Urinary protein changes during the short-term growth and development of rats.

Can the urine proteome reflect short-term changes in the growth and development of animals? Do short-term developmental effects on urinary protein need to be considered when performing urine marker studies using model animals with faster growing periods? In this study, urine samples were collected from 10 Wistar rats aged 6-8 weeks 3 and 6 days apart. The results showed that the urine proteome could sensitively reflect short-term growth and development in rats. For example, comparing the urine proteome of Day 0 and Day 6, 195 differential proteins were identified after screening (FC ≥ 1.5 or ≤ 0.67, P < 0.05), and verified by randomization, the average number of randomly generated differential proteins was 17.99. At least 90.77 % of the differential proteins were not randomly generated. This finding demonstrates that the differential proteins identified in the samples collected at different time points were not randomly generated. A large number of biological processes and pathways related to growth and development were enriched, which shows that the urine proteome reflects the short-term growth and development of rats, and provides a means for in-depth and meticulous study of growth and development. Moreover, an interfering factor in animal experiments using 6- to 8-week-old rats to construct models was identified. The results of this study demonstrated that there were differences in the urinary proteome in rats aged 6-8 weeks only 3-6 days apart, which suggests that the sensitivity of urinary proteomics is high and shows the sensitive and precise response of the urinary proteome to body changes.

Urine proteomic analysis of the rat e-cigarette model.

Background: We were curious if the urinary proteome could reflect the effects of e-cigarettes on the organism. Methods: Urine samples were collected from a rat e-cigarette model before, during, and after two weeks of e-cigarette smoking. Urine proteomes before and after smoking of each rat were compared individually, while the control group was set up to rule out differences caused by rat growth and development. Results: Fetuin-B, a biomarker of chronic obstructive pulmonary disease (COPD), and annexin A2, which is recognized as a multiple tumour marker, were identified as differential proteins in five out of six smoking rats on day 3. To our surprise, odourant-binding proteins expressed in the olfactory epithelium were also found and were significantly upregulated. Pathways enriched by the differential proteins include the apelin signalling pathway, folate biosynthesis pathway, arachidonic acid metabolism, chemical carcinogenesis-DNA adducts and chemical carcinogenesis-reactive oxygen species. They have been reported to be associated with immune system, cardiovascular system, respiratory system, etc. Conclusions: Urinary proteome could reflect the effects of e-cigarettes in rats.

Early urinary candidate biomarkers and clinical outcomes of intervention in a rat model of experimental autoimmune encephalomyelitis.

Multiple sclerosis is a chronic autoimmune demyelinating disease of the central nervous system and is difficult to diagnose in early stages. Without homeostatic control, urine was reported to have the ability to accumulate early changes in the body. We expect that urinary proteome can reflect early changes in the nervous system. The early urinary proteome changes in a most employed multiple sclerosis rat model (experimental autoimmune encephalomyelitis) were analysed to explore early urinary candidate biomarkers, and early treatment of methylprednisolone was used to evaluate the therapeutic effect. Twenty-five urinary proteins were altered at day 7 when there were no clinical symptoms and obvious histological changes. Fourteen were reported to be differently expressed in the serum/cerebrospinal fluid/brain tissues of multiple sclerosis patients or animals such as angiotensinogen and matrix metallopeptidase 8. Functional analysis showed that the dysregulated proteins were associated with asparagine degradation, neuroinflammation and lipid metabolism. After the early treatment of methylprednisolone, the incidence of encephalomyelitis in the intervention group was only 1/13. This study demonstrates that urine may be a good source of biomarkers for the early detection of multiple sclerosis. These findings may provide important information for early diagnosis and intervention of multiple sclerosis in the future.

A multi-omics dataset of human transcriptome and proteome stable reference.

The development of high-throughput omics technology has greatly promoted the development of biomedicine. However, the poor reproducibility of omics techniques limits their application. It is necessary to use standard reference materials of complex RNAs or proteins to test and calibrate the accuracy and reproducibility of omics workflows. The transcriptome and proteome of most cell lines shift during culturing, which limits their applicability as standard samples. In this study, we demonstrated that the human hepatocellular cell line MHCC97H has a very stable transcriptome (r = 0.983~0.997) and proteome (r = 0.966~0.988 for data-dependent acquisition, r = 0.970~0.994 for data-independent acquisition) after 9 subculturing generations, which allows this steady standard sample to be consistently produced on an industrial scale in long term. Moreover, this stability was maintained across labs and platforms. In sum, our study provides omics standard reference material and reference datasets for transcriptomic and proteomics research. This helps to further standardize the workflow and data quality of omics techniques and thus promotes the application of omics technology in precision medicine.

