight interactions were carried out, and all presented precipitation, observing through the electrophoresis technique a decrease in the bands corresponding to the protein; these results were analyzed with HPLC observing a decrease in the size of the area of the peaks in the chromatograms.Conclusion: The results obtained in this study suggest an agglomeration of the protein, generating a precipitate that could benefit the protein's inactivation as a detoxification process.

Introduction: Ovarian cancer is a common gynecological malignancy. It is one of the leading causes of death among women worldwide. The incidence of ovarian cancer ranks third, and mortality is the first among gynecological malignant tumors. CCL2 (Chemokine C-C motif Ligand 2) is associated with the progression of a variety of tumors, including ovarian cancer. However, the mechanism of CCL2 in A2780 cell growth has not been clarified.Method: In this study, we found that exogenous CCL2 promoted A2780 cell activity. RNA sequencing was used to identify the transcriptomic changes in CCL2-treated A2780 cells. Based on a p-value less than 0.05 and |log2 Fold Change| greater than 1, 190 differentially expressed genes were selected. Of these genes, 82 were observed to be upregulated and 108 downregulated.Result: The GO (gene ontology) analysis of differentially expressed genes was used to identify the underlying functions and biological processes. In addition, the expression of the topmost upregulated genes was verified by qPCR.Conclusion: This work may provide new markers and reveal the underlying mechanism of exogenous CCL2 in A2780 cell proliferation.

Background: Neonate lung injury is a common phenomenon after perinatal asphyxia.Objective: To evaluate proteomic profiles of exosomes isolated from lung injury offspring serum after perinatal asphyxia.Methods: Serum samples were collected at 12 h, 24 h, and 72 h after birth in neonates with perinatal asphyxia-induced lung injury. Exosomes were isolated, and the concentration and size distribution were assessed. The exosome surface markers CD9, CD63, CD81, HSP70, and TSG101 were detected by Western blot. The exosome proteins were evaluated by quantitative proteomics using a tandem mass tag (TMT). All the identified proteins were submitted to the Weighted Gene Co-Expression Network Analysis (WGCNA), GO function, and KEGG pathway analysis. A protein-protein interaction network (PPI) was utilized to identify hub proteins with the Cytohubba plugin of Cytoscape.Results: The exosomes were round or oval vesicular structures at a diameter range of 100-200 nm, and the size distribution was standard and consistent. Exosome surface markers CD9, CD63, CD81, HSP70, and TSG101 were detected. 444 out of 450 proteins were mapped with gene names. A brown module containing 71 proteins was highly linked with the 12 h phenotype and was predominantly concentrated in lipoprotein and complement activation. The top 10 proteins, APOA1, APOB, APOE, LPA, APOA2, CP, C3, FGB, FGA, and TF, were determined as hub proteins.Conclusion: The present study demonstrates comprehensive information for understanding molecular changes of lung injury following perinatal asphyxia, which provides a reliable basis for screening potential biomarkers and therapeutic targets in the clinic.