Youhe Gao

Early urinary candidate biomarkers in a rat model of experimental autoimmune encephalomyelitis

Biomarker is the change associated with the disease. Without homeostatic control, urine can accumulate early changes in the body. We expect that urinary proteome can also reflect early changes in the nervous system and urine is a better biomarker source for nervous system diseases. Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system and is difficult to diagnose in early stages. In this study, a tandem mass labeling approach coupled with high-resolution mass spectrometry was used to analyze seven-day urinary proteome changes in a rat model of experimental autoimmune encephalomyelitis when the clinical scores in the EAE group were “0” and no obvious histological changes were observed. Thirty-one urinary proteins were altered, based on Ingenuity Pathway Analysis, seventeen of these proteins were associated with neurological functions. The top canonical pathways represented by these dysregulated proteins included the acute phase response and metabolic processes. The acute phase response was characterized by an increase in inflammatory factors that are known to cause multiple sclerosis. Additionally, lipid or glucose metabolic alterations may provide clues for future mechanistic studies on multiple sclerosis. Fourteen proteins were identified to have catalytic activities that may contribute to neuronal damage. Furthermore, among the seven proteins that were most affected, six were reported to be expressed in the serum/cerebrospinal fluid/brain tissue of multiple sclerosis patients, thereby indicating that urine can be a good source of biomarkers for the early detection of multiple sclerosis.

Urine proteome changes in a TNBS-induced colitis rat model

Urine is an important resource for biomarker research. Without homeostasis, urine accumulates markers of all the changes in the body. Urine proteins reflect not only renal diseases but also changes in other organs in the body. However, urine has rarely been used to reflect inflammatory bowel disease. In the present study, a trinitrobenzene sulfonic acid (TNBS)-induced colitis rat model was used to mimic the human inflammatory bowel disease Crohn’s disease (CD). Urine samples from a control group (n=3), a TNBS 2-day group (n=3) and a TNBS 7-day group (n=3) were analyzed for candidate biomarker discovery by label-free and TMT-labeled proteomic quantitative methods. Seventy-seven urinary proteins were significantly changed in the colitis rats compared with that in the controls. These proteins were further validated by parallel reaction monitoring (PRM) targeted proteomic quantitative methods. Urine samples from the control group (n=8), the TNBS 2-day group (n=11) and the TNBS 7-day group (n=11) were analyzed by PRM. This led to the identification of 9 significantly differential expressed urinary proteins: CAH1, G3P, MMP-8, MANBA, NGAL, RNS1G, SLC31, S6A18, and TMM27. Based on the human protein tissue atlas, CAH1, RNS1G and SLC31 are highly enriched in the gastrointestinal tract. Among the 9 PRM-validated proteins, CAH1, MMP-8 and NGAL were previously reported as IBD-associated proteins (all exhibiting consistent trends with our observation), whereas the others are newly discovered by this study. Our results provide valuable clues for future study of urine biomarker of inflammatory bowel disease and Crohn’s disease.

Unilateral relapse of Behcet\'s disease-associated uveitis does not appear to cause asymmetric tear protein profiles

Purpose: To explore whether unilateral relapse of Bechet’s disease uveitis (BDU) causes differences in the tear proteome between the diseased and the contralateral quiescent eye. Experimental design: To minimize interindividual variations, bilateral tear samples were collected from the same patient (n=15) with unilateral relapse of BDU. A data-independent acquisition (DIA) strategy was used to identify proteins that differed between active and quiescent eyes. Results: A total of 1,797 confident proteins were identified in the tear samples, of which 371 are also highly expressed in various tissues and organs. Sixty-two (3.5%) proteins differed in terms of expression between tears in active and quiescent eyes, similar to the number of differentially expressed proteins (74, 4.1%) identified in a randomized grouping strategy. Furthermore, the intrapair trend of the differentially expressed proteins was not consistent and none of the proteins showed the same trend in more than 9 pairs of eyes. Conclusions and clinical relevance: Unilateral relapse of BDU does not appear to cause asymmetric changes in the tear proteome between active and contralateral quiescent eyes. Tear fluid is a valuable source for biomarker studies of systemic diseases. Statement of clinical relevance Tears are an easily, noninvasively accessible body fluid that is a valuable source of biomarkers for various diseases. Behcet’s disease uveitis (BDU) has high potential to cause blindness and represents the leading cause of morbidity in BD patients, especially in frequently relapsing cases. Here, we adopted a method combining a “dry” method for tear preservation and nano-LC-DIA-MS/MS system to explore whether unilateral relapse of BDU causes differences in the tear proteome between the diseased and the contralateral quiescent eye, with the aim of evaluating tear fluid as a source for biomarker studies of uveitis relapse.

