Protocol optimization for proteomic analysis of tears in patients with oral potentially malignant lesions and mouth cancer

Author(s)

Erison Santana dos Santos

• Document type: Master's Dissertation
• Press: Piracicaba, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Odontologia de Piracicaba
• Defense date: 2020-07-27

Examining board members

Adriana Franco Paes Leme; Ariane Fidelis Busso Lopes; Pablo Agustin Vargas

• Advisor: Adriana Franco Paes Leme

Abstract

Oral squamous cell carcinoma (OSCC) is the main histological type of mouth cancer. This neoplasm may arise from lesions known as potentially malignant oral disorders, such as oral leukoplakia (OL) and proliferative verrucous leukoplakia (PVL) that show a high frequency and potential for malignancy, respectively. However, the process of malignant transformation of these lesions is not completely understood. Therefore, the search for molecular signatures that may assist the early diagnosis and improve the prognosis of patients with oral cancer. Liquid biopsy, such as lacrimal fluids, is a promising non-invasive source of molecular signatures. This study aimed to optimize a protocol of collection, extraction, and digestion of proteins of tears to further search for molecular signatures of malignant transformation. This study was performed in two phases. Firstly, we tested 12 protocols in which the proteins of lacrimal fluid from healthy patients were solubilized in chaotropic agent, detergent, surfactant, denaturing agent.We performed in-solution and in-gel digestion. The liquid chromatography–mass spectrometry (LC-MS/MS) was performed using at least two gradients of liquid chromatography using different mass spectrometers. The data were analyzed using the softwares MaxQuant or Mascot, and InteractiVenn, The Human Protein Atlas, Uniprot, Enrichr, R-Studio, SignaIP, TMHMM and String. Among the tested protocols, the protocol named P11 outperformed the others. In this protocol, the proteins were solubilized in urea and submitted to in-solution trypsin digestion, with double digestion, and the resulting peptides were separated in a 60-min gradient of liquid chromatography in the LTQ Orbitrap Velos. This protocol identified 209 proteins, 72 presented signal peptides, and 23 presented predictors of a transmembrane helix. The biological process most associated with these proteins was retina homeostasis. The keratins presented the highest evidence of protein-protein interaction. In the second phase, we collected and prepared samples of lacrimal fluids from patients with OL, PVL, and OSCC and healthy patients for further analysis by LC-MS/MS. In summary, this study shows for the first time the optimization of a protocol for the analysis of lacrimal fluid proteins aiming the search for molecular signatures of malignant transformation to anticipate the progression to oral cancer (AU)

• FAPESP's process: 18/19922-9 - Proteomic analysis of tears in patients with oral potentially malignant lesions and oral squamous cell carcinoma
• Grantee: Erison Santana dos Santos
• Support Opportunities: Scholarships in Brazil - Master