Urinary biomarkers for the diagnosis of cervical cancer by quantitative label‑free mass spectrometry analysis

Abstract

Due to the invasive procedure associated with Pap smears for diagnosing cervical cancer and the conservative culture of developing countries, identifying less invasive biomarkers is of great interest. Quantitative label-free mass spectrometry was performed to identify potential biomarkers in the urine samples of patients with cervical cancer. This technique was used to study the differential expression of urinary proteomes between normal individuals and cancer patients. The alterations in the levels of urinary proteomes in normal and cancer patients were analyzed by Progenesis label-free software and the results revealed that 60 proteins were upregulated while 73 proteins were downregulated in patients with cervical cancer. This method could enrich high molecular weight proteins from 100 kDa. The protein-protein interactions were obtained by Search Tool for the Retrieval of Interacting Genes/Proteins analysis and predicted the biological pathways involving various functions including cell-cell adhesion, blood coagulation, metabolic processes, stress response and the regulation of morphogenesis. Two notable upregulated urinary proteins were leucine-rich α-2-glycoprotein (LRG1) and isoform-1 of multimerin-1 (MMRN1), while the 3 notable downregulated proteins were S100 calcium-binding protein A8 (S100A8), serpin B3 (SERPINB3) and cluster of differentiation-44 antigen (CD44). The validation of these 5 proteins was performed by western blot analysis and the biomarker sensitivity of these proteins was analyzed individually and in combination with receiver operator characteristic curve (ROC) analysis. Quantitative mass spectrometry analysis may allow for the identification of urinary proteins of high molecular weight. The proteins MMRN1 and LRG1 were presented, for the first time, to be highly expressed urinary proteins in cervical cancer. ROC analysis revealed that LRG1 and SERPINB3 could be individually used, and these 5 proteins could also be combined, to detect the occurrence of cervical cancer.

Identification of potential cervical cancer serum biomarkers in Thai patients

“…MMRN1 has 2 isoforms, each measuring 138 and 58 kDa, due to alternative splicing. Isoform-1 of MMRN-1 has been identified as a potential serum biomarker for multiple myeloma and lung cancer (49) and urinary biomarker for cervical cancer (57). In the present study, serum levels of isoform-2 of MMRN-1, as confirmed by mass spectrometry, were decreased in the serum of HPV positive normal controls and in patients with early and late stage cervical cancer.…”

Identification of Candidate Protein Biomarkers for CIN2+ Lesions from Self-Sampled, Dried Cervico–Vaginal Fluid Using LC-MS/MS

“…Ras-related protein Rab-6A was also listed as an unfavourable biomarker for endometrial, liver and pancreatic cancers, Ubiquitin-40S ribosomal protein S27a as unfavourable biomarker for renal and liver cancer, and Tripartite motif-containing protein 29 as an unfavourable biomarker for endometrial, stomach and pancreatic cancer. Previous studies have reported Ubiquitin-40S ribosomal protein S27a as being present in higher amounts in the urine of cervical cancer patients compared to controls when measured with mass spectrometry [ 28 ] and an overexpression of Tripartite motif-containing protein 29 (both mRNA and protein) in cervical cancer tissue compared to matched adjacent cervical tissues [ 29 ].…”

Aberrant proteins expressed in skin fibroblasts of Parkinson's disease patients carrying heterozygous variants of glucocerebrosidase and parkin genes

“…In-solution tryptic-digestion was performed as described previously with certain modifications ( 23 ). Briefly, 10 µg protein from fibroblast lysates (pooled healthy controls and individual patient samples) was reduced in 10 mM DTT for 5 min at 95˚C and alkylated with 1/10 volume of 20 mM iodoacetamide, prior to incubation for 30 min in the dark at room temperature.…”

“…A nanoACQUITY UPLC system (Waters Corporation) coupled with an amaZon speed Ion Trap mass spectrometer (Bruker Corporation) was used to identify peptides/proteins, as described previously ( 23 ). The tryptic-digested peptides were dissolved in 0.1% formic acid in H 2 O and injected into a nanoACQUITY UPLC column (1.7 µm BEH, 75 µm x 200 mm C 18 , Water Corporation) at a flow rate of 300 nl/min.…”

“…Progenesis label-free LC-MS version 3.1 (Nonlinear Dynamics, Ltd.) was used to process the raw data obtained from LC-MS/MS and to compare the significant changes in protein expression levels. The method was applied to quantify peptides/proteins, as described previously, with certain modifications ( 23 ). Briefly, two sets of LC-MS/MS were performed.…”