Urine screening by surface-enhanced laser desorption/ionization-time of flight (SELDI-TOF) mass spectrometry followed by biomarker identification, "Beyond the Abstract," by Gilda Alves, PhD and Antonio Augusto Ornellas, MD, PhD

BERKELEY, CA (UroToday.com) - Kidney cancer is the most lethal urologic malignancy since over 40% of patients will die from the disease, despite the emergence of new drugs for treatment.[1] Patients could benefit from early detection since metastases are rare in tumors of less than 4 cm in diameter. More than 30% of patients show locally advanced or metastatic disease at diagnosis. Furthermore, approximately 40% of patients undergoing curative surgery for localized disease relapse.

Urine is especially attractive for biomarker discovery in urological diseases. In search of possible biomarkers, we have screened proteins/peptides in individual samples of urine from 61 Brazilian patients with renal cell carcinoma (RCC). Patients had the two most common histological subtypes, clear cell and papillary RCC.

The technology of SELDI-TOF (surface-enhanced laser desorption ionization time of flight) is a kind of mass spectrometer (MS) that measures the mass/charge (m/z) of charged particles. The SELDI-TOF instrument enables the separation of proteins and peptides by their physical properties (hydrophobic, hydrophilic, acidic, basic, with affinity for metal etc.) on a solid surface called a chip. The time-of-flight (TOF) analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they take to reach the detector. Ions’ velocities will depend on their masses. Lighter ions will reach the detector first. The SELDI-TOF has advantages such as detection of proteins with the sensitivity of femtomoles and the ability to detect proteins in fluids like urine without previous treatment.

Sixty-one samples of urine from clear cell RCC and papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a Protein Chip Reader PCS 4000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (clear cell and papillary). For identification, urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound.

The proteins that were identified as over-secreted in the urine of the controls were: immunoglobulin light chain, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin is quite common by proteomics methods and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while the role of vitellite membrane outer layer 1 is unknown since it was never purified in humans.

The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine makers and should be validated in a bigger cohort, including the other less common histological RCC subtypes. Our new unpublished results reinforce that down secreted MASP2 could be a good biomarker in the urine of RCC patients.

Reference:

  1. Ather MH, Masood N, Siddiqui T. Current management of advanced and metastatic renal cell carcinoma. Urol J. 2010 Winter;7(1):1-9.

Written by:
Gilda Alves, PhDa and Antonio Augusto Ornellas, MD, PhDb, c as part of Beyond the Abstract on UroToday.com. This initiative offers a method of publishing for the professional urology community. Authors are given an opportunity to expand on the circumstances, limitations etc... of their research by referencing the published abstract.

aLaboratório de Genética Aplicada, Serviço de Hematologia, Instituto Nacional de Câncer, Pça. da Cruz Vermelha, 23, Rio de Janeiro, RJ, Brazil. 
bServiço de Urologia, Instituto Nacional de Câncer, Pça. da Cruz Vermelha, 23, , Rio de Janeiro, RJ, Brazil
cServiço de Urologia, Hospital Mário Kroeff, Rua Magé, 326, Penha Circular, Rio de Janeiro, RJ, Brazil.

Urine screening by SELDI-TOF, followed by biomarker identification, in a Brazilian cohort of patients with renal cell carcinoma (RCC) - Abstract

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