Changes of Development from Childhood to Late Adulthood in Rats Tracked by Urinary Proteome.

To date, studies of development have mainly focused on the embryonic stage and a short time thereafter. There has been little research on the whole life of an individual from childhood to aging and death. For the first time, we used noninvasive urinary proteome technology to track changes in several important developmental time points in a group of rats, covering 10 time points from childhood, adolescence, young adulthood, middle adulthood, and near-death in old age. Similar to previous studies on puberty, proteins were detected and they are involved in sexual or reproductive maturation, mature spermatozoa in seminiferous tubules (first seen), gonadal hormones, decline of estradiol, brain growth, and central nervous system myelination, and our differential protein enrichment pathways also included reproductive system development, tube development, response to hormone, response to estradiol, brain development, and neuron development. Similar to previous studies in young adults, proteins were detected and they are involved in musculoskeletal maturity, peak bone mass, development of the immune system, and growth and physical development, and our differential proteins enrichment pathways also included skeletal system development, bone regeneration, system development, immune system processes, myeloid leukocyte differentiation, and developmental growth. Studies on aging-related changes in neurons and neurogenesis have been reported, and we also found relevant pathways in aged rats, such as regulation of neuronal synaptic plasticity and positive regulation of long-term neuronal synaptic plasticity. However, at all time points throughout life, there were many biological pathways revealed by differential urinary protein enrichment involving multiple organs, tissues, systems, etc. that have not been mentioned in existing studies. This study shows comprehensive and detailed changes in rat lifetime development through the urinary proteome, helping to fill the gap in development research. Moreover, it provides a new approach to monitoring changes in human health and diseases of aging using the urinary proteome.

Comparison of urine proteomes from tumor-bearing mice with those from tumor-resected mice.

Objective: This study aimed to address on the most important concern of surgeons-whether to completely resect tumor. Urine can indicate early changes associated with physiological or pathophysiological processes. Based on these ideas, we conducted experiments to explore changes in the urine proteome between tumor-bearing mice and tumor-resected mice. Method: The tumor-bearing mouse model was established with MC38 mouse colon cancer cells, and the mice were divided into the control group, tumor-resected group, and tumor-bearing group. Urine was collected 7 and 30 days after tumor resection. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify the urine proteome, which was analyzed for differentially expressed proteins and functional annotation. Results: (1) Seven days after tumor resection, 20 differentially expressed proteins distinguished the tumor-resected group and the tumor-bearing group. The identified biological processes included circadian rhythm, Notch signaling pathway, leukocyte cell-cell adhesion, and heterophilic cell-cell adhesion via plasma membrane cell adhesion molecules. (2) Thirty days after tumor resection, 33 differentially expressed proteins distinguished the tumor-resected group and the tumor-bearing group. The identified biological processes included cell adhesion; complement activation, the alternative pathway; the immune system process; and angiogenesis. (3) The difference in the urine proteome between the tumor-resected group and the healthy control group was smaller 30 days after tumor resection. Conclusion: Changes in the urinary proteome can reflect the complete resection of MC38 tumors.

Changes in the urinary proteome of rats after short-term intake of magnesium L-threonate(MgT).

Introduction: Magnesium (Mg) is an important mineral in living organisms. Magnesium has multiple functions in the human body, wherein it plays an important therapeutic and preventive role in a variety of diseases. Methods: Urine samples of rats before and after gavage of magnesium L-threonate (MgT) were collected, and the urinary proteome was identified using the LC-MS/MS technique and analyzed using various databases. Results and discussion: The results illustrated that the urinary proteome of rats was significantly altered after short-term intake of magnesium supplements and that the differential proteins and the biological functions were related to magnesium. This study innovatively establishes a method to study nutrients from the perspective of urine proteomics. This work demonstrates that the urinary proteome is capable of reflecting the effects of nutrient intake on the organism in a more systematic and comprehensive manner and has the potential to provide clues for clinical nutrition research and practice.

