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DNA/RNA-based classification of bladder cancer (BC) supports the existence of multiple molecular subtypes, while investigations at the protein level are scarce. Here, we aimed to investigate if Nonmuscle Invasive Bladder Cancer (NMIBC) can be stratified to biologically meaningful groups based on the proteome. Tissue specimens from 117 patients at primary diagnosis (98 with NMIBC and 19 with MIBC), were processed for high-resolution proteomics analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The proteomics output was subjected to unsupervised consensus clustering, principal component analysis (PCA) and investigation of subtype-specific features, pathways, and gene sets. NMIBC patients were optimally stratified to three NMIBC proteomic subtypes (NPS), differing in size, clinicopathologic and molecular backgrounds: NPS1 (mostly high stage/grade/risk samples) was the smallest in size (17/98) and overexpressed proteins reflective of an immune/inflammatory phenotype, involved in cell proliferation, unfolded protein response and DNA damage response, whereas NPS2 (mixed stage/grade/risk composition) presented with an infiltrated/mesenchymal profile. NPS3 was rich in luminal/differentiation markers, in line with its pathological composition (mostly low stage/grade/risk samples). PCA revealed a close proximity of NPS1 and conversely, remoteness of NPS3 to the proteome of MIBC. Proteins distinguishing these two extreme subtypes were also found to consistently differ at the mRNA levels between high and low-risk subtypes of the UROMOL and LUND cohorts. Collectively, our study identifies three proteomic NMIBC subtypes and following a cross-omics validation in two independent cohorts, shortlists molecular features meriting further investigation for their biomarker or potentially therapeutic value.

A major requirement in the application of proteins as clinical biomarkers is that they provide a highly sensitive and specific result in disease assessment. Since single biomarkers are generally of limited accuracy, a group or panel of well characterized biomarkers appears appropriate, providing a more robust and sensitive mass-spectrometry-based analytical platform. Capillary electrophoresis coupled to MS has been successfully used in biomarker discovery and application, as it enables the selective detection of peptides and small proteins, combining the high separation capacity of CE with the advanced sensitivity of MS. CE-MS allows the characterization of highly complex samples (such as urine, plasma and other biofluids) in a consistent and reproducible way. It has a range of applications, many focusing especially in studies on urinary peptide biomarkers in kidney and cardiovascular diseases. Another major field of interest has been malignancy of the genitourinary system. In the first part of this review we cover technical aspects and performance characteristics of CE-MS, with special focus on the requirements for biomarker discovery and clinical application. In the second part, we review the potential and development of CE-MS in the management of genitourinary cancers, especially bladder cancer (BCa). CE-MS has been employed in several studies aimed at discovering biomarkers for BCa that may be useful in diagnosis, monitoring for recurrence, and prediction of the risk for the muscle-invasive stage. In the last part of the review we discuss current challenges and provide an outlook for ongoing and possible future developments.

Purpose Urine is a rich source of potential biomarkers, including glycoproteins. Glycoproteomic analysis remains difficult due to the high heterogeneity of glycans. Nevertheless, recent advances in glycoproteomics software solutions facilitate glycopeptide identification and characterization. The aim is to investigate intact glycopeptides in the urinary peptide profiles of normal subjects using a novel PTM-centric software-Byonic. Experimental design The urinary peptide profiles of 238 normal subjects, previously analyzed using CE-MS and CE-MS/MS and/or LC-MS/MS, are subjected to glycopeptide analysis. Additionally, glycopeptide distribution is assessed in a set of 969 patients with five different cancer types: bladder, prostate and pancreatic cancer, cholangiocarcinoma, and renal cell carcinoma. Results A total of 37 intact O-glycopeptides and 23 intact N-glycopeptides are identified in the urinary profiles of 238 normal subjects. Among the most commonly identified O-glycoproteins are Apolipoprotein C-III and insulin-like growth factor II, while titin among the N-glycoproteins. Further statistical analysis reveals that three O-glycopeptides and five N-glycopeptides differed significantly in their abundance among the different cancer types, comparing to normal subjects. Conclusions and clinical relevance Through the established glycoproteomics workflow, intact O- and N-glycopeptides in human urine are identified and characterized, providing novel insights for further exploration of the glycoproteome with respect to specific diseases.

Clinical proteomics, the application of proteome analysis to serve a clinical purpose, represents a major field in the area of proteome research. Over 1000 manuscripts on this topic are published each year, with numbers continuously increasing. However, the anticipated outcome, the transformation of the reported findings into improvements in patient management, is not immediately evident. In this article, the value and validity of selected clinical proteomics findings are investigated, and it is assessed how far implementation has progressed. A main conclusion from this assessment is that to achieve implementation, well‐powered clinical studies are required in the appropriate population, addressing a specific clinical need and with a clear context‐of‐use. Efforts toward implementation, to be feasible, must be supported by the key players in science: publishers and funders. The authors propose a change on objectives, from additional discovery studies toward studies aiming at validation of the plethora of potential biomarkers that have been described, to demonstrate practical value of clinical proteomics. All elements required, potential biomarkers, technologies, and bio‐banked samples are available (based on today's literature), hence a change in focus from discovery toward validation and application is not only urgently necessary, but also possible based on resources available today.

Biomarker research is continuously expanding in the field of clinical proteomics. A combination of different proteomic–based methodologies can be applied depending on the specific clinical context of use. Moreover, current advancements in proteomic analytical platforms are leading to an expansion of biomarker candidates that can be identified. Specifically, mass spectrometric techniques could provide highly valuable tools for biomarker research. Ideally, these advances could provide with biomarkers that are clinically applicable for disease diagnosis and/ or prognosis. Unfortunately, in general the biomarker candidates fail to be implemented in clinical decision making. To improve on this current situation, a well‐defined study design has to be established driven by a clear clinical need, while several checkpoints between the different phases of discovery, verification and validation have to be passed in order to increase the probability of establishing valid biomarkers. In this review, we summarize the technical proteomic platforms that are available along the different stages in the biomarker discovery pipeline, exemplified by clinical applications in the field of bladder cancer biomarker research.