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Hepatocellular carcinoma (HCC) is the most prevalent form of primary liver cancer and a major cause of cancer-related mortality worldwide. Small extracellular vesicles (sEVs) are heterogeneous populations of membrane-structured vesicles that can be found in many biological fluids and are currently considered as a potential source of disease-associated biomarkers for diagnosis. The purpose of this study was to define the proteomic and phosphoproteomic landscape of urinary sEVs in patients with HCC. Mass spectrometry-based methods were used to detect the global proteome and phosphoproteome profiles of sEVs isolated by differential ultracentrifugation. Label-free quantitation analysis showed that 348 differentially expressed proteins (DEPs) and 548 differentially expressed phosphoproteins (DEPPs) were identified in the HCC group. Among them, multiple phosphoproteins related to HCC, including HSP90AA1, IQGAP1, MTOR, and PRKCA, were shown to be upregulated in the HCC group. Pathway enrichment analysis indicated that the upregulated DEPPs participate in the regulation of autophagy, proteoglycans in cancer, and the MAPK/mTOR/Rap1 signaling pathway. Furthermore, kinase-substrate enrichment analysis revealed activation of MTOR, AKT1, MAP2Ks, and MAPKs family kinases in HCC-derived sEVs, indicating that dysregulation of the MAPK and mTOR signaling pathways may be the primary sEV-mediated molecular mechanisms involved in the development and progression of HCC. This study demonstrated that urinary sEVs are enriched in proteomic and phosphoproteomic signatures that could be further explored for their potential use in early HCC diagnostic and therapeutic applications.

Many phosphoprotein biomarkers have been proved to exist in body fluids such as serum and urine, however, there is absence of rapid and efficient separation and identification method. In this study, we proposed to combine metal oxide affinity chromatography (MOAC), molecular imprinting technology (MIT) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to establish an effective approach to solve this problem. To verify the feasibility of this approach, we selected a typical phosphoprotein lysozyme (Lys) as template and magnetic TiO 2 as substrate to prepare the molecularly imprinted nanoparticles (denoted as Fe 3 O 4 @TiO 2 @Lys MIPs). A point worth noting is that polydopamine (PDA) as polymer layer made Fe 3 O 4 @TiO 2 @Lys MIPs more hydrophilic and biocompatible. Thanks to the recognition sites of phosphate and the template-shaped cavities, Fe 3 O 4 @TiO 2 @Lys MIPs showed great sensitivity (0.01 ng*μL -1 ) and selectivity (Lysozyme: BSA: β-casein = 1:100:100, mass ratio) in standard phosphoprotein solution. At the end, the Fe 3 O 4 @TiO 2 @Lys MIPs showed great separation ability to lysozyme phosphoprotein in both human serum and urine samples. Therefore, the MOAC-based molecularly imprinted approach is worthy to be expected in effective separation of phosphoprotein biomarker in complex body fluid, which will be a promising one in future., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Extracellular vesicles (EVs) have emerged as important carriers for intercellular communication and biological sources for diagnosis and therapeutics. Low efficiency in EV isolation from biofluids, however, severely restricts their downstream characterization and analysis. Here, we introduced a novel strategy for EV isolation from urine for prostate cancer diagnosis using bifunctionalized magnetic beads through high affinity Ti(IV) ions and the insertion of a phospholipid derivative, 1,2-distearoyl- sn -glycero-3-phosphoethanolamine, into the EV membrane synergistically. We demonstrated its efficient isolation of EVs from urine samples with low contamination, high recovery (>80%), and short separation time (within 1 h), resulting in the identification of 36,262 unique EV peptides corresponding to 3302 unique proteins and 3233 unique phosphopeptides representing 1098 unique phosphoproteins using only 100 μL and 5 mL urine samples, respectively. Coupled with trapped ion mobility spectrometry and parallel accumulation-serial fragmentation for phosphosite-specific resolution, quantitative phosphoproteomics of urine samples from prostate cancer patients and healthy individuals revealed 121 upregulated phosphoproteins in cancer patients in contrast to the healthy group. These particular advantages indicate that the novel bifunctional material enables sensitive EV phosphoproteomic analysis for noninvasive biomarker screening and early cancer diagnosis.

