Bladder cancer (BC) is one of the most common tumor with high incidence. Relative to other cancers, BC has a high rate of recurrence, which results in increased mortality. As a result, early diagnosis and life-long monitoring are clinically significant for improving the long-term survival rate of BC patients. At present, the main methods of BC detection are cystoscopy and biopsy; however, these procedures can be invasive and expensive. This can lead to patient refusal and reluctance for monitoring. There are several BC biomarkers that have been approved by the FDA, but their sensitivity, specificity, and diagnostic accuracy are not ideal. More research is needed to identify suitable biomarkers that can be used for early detection, evaluation, and observation. There has been heavy research in the proteomics and genomics of BC and many potential biomarkers have been found. Although the advent of metabonomics came late, with the recent development of advanced analytical technology and bioinformatics, metabonomics has become a widely used diagnostic tool in clinical and biomedical research. It should be emphasized that despite progress in new biomarkers for BC diagnosis, there remains challenges and limitations in metabonomics research that affects its translation into clinical practice. In this chapter, the latest literature on BC biomarkers was reviewed.Cardiac troponin T (cTnT) is a sensitive and specific biomarker for detecting cardiac muscle injury. Its concentration in blood can be significantly elevated outside the normal reference range under several pathophysiological conditions. The classical analytical method in routine clinical analysis to detect cTnT in serum or plasma is a single commercial immunoassay, which is designed to quantify the intact cTnT molecule. The targeted epitopes are located in the central region of the cTnT molecule. However, in blood cTnT exists in different biomolecular complexes and proteoforms bound (to cardiac troponin subunits or to immunoglobulins) or unbound (as intact protein or as proteolytic proteoforms). While proteolysis is a principal posttranslational modification (PTM), other confirmed PTMs of the proteoforms include N-terminal initiator methionine removal, N-acetylation, O-phosphorylation, O-(N-acetyl)-glucosaminylation, N(ɛ)-(carboxymethyl)lysine modification and citrullination. The immunoassay probably detects several of those cTnT biomolecular complexes and proteoforms, as long as they have the centrally targeted epitopes in common. While analytical cTnT immunoreactivity has been studied predominantly in blood, it can also be detected in urine, although it is unclear in which proteoform cTnT immunoreactivity is present in urine. This review presents an overview of the current knowledge on the pathophysiological lifecycle of cTnT. It provides insight into the impact of PTMs, not only on the analytical immunoreactivity, but also on the excretion of cTnT in urine as one of the waste routes in that lifecycle. Accordingly, and after isolating the proteoforms from urine of patients suffering from proteinuria and acute myocardial infarction, the structures of some possible cTnT proteoforms are reconstructed using mass spectrometry and presented.Extracellular vesicles (EV) are small membrane-coated structures secreted by all cells of the body and can be detected in all bodily fluids, including urine. EV contents (e.g. proteins and distinct RNA classes) reflect the physiological state of their cells of origin, offering a new source of biomarkers. Accordingly, urinary Extracellular Vesicles (uEVs) are emerging as a source for early biomarkers of kidney damage and beyond, holding the potential to replace the conventional invasive techniques including kidney biopsy. However, the lack of standardization and sample collection and isolation methods, and the influence of factors such as inter- and intra-individual variability create difficulties in interpreting current results. Here we review recent results and reported uses of especially urinary EVs and also pinpoint approaches to be considered when designing experiments.Kidney diseases are conditions that increase the morbidity and mortality of those afflicted. Diagnosis of these conditions is based on parameters such as the glomerular filtration rate (GFR), measurement of serum and urinary creatinine levels and equations derived from these measurements (Wasung, Chawla, Madero. Clin Chim Acta 438350-357, 2015). However, serum creatinine as a marker for measuring renal dysfunction has its limitations since it is altered in several other physiological situations, such as in patients with muscle loss, after intense physical exercise or in people on a high protein diet (Riley, Powers, Welch. Res Q Exerc Sport 52(3)339-347, 1981; Juraschek, Appel, Anderson, Miller. Am J Kidney Dis 61(4)547-554, 2013). Besides the fact that serum creatinine is a marker that indicates glomerular damage, it is necessary the discovery of new biomarkers that reflect not only glomerular damage but also tubular impairment. Recent advances in Molecular Biology have led to the generation or identification of new biomarkers for kidney diseases such as Acute Kidney Failure (AKI), chronic kidney disease (CKD), nephritis or nephrotic syndrome. There are recent markers that have been used to aid in diagnosis and have been shown to be more sensitive and specific than classical markers, such as neutrophil gelatinase associated lipocalin (NGAL) or kidney injury molecule-1 (KIM-1) (Wasung, Chawla, Madero. Clin Chim Acta 438350-357, 2015; George, Gounden. Adv Clin Chem 8891-119, 2019; Han, Bailly, Abichandani, Thadhani, Bonventre. Kidney Int 62(1)237-244, 2002; Fontanilla, Han. Expert Opin Med Diagn 5(2)161-173, 2011). However, early diagnostic biomarkers are still necessary to assist the intervention and monitor of the progression of these conditions.Proteomics analysis of urine samples allows for studying the impact of system perturbation. check details However, meaningful proteomics-based biomarker discovery projects often require the analysis of large patient cohorts with hundreds of samples to describe the biological variability. Thus, robust high-throughput sample processing methods are a prerequisite for clinical proteomics pipelines that minimize experimental bias due to individual sample processing methods. Herein we describe a high-throughput method for parallel 96-well plate-based processing of urine samples for subsequent LC/MS-based proteomic analyses. Protein digestion and subsequent sample processing steps are efficiently performed in 96-well polyvinylidene fluoride (PVDF) membrane plate allowing for the use of vacuum manifolds for rapid liquid transfer, and multichannel pipettes and/or liquid handing robots. In this chapter we make available a detailed step-by-step protocol for our 'MStern blotting' sample processing strategy applied to patient urine samples followed by mass spectrometry-based proteomics analysis.