Label-Free Semiquantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics Analysis of Laryngeal/Hypopharyngeal Squamous Cell Carcinoma on Formalin-Fixed, Paraffin-Embedded Tissue Samples - a Pilot Study

Abstract

Squamous cell carcinoma (SCC) of the head and neck region is the sixth most frequent malignancy with high mortality rate. Due to its poor prognosis it is considered a growing public health problem worldwide inspite of existing treatment modalities. Thus, early diagnosis of new diseases and recurrences is emerging on one hand, but on the other hand troublesome in the lack of reliable tumor markers in this field. The rapid development of proteomics has opened new perspectives in tumor marker discovery. Liquid chromatography/mass spectrometry (LC/MS) as the gold standard in proteomics enables the semi-quantitative analysis of proteins within various tissues. Abundance differences between tumor and normal tissue also can be interpreted as tumor specific changes. The aim of this study was to identify potential tumor markers of laryngeal/hypopharyngeal SCC by revealing abundance changes between cancerous and the surrounding phenotypically healthy tissue. After separating the phenotypically cancerous and healthy parts of formalin-fixed paraffin-embedded tissues, each sample underwent protein recovery process and tryptic digestion for label-free semi-quantitative LC/MS analysis. Eight proteins showed significantly higher abundance in tumor including tenascin, transmembrane emp24 domain-containing protein 2, cytoplasmic dynein light chain 1, coactosin-like protein, small proline-rich protein 2D, nucleolin, U5 small nuclear RNP 200-kDa helicase and fatty aldehyde dehydrogenase. Desmoglein-1 and keratin type I cytoskeletal 9 were down-regulated in tumor. Using Ingenuity Pathway Analysis we mapped the signaling pathways these proteins play role in regarding other tumors. Based on these findings these proteins may serve as promising biomarkers in the fight against laryngeal/hypopharyngeal SCCs.

Proteomics analysis of cancer tissues identifies IGF2R as a potential therapeutic target in laryngeal carcinoma

“…Among existing proteomic analysis-related research articles, reports exclusively addressing LC are scarce. Burian A et al (14) determined that 8 proteins were significantly more abundant and 9 were downregulated in formalin-fixed paraffin-embedded tissue samples from tumors. Our study differs in its use of fresh surgical specimens, which theoretically diminishes protein degradation to the fullest extent and ensures the accuracy of the results.…”

Advancements in Oncoproteomics Technologies: Treading toward Translation into Clinical Practice

“…In a typical pipeline of proteome analysis, the extracted or purified protein products can be either fractionated directly (a top-down approach) or after protease (usually tryptic) digestion (a bottom-up approach), and analyzed using mass spectrometry (MS) to identify proteins, and the data can be interpreted using a proteome database [ 22 , 23 , 24 , 25 , 26 ]. In clinical research, label-based and label-free MS approaches are utilized for quantitative analysis [ 27 , 28 , 29 , 30 ]. Multiplex and innovative technologies like protein-, antibody-, tissue microarray, proximity extension assay, nanoproteomics and single-cell proteomics have significantly improved protein purification and automation in the identification of protein traces in minuscule samples [ 31 , 32 , 33 ].…”