Changes to Urinary Proteome in High-Fat-Diet ApoE-/- Mice.

Cardiovascular disease is currently the leading cause of death worldwide. Atherosclerosis is an important pathological basis of cardiovascular disease, and its early diagnosis is of great significance. Urine bears no need nor mechanism to be stable, so it accumulates many small changes and is therefore a good source of biomarkers in the early stages of disease. In this study, ApoE-/- mice were fed a high-fat diet for 5 months. Urine samples from the experimental group and control group (C57BL/6 mice fed a normal diet) were collected at seven time points. Proteomic analysis was used for comparison within the experimental group and for comparison between the experimental group and the control group. The results of the comparison within the experimental group showed a significant difference in the urinary proteome before and after a one-week high-fat diet, and several of the differential proteins have been reported to be associated with atherosclerosis and/or as biomarker candidates. The results of the comparison between the experimental group and the control group indicated that the biological processes enriched by the GO analysis of the differential proteins correspond to the progression of atherosclerosis. The differences in chemical modifications of urinary proteins have also been reported to be associated with the disease. This study demonstrates that urinary proteomics has the potential to sensitively monitor changes in the body and provides the possibility of identifying early biomarkers of atherosclerosis.

Changes in the urinary proteome before and after quadrivalent influenza vaccine and COVID-19 vaccination.

The proteome of urine samples from quadrivalent influenza vaccine cohort were analyzed with self-contrasted method. Significantly changed urine protein at 24 hours after vaccination was enriched in immune-related pathways, although each person's specific pathways varied. We speculate that this may be because different people have different immunological backgrounds associated with influenza. Then, urine samples were collected from several uninfected SARS-CoV-2 young people before and after the first, second, and third doses of the COVID-19 vaccine. The differential proteins compared between after the second dose (24h) and before the second dose were enriched in pathways involving in multicellular organismal process, regulated exocytosis and immune-related pathways, indicating no first exposure to antigen. Surprisingly, the pathways enriched by the differential urinary protein before and after the first dose were similar to those before and after the second dose. It is inferred that although the volunteers were not infected with SARS-CoV-2, they might have been exposed to other coimmunogenic coronaviruses. Two to four hours after the third vaccination, the differentially expressed protein were also enriched in multicellular organismal process, regulated exocytosis and immune-related pathways, indicating that the immune response has been triggered in a short time after vaccination. Multicellular organismal process and regulated exocytosis after vaccination may be a new indicator to evaluate the immune effect of vaccines. Urinary proteome is a terrific window to monitor the changes in human immune function.

[Changes in the urine proteome in an acute hypoxic rat model].

The purpose of this study was to explore the effect of acute hypoxia on urine proteome in rats. In this study, rats were placed in a hypoxic chamber simulating a plateau environment at an altitude of 5 000 m for 24 hours. Urine samples were collected at 0, 12, and 24 h after hypoxia. Urinary proteins were profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Compared with the control (before hypoxia), a total of 144 differentially expressed proteins were identified in the hypoxia 12 h group, and 129 differentially expressed proteins were identified in the hypoxia 24 h group. Functional annotation analysis revealed that these differentially expressed proteins were involved in a series of biological pathways related to hypoxic stress, such as anti-oxidative stress, glycolysis, complement and coagulation cascade. Our results suggest that the urinary proteome can reflect significant changes upon acute hypoxic stimulation. These findings may provide an approach to judge the hypoxia state of the body and help to assist the detection of hypoxia state.

[Effect of methotrexate on the urinary proteome of rats].

Methotrexate (MTX) is a widely used immunosuppressive drug. Large-dose of MTX is used for the treatment of cancer while low-dose is used for the treatment of rheumatoid arthritis (RA). This study aimed to explore the effect of MTX on the urinary proteome of rats. MTX was given to rats orally to construct an MTX intragastric administration rat model. The urine of the rats were collected within 10 hours after giving MTX, and the urine proteins of the rats were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). A total of 31 differential proteins were identified, of which 7 proteins were related to the effect MTX and the symptom of RA. The biological processes of some rats reflected the effect of MTX on the body's glutathione metabolism and the JAK/STAT signaling pathway, which indicated that urine proteins have the ability to reflect the effects of MTX on the body of rats. The spectrum of the differential proteins of each single rat showed that different individuals respond to the drug quite differently.