Introduction and Objective: Fibroblast growth factor (FGF-23) and matrix extracellular phosphoglycoprotein (MEPE) are bone-related factors and their role in physiologic conditions and in different life stages are unknown. We aimed to evaluate age- and pregnancy-related changes in MEPE and FGF-23 levels and their correlations with calcium (Ca)-phosphate (PO4) metabolism., Methods: The study population included 96 healthy children (50 females) and 31 women (11 healthy, 10 pregnant, and 10 lactating). Intact FGF-23 (iFGF-23), MEPE, ferritin, parathyroid hormone (PTH), 25-OH vitamin D, alkaline phosphatase (ALP), IGF-I, IGFBP-3 and, Ca, PO4 and creatine (Cre) in serum (S) and urine (U) samples were determined. The renal phosphate threshold (TmPO4/GFR) and z-scores for the parameters that show age-related changes were calculated., Results: Serum iFGF-23 concentrations showed nonsignificant changes with age; however, MEPE decreased with age, reaching the lowest levels after 7 years. Additionally, higher serum MEPE concentrations were observed during pregnancy. Other than ALP, all other examined parameters demonstrated age-related changes. ALP, BUN, S-Cre, and U-Ca/Cre showed puerperal and pregnancy related changes together with MEPE. iFGF-23 was positively correlated with S-PO4 and TmPO4/GFR. MEPE was positively correlated with S-Ca, S-PO4 and TmPO4/GFR and negatively correlated with PTH, IGF-1, and IGFBP-3., Conclusion: Not iFGF-23 but MEPE showed age-dependent changes and was affected by pregnancy. Although, MEPE and iFGF-23 did not correlate with each other, they seem to affect serum and urinary phosphate in the same direction. Additionally, we found evidence that ferritin and growth factors might have a role in serum calcium and phosphate regulation., (© 2020 S. Karger AG, Basel.)

Analysis of protein phosphorylation in extracellular vesicles (EVs) offers an unprecedented potential for understanding cancer signaling and early stage disease diagnosis. However, prior to the phosphoproteome analysis step, the isolation of EVs from biofluids remains a challenging issue to overcome due to the low yield and impurity from current isolation methods. Here, we carry out an extensive assessment of several EV isolation methods including a novel rapid isolation method EVTRAP for highly efficient capture of extracellular vesicles from human urine sample. We demonstrate that over 95% recovery yield can be consistently achieved by EVTRAP, a significant improvement over current standard techniques. We then applied EVTRAP to identify over 16 000 unique peptides representing 2000 unique EV proteins from 200 μL urine sample, including all known EV markers with substantially increased recovery levels over ultracentrifugation. Most importantly, close to 2000 unique phosphopeptides were identified from more than 860 unique phosphoproteins using 10 mL of urine. The data demonstrated that EVTRAP is a highly effective and potentially widely implementable clinical isolation method for analysis of EV protein phosphorylation.

Liquid biopsies using body fluids have gained much attention in recent years due to their multiple advantages in clinical diagnosis, such as less/non-invasive collection, suitability for longitudinal disease monitoring, and better representation of tumor heterogeneity. As an attractive choice for liquid biopsy, urine proteins and their post-translational modifications (PTMs) have the potential to offer significant insights into physiological variations and pathological changes in the human body. However, due to the intrinsically large variability of urine proteins and their PTMs among different individuals, there is a high demand for strategies for high-throughput analysis of a large number of samples to obtain a comprehensive view and a reliable reference interval of the urine proteome. In this work, we proposed a new urine phosphoproteome sample processing strategy that combines fast protein extraction, efficient multiple immobilized-proteases digestion, and tandemly connected centrifugal tips device-based facile phosphopeptide enrichment & fractionation. This strategy is capable of paralleled sample processing with an approximate five-fold reduction in processing time and is therefore particularly suitable for handling a large number of urine samples. Totally, we identified 4196 phosphosites in human urine proteins by mass spectrometry in replicated tests, a number which is dozens of times larger than those previously reported. Therefore, this strategy may have great potential in urine-based phosphoprotein biomarker screening and drug response studies., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: A noninvasive, highly sensitive and specific urine test is needed for bladder cancer (BC) diagnosis and surveillance in addition to the invasive cystoscopy. We previously described the diagnostic effectiveness of urinary tyrosine-phosphorylated proteins (UPY) and a new assay (UPY-A) for their measurement in a pilot study. The aim of this work was to evaluate the performances of the UPY-A using an independent cohort of 262 subjects., Methods: Urinary tyrosine-phosphorylated proteins were measured by UPY-A test. The area under ROC curve, cutoff, sensitivity, specificity and predictive values of UPY-A were determined. The association of UPY levels with tumour staging, grading, recurrence and progression risk was analysed by Kruskal-Wallis and Wilcoxon's test. To test the probability to be a case if positive at the UPY-A, a logistic test adjusted for possible confounding factor was used., Results: Results showed a significant difference of UPY levels between patients with BC vs healthy controls. For the best cutoff value, 261.26 Standard Units (SU), the sensitivity of the assay was 80.43% and the specificity was 78.82%. A statistically significant difference was found in the levels of UPY at different BC stages and grades between Ta and T1 and with different risk of recurrence and progression. A statistically significant increased risk for BC at UPY-A ⩾261.26 SU was observed., Conclusions: The present study supplies important information on the diagnostic characteristics of UPY-A revealing remarkable performances for early stages and allowing its potential use for different applications encompassing the screening of high-risk subjects, primary diagnosis and posttreatment surveillance.