Changes in the urinary proteome in a Patient-Derived Xenograft model

In this report, the urinary proteome from a patient-derived xenograft (PDX) model was compared at the peptide level to study the origins of urinary proteins in tumor-bearing nude mice. Urine was collected from the PDX mice before and after tumor implantation. A total of 515 mouse proteins were identified, of which 8 were differential proteins. Seventy-eight unambiguous human peptides from 42 human proteins were identified in the tumor-bearing group. Compared with the differential urinary proteins from the tumor-bearing immuno-competent rats, the differential proteins in the urine from the PDX model had no host immune response proteins in the very early stage urine in the tumor-bearing immuno-competent rat model.

Urinary protein changes in the early phase of smoking-induced Chronic Obstructive Pulmonary Disease in a rat model

Chronic obstructive pulmonary disease (COPD) is a group of severe respiratory diseases. Identifying COPD through early urinary biomarkers by proteomics technology may help to reduce the mortality rate of the disease, improve the quality of life of patients and reduce the burden on society. Urine samples from a COPD rat model induced by smoking were taken at week 2, week 4 and week 8. By LC-MS/MS, 15 differential proteins with human orthologs were identified. After smoking for 2 weeks when there were no significant pathological changes, 8 differential proteins were identified: 2 proteins had been reported to be markers of COPD, while 4 proteins were associated with COPD. After smoking for 4 weeks, which is when slight pathological changes were observed, 7 differential proteins were identified: 3 of them were reported to be associated with COPD, while 1 protein had been reported to be a marker of COPD. After smoking for 8 weeks, there were significant pathological changes: 5 differential proteins were identified, 3 of which were reported to be associated with COPD. The results of this study suggest that differential urinary proteins may provide important clues for the early diagnosis of COPD.

Early candidate biomarkers in urine of Walker-256 lung metastasis rat model.

Cancer metastasis accounts for the majority of deaths by cancer. Detection of cancer metastasis at its early stage is important for the management and prediction of cancer progression. Urine, which is not regulated by homeostatic mechanisms, reflects systemic changes in the whole body and can potentially be used for the early detection of cancer metastasis. In this study, a lung metastasis of a Walker-256 rat model was established by tail-vein injection of Walker-256 cells. Urine samples were collected at days 2, 4, 6 and 9 after injection, and the urinary proteomes were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The urinary protein patterns changed significantly with the development of Walker-256 lung metastasis. On the fourth day, lung metastasis nodules appeared. On the sixth day, clinical symptoms started. On days 2, 4, 6 and 9, 11, 25, 34 and 44 differential proteins were identified in 7 lung metastatic rats by LC-MS/MS. Seventeen of these 62 differential proteins were identified on the second day, and 18 of them were identified on the fourth day. The differential urinary proteins changed significantly two days before lung metastasis nodules appeared. Differential urinary proteins differed in Walker-256 lung metastasis rat models and Walker-256 subcutaneous rat models. A total of 9 differential proteins (NHRF1, CLIC1, EZRI, AMPN, ACY1A, HSP7C, BTD, NID2, and CFAD) were identified in 7 lung metastatic rats at one or more common time points, and these 9 differential proteins were not identified in the subcutaneous rat model. Seven of these 9 differential proteins were associated with both breast cancer and lung cancer, eight of the nine were identified on the second day, and 8 of the nine can be identified on the fourth day; these early changes in urine were also identified with differential abundances at late stages of lung metastasis. Our results indicate that (1) the urine proteome changed significantly, even on the second day after tail-vein injection of Walker-256 cells and that (2) the urinary differential proteins were different in Walker-256 lung metastatic tumors and Walker-256 subcutaneous tumors. Our results provide the potential to detect early breast cancer lung metastasis, monitor its progression and differentiate it from the same cancer cells grown at other locations.