Proteome analysis of urinary biomarkers in a cigarette smoke-induced COPD rat model.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory airway disease caused by inhalation of cigarette smoke (CS) and other harmful gases and particles. METHODS: This study aimed to explore potential urinary biomarkers for CS-induced COPD based on LC-MS/MS analysis. RESULTS: A total of 340 urinary proteins were identified, of which 79 were significantly changed (30, 31, and 37 at week 2, 4 and 8, respectively). GO annotation of the differential urinary proteins revealed that acute-phase response, response to organic cyclic compounds, complement activation classical pathway, and response to lead ion were significantly enriched at week 2 and 4. Another four processes were only enriched at week 8, namely response to oxidative stress, positive regulation of cell proliferation, thyroid hormone generation, and positive regulation of apoptotic process. The PPI network indicated that these differential proteins were biologically connected in CS-exposed rats. Of the 79 differential proteins in CS-exposed rats, 56 had human orthologs. Seven proteins that had changed at week 2 and 4 when there were no changes of pulmonary function and pathological morphology were verified as potential biomarkers for early screening of CS-induced COPD by proteomic analysis. Another six proteins that changed at week 8 when obvious airflow obstruction was detected were verified as potential biomarkers for prognostic assessment of CS-induced COPD. CONCLUSIONS: These results reveal that the urinary proteome could sensitively reflect pathological changes in CS-exposed rats, and provide valuable clues for exploring COPD biomarkers.

Many kinds of oxidized proteins are present more in the urine of the elderly.

BACKGROUND: Many studies have shown an association between aging and oxidation. To our knowledge, there have been no studies exploring aging-related urine proteome modifications. The purpose of this study was to explore differences in global chemical modifications of urinary protein at different ages. METHODS: Discovery (n=38) cohort MS data including children, young and old groups were downloaded from three published studies, and this data was analyzed using open-pFind for identifying modifications. Verification cohort human samples (n=28) including young, middle-aged, and old groups, rat samples (n=7) at three-time points after birth, adulthood, and old age were collected and processed in the laboratory simultaneously based on label-free quantification combined with pFind. RESULTS: Discovery cohort: there were 28 kinds of differential oxidations in the old group that were higher than those in the young or children group in. Verification cohort: there were 17 kinds of differential oxidations of 49 oxidized proteins in the middle and old groups, which were significantly higher than those in the young group. Both oxidations and oxidized proteins distinguished different age groups well. There were also 15 kinds of differential oxidations in old age higher than others in the rat cohort. The results showed that the validation experiment was basically consistent with the results of the discovery experiment, showing that the level of oxidized proteins in urine increased significantly with age. CONCLUSIONS: Our study is the first to show that oxidative proteins occur in urine and that oxidations are higher in older than younger ages. Perhaps improving the degree of excretion of oxidative protein in vivo through the kidney is helpful for maintaining the homeostasis of the body's internal environment, delaying aging and the occurrence of senile diseases.

Proteome Analysis of Urinary Biomarkers in a Bovine IRBP-Induced Uveitis Rat Model via Data-Independent Acquisition and Parallel Reaction Monitoring Proteomics.

Uveitis, a group of intraocular inflammatory diseases, is one of the major causes of severe visual impairment among the working-age population. This study aimed to screen potential urinary biomarkers for uveitis based on proteome analysis. An experimental autoimmune uveitis (EAU) rat model induced by bovine interphotoreceptor retinoid-binding protein (IRBP) was used to mimic uveitis. In discovery phase, a total of 704 urinary proteins were identified via data-independent acquisition (DIA) proteomic technique, of which 76 were significantly changed (34, 36, and 37 on days 5, 8, and 12, respectively, after bovine IRBP immunization). Gene Ontology annotation of the differential proteins indicates that acute-phase response, innate immune response, neutrophil aggregation, and chronic inflammatory response were significantly enriched. Protein-protein interaction network indicates that these differential urinary proteins were biologically connected in EAU, as a group. In validation phase, 17 proteins having human orthologs were verified as the potential markers associated with uveitis by parallel reaction monitoring (PRM) targeted quantitative analysis. Twelve differential proteins changed even when there were no clinical manifestations or histopathological ocular damage. These 12 proteins are potential biomarkers for early diagnosis of uveitis to prevent the development of visual impairment. Five differential proteins changed at three time-points and showed progressive changes as the uveitis progressed, and another five differential proteins changed only on day 12 when EAU severity peaked. These 10 proteins may serve as potential biomarkers for prognostic evaluation of uveitis. Our findings revealed that the urinary proteome could sensitively reflect dynamic pathophysiological changes in EAU, and represent the first step towards the application of urinary protein biomarkers for uveitis.