Urine, by accumulating all kinds of changes, was proposed to be a better source for biomarker discovery. As one of the most common post-translational modifications, phosphorylation plays a vital role in many biological activities. However, the urine phosphoproteome has been largely neglected due to the low abundance of phosphoproteins and the presence of various phosphatases in urine. The low level of background phosphorylation in urine is actually advantageous, as urinary phosphopeptides/proteins that are stable to the phosphatases present in urine have the potential to serve as valuable disease biomarkers. Using a TiO2 enrichment strategy, this study aimed to create a comprehensive proteomic profile of human urinary phosphoproteins and to characterize the changes in the urine phosphoproteome after incubation of urine with renal carcinoma cell lysates. In total, 106 urine phosphorylation sites corresponding to 64 proteins, including 80 previously unidentified human urine protein phosphorylation sites, were identified by mass spectrometry. Fifteen phosphopeptides, together averaging 47% of the total phosphopeptides, were found in samples from three individuals. Cellular proteins are potential source of biomarker in urine phosphorylated proteins. Addition of renal carcinoma cellular proteins to urine did not significantly change the phosphorylation level of urine proteins. But there were still a few phosphopeptides from cell lysates survived urinary phosphatases; such phosphopeptides represent potential biomarkers in urine.

Background: Progress in the fields of protein separation and identification technologies has accelerated research into biofluids proteomics for protein biomarker discovery. Urine has become an ideal and rich source of biomarkers in clinical proteomics. Here we performed a proteomic analysis of urine samples from pregnant and non-pregnant patients using gel electrophoresis and high-resolution mass spectrometry. Furthermore, we also apply a non-prefractionation quantitative phosphoproteomic approach using mTRAQ labeling to evaluate the expression of specific phosphoproteins during pregnancy comparison with non-pregnancy., Results: In total, 2579 proteins (10429 unique peptides) were identified, including 1408 from the urine of pregnant volunteers and 1985 from the urine of non-pregnant volunteers. One thousand and twenty-three proteins were not reported in previous studies at the proteome level and were unique to our study. Furthermore, we obtained 237 phosphopeptides, representing 105 phosphoproteins. Among these phosphoproteins, 16 of them were found to be significantly differentially expressed, of which 14 were up-regulated and two were down-regulated in urine samples from women just before vaginal delivery., Conclusion: Taken together, these results offer a comprehensive urinary proteomic profile of healthy women during before and after vaginal delivery and novel information on the phosphoproteins that are differentially regulated during the maintenance of normal pregnancy. Our results may provide a better understanding of the mechanisms of pregnancy maintenance, potentially leading to the development of biomarker-based sensitive assays for understanding pregnancy.

Background: Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine., Methods: The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated., Results: Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform., Conclusions: A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker., General Significance: The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The urinary proteome is known to be a valuable field of study related to human physiological functions because many components in urine provide an alternative to blood plasma as a potential source of disease biomarkers useful in clinical diagnosis and therapeutic application. Due to the variability and complexity of urine, sample preparation is very important for decreasing the dynamic range of components and isolating specific urinary proteins prior to analysis. We discuss many useful sample preparation methods in this chapter, including those of lung cancer urine samples. In addition, protein detection methods are also crucial in visualizing protein profiles and for quantification of protein content in urine samples from both normal donor and lung cancer patients. This chapter also provides alternative choices of urine sample preparation and detection methods for selective use in urinary proteome analysis and for identifying urinary protein markers in lung cancer and other diseases.

Background: Prostate cancer is the second leading cause of cancer death in men, and early detection is essential to reduce mortality and increase survival. delta-Catenin is a unique beta-catenin superfamily protein primarily expressed in the brain but is upregulated in human prostatic adenocarcinomas. Despite its close correlation with the disease, it is unclear whether delta-catenin presents the potential in prostate cancer screening because it is an intracellular protein. In this study, we investigated the hypothesis of delta-catenin accumulation in the urine of prostate cancer patients and its potential pathways of excretion into extracellular milieu., Methods: Prostate cancer cell cultures, human tissue biopsies, and voided urines were characterized to determine extracellular delta-catenin accumulation and co-isolation with exosomes/prostasomes., Results: We identified delta-catenin in culture media and in the stroma of human prostate cancer tissues. In PC-3 cells in culture, delta-catenin was partially co-localized and co-isolated with raft-associated membrane protein caveolin-1 and glycosylphosphatidylinositol-anchored protein CD59, suggesting its potential excretion into extracellular milieu through exosome/prostasome associated pathways. Interference with endocytic pathway using wortmannin did not block prostasome excretion, but delta-catenin overexpression promoted the extracellular accumulation of caveolin-1. delta-Catenin, caveolin-1, and CD59 were all detected in cell-free human voided urine prostasomes. delta-Catenin immunoreactivity was significantly increased in the urine of prostate cancer patients (P < 0.0005)., Conclusions: This study demonstrated, for the first time, the extracellular accumulation of delta-catenin in urine supporting its potential utility for non-invasive prostate cancer detection., (2008 Wiley-Liss, Inc.)

