PROTEOMICS – Clinical Applications最新文献

Purpose: Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence of Tf protein variants within the general population. Although reports describe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic.

Experimental design: Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot, N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing of PMM2 was performed.

Results: In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies. PMM2 causative variants were identified in both patients.

Conclusion and clinical relevance: We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.

Aim: Hypoxic Ischemic Encephalopathy (HIE) is one of the principal causes of neonatal mortality and long-term morbidity worldwide. The neonatal signs of mild cerebral injury are subtle, making an early precise diagnosis difficult. Delayed detection, poor prognosis, and lack of specific biomarkers for the disease are increasing mortality rates. In this study, we intended to identify specific biomarkers using comparative proteomic analysis to predict the severity of perinatal asphyxia so that its outcome can also be prevented.

Experimental design: A case-control study was conducted on 38 neonates, and urine samples were collected within 24 and 72 h of life. A tandem mass spectrometry-based quantitative proteomics approach, followed by validation via sandwich ELISA, was performed.

Results: The LC-MS/MS-based proteomics analysis resulted in the identification of 1201 proteins in urine, with 229, 244, and 426 being differentially expressed in HIE-1, HIE-2, and HIE-3, respectively. Axon guidance, Diseases of programmed cell death, and Detoxification of reactive oxygen species pathways were significantly enriched in mild HIE versus severe HIE. Among the differentially expressed proteins in various stages of HIE, we chose to validate four proteins - APP, AGT, FABP1, and FN1 - via sandwich ELISA. Individual and cumulative ROC curves were plotted. AGT and FABP1 together showed high sensitivity, specificity, and accuracy as potential biomarkers for early diagnosis of HIE.

Conclusion: Establishing putative urinary biomarkers will facilitate clinicians to more accurately screen neonates for brain injury and monitor the disease progression. Prompt treatment of neonates may reduce mortality and neurodevelopmental impairment.

Purpose: The discovery of specific and sensitive disease-associated biomarkers for early diagnostic purposes of many diseases is still highly challenging due to various complex molecular mechanisms triggered, high variability of disease-related interactions, and an overlap of manifestations among diseases. Human peripheral blood mononuclear cells (PBMCs) contain protein signatures corresponding to essential immunological interplay. Certain diseases stimulate PBMCs and contribute towards modulation of their proteome which can be effectively identified and evaluated via the comparative proteomics approach.

Experimental design: In this review, we made a detailed survey of the PBMCS-derived protein biomarker candidates for a variety of diseases, published in the last 15 years. Articles were preselected to include only comparative proteomics studies.

Results: PBMC-derived biomarkers were investigated for cancer, glomerular, neurodegenerative/neurodevelopmental, psychiatric, chronic inflammatory, autoimmune, endocrinal, infectious, and other diseases. A detailed review of these studies encompassed the proteomics platforms, proposed candidate biomarkers, their immune cell type specificity, and potential clinical application.

Conclusions: Overall, PBMCs have shown a solid potential in giving early diagnostic and prognostic biomarkers for many diseases. The future of PBMC biomarker research should reveal its full potential through well-designed comparative studies and extensive testing of the most promising protein biomarkers identified so far.

Purpose: Urine proteome is a valuable reservoir of biomarkers for disease diagnosis and monitoring. Following formation as the plasma filtrate in the kidney, urine is progressively modified by the active reabsorption and secretion of the urinary tract. However, little is known about how the urine proteome changes as it passes along the urinary tract.

Experimental design: To investigate this, we compared the proteome composition of the renal pelvis urine (RPU) and individually self-voided bladder urine (BU) collected from seven unilateral urinary tract obstruction male patients by LC-MS/MS screening. To our knowledge, this is the first proteomic comparison of RPU and BU samples from the same individual.

Results: Overall, RPU and BU proteomes did not exhibit proteins that were exclusively present in all samples of one urine type while in none of the other type. Nonetheless, BU had more overrepresented proteins that were observed at a higher frequency than RPU. Label-free quantitative analyses revealed BU-RPU differential proteins that are enriched in exosomes and extracellular proteins. However, the differences were not significant after corrections for multiple testing. Interestingly, we observed a significant increase of collagen peptides with hydroxyproline modifications in the BU samples, suggesting differences in protein modifications.

Conclusions and clinical relevance: Our study revealed no substantial differences at the protein level between the BU and RPU samples. Future investigations with expanded cohorts would provide more insights about the urothelial-urinary interactions.

Purpose: Advances in mass spectrometry-based quantitative proteomic analysis have successfully demonstrated the in-depth detection of protein biomarkers in bronchoalveolar lavage fluid (BALF) from patients with lung cancers. Recently, ion mobility technology was incorporated into the mass spectrometers escalating the sensitivity and throughput. Utilizing these advantages, herein, we employed the parallel accumulation-serial fragmentation (PASEF) implanted in a timsTOF Pro mass spectrometer to examine the alteration of BALF proteomes in patients with nonsmall cell lung cancers (NSCLCs).

Experimental design: BALF proteins were processed from patients with NSCLC and analyzed in a timsTOF Pro mass spectrometer with the PASEF method using a peptide input of 100 ng. Label-free mass spectrometry data were analyzed in the FragPipe platform.

Results: We quantitated over 1400 proteins from a single injection of 100 ng of peptides per sample with a median of ∼2000 proteins. We were able to find a few potential biomarker proteins upregulated in NSCLC.

Conclusions and clinical relevance: The alterations of the BALF proteome landscape vary among patients with NSCLC as previously observed in patients with small-cell lung cancers. The PASEF method has significantly enhanced the sensitivity and throughput, demonstrating its effectiveness in clinical research and application.

Purpose: Despite recent advancements in our understanding of driver gene mutations and heterogeneity within brain tumors, whether primary or metastatic (also known as secondary), our comprehension of proteomic changes remains inadequate. The aim of this study is to provide an informative source for brain tumor researches, and distinguish primary brain tumors and secondary brain tumors from extracranial origins based on proteomic analysis.

Experimental design: We assembled the most frequent brain tumors as follows: gliomas from WHO grade 2 to 4, with IDH1 mutations and wildtypes; brain metastases (BrMs) originating from lung cancer (LC), breast cancer (BC), ovarian cancer (OC), and colorectal cancer (CC). A total of 29 tissue samples were analyzed by label free quantitative mass spectrometry-based proteomics.

Results: In total, 8165 protein groups were quantified, of which 4383 proteins were filtered at 50% valid intensity values for downstream analysis. Proteomic analysis of BrMs reveals conserved features shared among multiple origins. While proteomic heterogeneities were found for discriminating different grades of gliomas, as well as IDH1 mutant and wildtype gliomas. In addition, notable distinctions were observed at the pathway level between BrMs and gliomas. Specifically, BrMs exhibited characteristic pathways focused on proliferation and immunomodulation after colonizing the brain, whereas gliomas primarily engaged in invasion processes.

Conclusions and clinical relevance: We characterized an extensive proteomic landscape of BrMs and gliomas. These findings have promising implications for the development of targeted therapies for BrMs and gliomas.