Candidate urine biomarker discovery from only five pairs of samples before and after tumor resection in glioma patients

Biomarkers are measurable changes associated with the disease. Without the control of homeostatic mechanisms, urine accumulates systemic body changes and thus serves as an excellent early biomarker source. However, urine is affected by many factors other than disease. Although many candidate biomarkers have been identified in animal models, a large number of clinical samples might still be required for the disease related changes. A self-controlled study should be able to avoid the interferences of individual differences among patients. Gliomas are the most common primary malignant brain tumors and have a very poor prognosis. Early diagnosis of gliomas and the monitoring of tumor recurrence are crucial to improve glioma patient outcomes. Here we set to try if biomarker candidates can be identified by comparing urine samples from five glioma patients collected at the time of tumor diagnosis and after surgical removal of the tumor. Using label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) quantification, twenty-seven urinary proteins were significantly changed after tumor resection (fold change ≥ 1.5, P-value < 0.05, and similar changes in all 5 patients), many of which have been previously associated with gliomas, such as CEACAM1, ANXA7, CALR, CRYAB, CD276, pIgR and cathepsin D. Functions of these proteins were significantly enriched in the regulation of tissue remodeling, autophagy, the inhibition of gene expression, the positive regulation of natural killer cell-mediated cytotoxicity and angiogenesis, which are associated with glioma development. Our results suggested that using the self-control of before and after tumor resection is an effective method to identify differential proteins associated with the disease, even with a small number of patients.

Effects of regional differences on the urinary proteomes of healthy Chinese individuals

Urine is a promising biomarker source for clinical proteomics studies. Although regional physiological differences are common in multi-center clinical studies, the presence of significant differences in the urinary proteomes of individuals from different regions remains unknown. In this study, morning urine samples were collected from healthy urban residents in three regions of China and urinary proteins were preserved using a membrane-based method (Urimem). The urine proteomes of 27 normal samples were analyzed using LC-MS/MS and compared among the three regions. We identified 1,898 proteins from Urimem samples using label-free proteome quantification, of which 62 urine proteins were differentially expressed among the three regions. Hierarchical clustering analysis showed that inter-regional differences caused less significant changes in the urine proteome than inter-sex differences. Of the 62 differentially expressed proteins, 10 have been reported to be disease biomarkers in previous clinical studies. Urimem facilitates urinary protein storage for large-scale urine sample collection, and thus accelerates biobank development and urine biomarker studies employing proteomics approaches. Regional differences are a confounding factor influencing the urine proteome and should be considered in future multi-center biomarker studies.

Effects of the glucocorticoid drug prednisone on urinary proteome and candidate biomarkers

Urine is a good source of biomarkers for clinical proteomics studies. However, one challenge in the use of urine biomarkers is that outside factors can affect the urine proteome. Prednisone is a commonly prescribed glucocorticoid used to treat various diseases in the clinic. To evaluate the possible impact of glucocorticoid drugs on the urine proteome, specifically disease biomarkers, this study investigated the effects of prednisone on the rat urine proteome. Urine samples were collected from control rats and prednisone-treated rats after drug administration. The urinary proteome was analyzed using liquid chromatography–tandem mass spectrometry (LC-MS/MS), and proteins were identified using label-free proteome quantification. Differentially expressed proteins and their human orthologs were analyzed with bioinformatics methods. A total of 523 urinary proteins were identified in rat urine. Using label-free quantification, 27 urinary proteins showed expression changes after prednisone treatment. A total of 16 proteins and/or their human orthologs have been previously annotated as disease biomarkers. After functional analysis, we found that the pharmacological effects of prednisone were reflected in the urine proteome. Thus, urinary proteomics has the potential to be a powerful drug efficacy monitoring tool in the clinic. Meanwhile, alteration of the urine proteome due to prednisone treatment should be considered in future disease biomarker studies.

A Dry Method For Preserving Tear Samples

Tears covering the ocular surface is an important bio-fluid containing thousands of molecules, including proteins, lipids, metabolites, nucleic acids, and electrolytes. Tears are valuable resources for biomarker research of ocular and even systemic diseases. For application in biomarker studies, tear samples should ideally be stored using a simple, low-cost, and efficient method along with the patient’s medical records. For this purpose, we developed a novel Schirmer’s strip-based dry method that allows for storage of tear samples in vacuum bags at room temperature. Using this method, tear protein patterns can also be preserved. Liquid chromatography-mass spectrometry/mass spectrometry analysis of proteins recovered from the dry method and traditional wet method showed no significant difference. Some tissue/organ enriched proteins were identified in tear, thus tear might be a good window for monitoring the change of these tissues or organs. This dry method facilitates sample transportation and enables the storage of tear samples on a large scale, increasing the availability of samples for studying disease biomarkers in tears.