To study the inhibitory effects of calcium phosphate-associated proteins on calcium oxalate crystallization and urinary concentrations of proteins in people who form stones and healthy controls. From 60 L of urine from healthy men, calcium phosphate-associated proteins (alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin) were obtained. The effects of the proteins on calcium oxalate (CaOx) crystallization were studied with a mixed suspension mixed product removal system. To examine urinary concentrations of the proteins, urine samples were collected from 17 healthy subjects and 15 stone formers and analyzed using anion-exchange chromatography and an enzyme immunoassay. Prothrombin fragment 1 (PTF1) and osteopontin (OPN) had strong inhibitory effects on CaOx crystallization, while alpha-2-HS-glycoprotein had a mild inhibitory effect. Urinary concentrations of PTF1 and OPN were lower in stone formers than in healthy controls. Low urinary concentrations of PTF1 and OPN might be one of the reasons for stone formation.

Osteopontin (OPN) is a phosphoprotein secreted by many cells of epithelial, mesenchymal, and hematopoietic origin. In the kidney, OPN is expressed in the renal tubules and collecting ducts and is excreted into the urine. A pathophysiologic role for urinary OPN has not been established. In this study, urinary excretion of OPN was analyzed in patients with primary glomerular diseases, including immunoglobulin A nephropathy (IgAN; n = 32), minimal change nephrotic syndrome (MCNS; n = 16), and membranous nephropathy (MN; n = 18). Compared with normal controls (n = 20), mean +/- SD of urinary OPN in IgAN patients was decreased significantly (21.4 +/- 6.2 versus 11.6 +/- 9.6 mg/g creatinine, P: < 0.001). In contrast, the levels of urinary OPN in patients with MCNS or MN did not differ significantly from normal values. Immunoblot analysis showed that OPN is present as a 55- to 60-kd molecule in normal urine. A 34-kd fragment of OPN was the major immunoreactive band in samples from IgAN patients. This fragment also was detectable in the urine from some patients with MCNS or MN but was absent in normal subjects. OPN has a thrombin-cleavage site near its central portion. Thrombin treatment of the urine from normal controls could result in 34-kd OPN fragments. Although the underlying mechanisms remain to be determined, these data provide evidence that secretion or processing (or both) of urinary OPN is altered in patients with IgAN.

Polyclonal antibodies against a 65 kDa tumor-associated phosphoprotein (p65) were used to develop an ELISA to analyze the presence of p65 in urine and serum of rats bearing N-methyl-N-nitrosourea-induced mammary gland adenocarcinomas. Highly purified rat p65 was added to normal urine and serum to establish a quantitative standard curve with the average correlation coefficient being 0.98 and 0.99 respectively. All samples of urine and serum obtained from different carcinoma-bearing rats showed p65 concentrations above the normal levels found in the control urine and sera. The correlation coefficient between tumor burden and p65 concentration in urine and serum was 0.65 and 0.77 respectively. The average levels of p65 in normal urine and normal serum were 37.0 +/- 32.0 and 48.0 +/- 38.0 ng/ml respectively. In the case of urine obtained from rats bearing mammary adenocarcinomas, the mean p65 level was 119.0 +/- 35.9 ng/ml and their serum level was 225.4 +/- 67.5 ng/ml. Sensitivity, specificity and predictive value for serum and urine marker elevation were 78.5, 70.0 and 78.5% respectively. Following in vitro phosphorylation of concentrated urinary proteins, isoelectrofocusing, SDS-PAGE and autoradiography, a phosphorylated form of the 65 kDa protein with a pI of 5.8 was identified in the urine of tumor-bearing rats. This phosphoprotein bound to an antiphosphotyrosine monoclonal antibody and an anti-p65 polyclonal as determined by Western blot analysis. Using the anti-p65 antibodies in an immunoprecipitation procedure, the main radio- and immunoactive band of 65 kDa and two lower mol. wt bands of 50 and 41 kDa, apparently representing degradation products of p65, were identified after in vitro and in vivo phosphorylation of urinary proteins obtained from mammary carcinoma-bearing rats.