Elsevier

Talanta

Volume 180, 1 April 2018, Pages 36-46
Talanta

Dithiothreitol-based protein equalization technology to unravel biomarkers for bladder cancer

https://doi.org/10.1016/j.talanta.2017.11.063Get rights and content

Highlights

  • We have found seven proteins differentially expressed belonging to patients with bladder cancer.

  • The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules.

  • Overall DTT-based protein equalization is a promising methodology in bladder cancer research.

Abstract

This study aimed to assess the benefits of dithiothreitol (DTT)-based sample treatment for protein equalization to assess potential biomarkers for bladder cancer. The proteome of plasma samples of patients with bladder carcinoma, patients with lower urinary tract symptoms (LUTS) and healthy volunteers, was equalized with dithiothreitol (DTT) and compared. The equalized proteomes were interrogated using two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry. Six proteins, namely serum albumin, gelsolin, fibrinogen gamma chain, Ig alpha-1 chain C region, Ig alpha-2 chain C region and haptoglobin, were found dysregulated in at least 70% of bladder cancer patients when compared with a pool of healthy individuals. One protein, serum albumin, was found overexpressed in 70% of the patients when the equalized proteome of the healthy pool was compared with the equalized proteome of the LUTS patients. The pathways modified by the proteins differentially expressed were analyzed using Cytoscape. The method here presented is fast, cheap, of easy application and it matches the analytical minimalism rules as outlined by Halls. Orthogonal validation was done using western-blot. Overall, DTT-based protein equalization is a promising methodology in bladder cancer research.

Introduction

Bladder cancer is the most common cancer of the urinary tract with two clinical phenotypes: non-muscle invasive bladder cancer and muscle-invasive bladder cancer [1]. Bladder cancer continues to pose a significant challenge to physicians and patients who attempt to balance the toxicity of over treating highly curable lower-risk disease while avoiding undertreating lethal high-grade lesions [2]. Besides that, requirements on cystoscopy and imaging to monitor bladder cancer has made this cancer the single most expensive solid tumor to treat from diagnosis to death [3] Bladder cancer diagnosis lacks in accuracy. Furthermore, recurrence of this cancer is high, which makes the five-year survival ratio for this disease one of the worst of all carcinomas [4]. Clinical and pathological parameters are used for bladder cancer stratification, such as, (i) number of tumors, (ii) tumor size, (iii) prior recurrence, (iv) T-stage, (v) presence of carcinoma in situ, (vi) tumor grade, (vii) lymph node status, and (viii) variant histology [1]. However, up to date, there are no effective protein biomarkers for monitoring therapy for this disease.

Most abundant proteins (MAPs) in plasma are a problem to deal with when searching for new or known biomarkers, as they mask or interfere with the detection of proteins belonging to the low-abundance protein fraction [5], [6]. There are many approaches to reduce the complexity of the plasma proteome for biomarkers discovery. However, they are expensive, tedious, time-consuming and/or labor intensive.

Protein equalization using dithiothreitol, DTT, for proteomics purposes was first reported by Warder et al. [7], and it was adapted soon by other researchers, including our team, with some modifications as described by Fernández et al. [6] and Araújo et al. [8], [9], [10]. Protein equalization using DTT is a cost-effective approach of easy implementation that meets the analytical minimalism rules as outlined by Halls [11], where each stage of the analysis is evaluated to minimize the time, cost, sample requirement, reagent consumption, energy requirements and production of waste products.

This work aims to explore the potential of the dithiothreitol-based sample treatment for protein equalization of plasma samples of bladder cancer patients to find potential biomarkers for diagnosis and prognosis. To this aim healthy individual and patients with bladder cancer and Lower Urinary Tract Symptoms (LUTS) were recruited for this study. LUTS it is characterized by several symptoms, whose symptoms can be common to multiple pathologies including bladder cancer [12], [13], [14], [15].

Section snippets

Reagents

All reagents used were HPLC grade or electrophoresis grade. Albumin, from bovine serum (BSA), (N, N, N′, N′-tetramethylethylene-diamine (TMED)), glycine, β-mercaptoethanol, glycerol 86–88%, Bradford reagent, coomassie blue G-250, DL-dithiothreitol (DTT), iodoacetamide (IAA), acrylamide/bis- acrylamide 30% solution (37.5:1), mineral oil, TWEEN®20, phosphate buffered saline (PBS), Fibrinogen from human plasma, haptoglobin from pooled human plasma, the primary antibodies (anti-FGG or anti-HPT),

Proteomic analysis

In the present study samples of 20 individuals, comprising three different groups, were collected as described in the Section 2.2. Clinical data of patients and healthy volunteers is described in Table SM1 (Supplementary material). Total protein content was quantified using Bradford protein assay. Then, protein equalization with Dithiothreitol, DTT, was performed for all samples, following the procedure described in Section 2.5. Protein equalization with Dithiothreitol has been previously

Healthy pool versus bladder cancer

When the healthy pool was compared with the seven bladder cancer patients, four spots (six proteins) were found differentially expressed in five patients (70%) (see Section 3.1 for further details).

Of these six proteins, two were found with levels consistently downregulated in six of the seven patients, namely, serum albumin and gelsolin. Serum albumin is a well-known multifunctional protein, with great binding and transport properties towards many different compounds. Furthermore, contributes

Conclusions

We have demonstrated that equalization of complex proteomes from bladder cancer samples is conveniently achieved with the DTT approach, a cheap method which allows plasma equalization in a quickly and straightforward manner. For healthy pool vs. bladder cancer and LUTS analysis, we identified a total of 96 spots differentially expressed that were excised and analyzed rendering a total of 36 unique proteins. From those unique proteins, 29 were detected dysregulated in bladder cancer and LUTS.

Acknowledgements

The PROTEOMASS Scientific Society is acknowledged by the funding provided to the Biological Mass Spectrometry Isabel Moura. Authors acknowledge the funding provided by UCIBIO, Unidade de Ciências Biomoleculares Aplicadas, which is financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01–0145-FEDER-007728) and to the Associate Laboratory for Green Chemistry LAQV which is financed by national funds from FCT/MEC (

Conflict of interest

The authors declare no conflict of interest.

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