Dynamic urine proteome changes in a rat model of simvastatin-induced skeletal muscle injury.

Statin-associated muscle symptoms (SAMS) are the main side effects of statins. Currently, there are no effective biomarkers for accurate clinical diagnosis. Urine is not subject to homeostatic control and therefore accumulates early changes, making it an ideal biomarker source. We therefore examined urine proteome changes associated with SAMS. Here, we established a SAMS rat model by intragastric intubation with simvastatin (80 mg/kg). Biochemical analyses and hematoxylin and eosin staining were used to evaluate the degree of muscle injury. The urine proteome on days 3, 6, 9 and 14 was profiled using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Differential proteins on day 14 of SAMS were mainly associated with glycolysis/gluconeogenesis, pyruvate metabolism, metabolism of reactive oxygen species and apoptosis, which were associated with the pathological mechanism of SAMS. Among the 14 differential proteins on day 3, Fibrinogen gamma chain (FIBG), Osteopontin (OSTP) and C-reactive protein (CRP) were associated with muscle damage, while EH domain-containing protein 1(EHD1), Cubilin (CUBN) and Fibronectin (FINC) were associated with the pathogenic mechanisms of SAMS. Our preliminary results indicated that the urine proteome can reflect early changes in the SAMS rat model, providing the potential for monitoring drug side effects in future clinical research. SIGNIFICANCE: This study demonstrate that the early muscle damage caused by simvastatin can be reflected in urinary proteins. The urine proteome also has the potential to reflect the pharmacology and toxicology of drugs in future clinical research.

Urinary Proteome Changes during Pregnancy in Rats.

Pregnancy involves a significant number of physiological changes. A normal pregnancy is essential to ensure healthy maternal and fetal development. We sought to explore whether the urinary proteome could reflect the pregnancy process. Urine samples were collected from pregnant and control rats on various gestational days. The urinary proteome was profiled by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), and differential proteins were obtained by comparing to the gestational day 1 of the same group at each time point. Many pathways related to embryo implantation and trophoblast differentiation were enriched in the early days in urine. Liver, kidney, and bone development started early to be enriched in the pregnant group, but not in the control group. Interestingly, the developmental processes of the fetal heart such as heart looping and endocardial cushion formation could be seen in urine of pregnant rats. Moreover, the timings were consistent with those of embryological studies. The timing of the surfactant appearance in urine was right before birth. The differential proteins related to pancreas development appeared in urine at the time during reported time of pancreatic cell proliferation and differentiation. These processes were enriched only in the pregnant group and not in the control group. Furthermore, coagulation-associated pathways were found to be increasingly prominent before labor. Our results indicated that the urine proteome of pregnant rats can reflect the process of pregnancy, even fetal embryonic development. Maternal urinary proteome detection was earlier than the developmental time point of tissue sections observed by microscopy.

Comparison of urine proteome among rat models by intraperitoneal injection with single bacteria and co-injection with two bacteria.

PURPOSE: To explore and compare urine proteome changes among rat models by intraperitoneal injection with single bacteria and co-injection with two bacteria. METHOD: Escherichia coli and Staphylococcus aureus are two common human pathogens. Three rat models were established: (i) the intraperitoneal co-injection of E. coli and S. aureus model (ES model), (ii) intraperitoneal injection of E. coli model (E model), and (iii) intraperitoneal injection of S. aureus model (S model). Urinary proteomes on days 0, 1 and 2 of the three models were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: A total of 111, 34 and 94 differential proteins were identified in the ES model, E model and S model, respectively. Among them, some differential proteins were reported to be associated with bacterial infection. Approximately 47% differential proteins in the E model overlapped with ES model, and 37% differential proteins in the S model overlapped with ES model. Compared with the E model and S model, a total of 71 unique differential proteins were identified in the ES model. CONCLUSION: Our results indicated that (1) the urine proteome could distinguish different bacterial intraperitoneal injections models and (2) the effects of co-injection with two bacteria on the urine proteome were not simple superposition of single injection.

Changes in the urinary proteome in rats with regular swimming exercise.

PURPOSE: Urine can sensitively reflect early pathophysiological changes in the body. The purpose of this study was to explore the changes of urine proteome in rats with regular swimming exercise. METHODS: In this study, experimental rats were subjected to daily moderate-intensity swimming exercise for 7 weeks. Urine samples were collected at weeks 2, 5, and 7 and were analyzed by using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). RESULTS: Unsupervised clustering analysis of all urinary proteins identified at week 2 showed that the swimming group was distinctively different from the control group. Compared to the control group, a total of 112, 61 and 44 differential proteins were identified in the swimming group at weeks 2, 5 and 7, respectively. Randomized grouping statistical analysis showed that more than 85% of the differential proteins identified in this study were caused by swimming exercise rather than random allocation. According to the Human Protein Atlas, the differential proteins that have human orthologs were strongly expressed in the liver, kidney and intestine. Functional annotation analysis revealed that these differential proteins were involved in glucose metabolism and immunity-related pathways. CONCLUSION: Our results revealed that the urinary proteome could reflect significant changes after regular swimming exercise. These findings may provide an approach to monitor the effects of exercise of the body.

Dynamic Urinary Proteome Changes in Ovalbumin-Induced Asthma Mouse Model Using Data-Independent Acquisition Proteomics.

BACKGROUND: In this work, we aim to investigate dynamic urinary proteome changes during asthma development and to identify potential urinary protein biomarkers for the diagnosis of asthma. METHODS: An ovalbumin (OVA)-induced mouse model was used to mimic asthma. The urinary proteome from asthma and control mice was determined using data-independent acquisition combined with high-resolution tandem mass spectrometry. RESULTS: Overall, 331 proteins were identified, among which 53 were differentially expressed (26, 24, 14 and 20 on days 2, 8, 15 and 18, respectively; 1.5-fold change, adjust P<0.05). Gene Ontology annotation of the differential proteins showed that the acute-phase response, innate immune response, B cell receptor signaling pathway, and complement activation were significantly enriched. Protein-protein interaction network revealed that these differential proteins were partially biologically connected in OVA-induced asthma, as a group. On days 2 and 8, after two episodes of OVA sensitization, six differential proteins (CRAMP, ECP, HP, F2, AGP1, and CFB) were also reported to be closely associated with asthma. These proteins may hold the potential for the early screening of asthma. On days 15 and 18, after challenged with 1% OVA by inhalation, seven differential proteins (VDBP, HP, CTSE, PIGR, AAT, TRFE, and HPX) were also reported to be closely associated with asthma. Thus, these proteins hold the potential to be biomarkers for the diagnosis of asthma attack. CONCLUSION: Our results indicate that the urinary proteome could reflect dynamic pathophysiological changes in asthma progression.

Label-Free Liquid Chromatography-Mass Spectrometry Proteomic Analysis of the Urinary Proteome for Measuring the Escitalopram Treatment Response From Major Depressive Disorder.

Major depressive disorder (MDD) is a common mental disorder that can cause substantial impairments in quality of life. Clinical treatment is usually built on a trial-and-error method, which lasts ~12 weeks to evaluate whether the treatment is efficient, thereby leading to some inefficient treatment measures. Therefore, we intended to identify early candidate urine biomarkers to predict efficient treatment response in MDD patients. In this study, urine samples were collected twice from 19 respondent and 10 non-respondent MDD patients receiving 0-, 2-, and 12-week treatments with escitalopram. Differential urinary proteins were subsequently analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Our two pilot tests suggested that the urine proteome reflects changes associated with major depressive disorder at the early stage of treatment measures. On week 2, 20 differential proteins were identified in the response group compared with week 0, with 14 of these proteins being associated with the mechanisms of MDD. In the non-response group, 60 differential proteins were identified at week 2, with 28 of these proteins being associated with the mechanisms of MDD. In addition, differential urinary proteins at week 2 between the response and non-response groups can be clearly distinguished by using orthogonal projection on latent structure-discriminant analysis (OPLS-DA). Our small pilot tests indicated that the urine proteome can reflect early effects of escitalopram therapy between the response and non-response groups since at week 2, which may provide potential early candidate urine biomarkers to predict efficient treatment measures in MDD patients.