Using De Novo Sequencing and Protein Identification Techniques to Study the Novel Anti-NSCLC Protein PFAP

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for about 85% of lung cancer cases. At the same time, more than 30% of NSCLC patients are diagnosed at a late stage. However, despite the existence of radiotherapy technology and new auxiliary immunotherapy, the survival rate of late-stage NSCLC patients has not achieved ideal results, and the five-year survival rate after radiotherapy remains between 10-15%.

 

Unlike traditional drugs, mushrooms have received attention due to their immunomodulatory, anti-inflammatory, and anti-tumor properties. Mushrooms can not only directly inhibit cancer cells, but also serve as an option for adjuvant therapy to help improve symptoms such as nausea, vomiting, diarrhea, fatigue, and insomnia caused by cancer, and enhance the function of the human immune system. Therefore, developing new drugs for NSCLC based on the anti-cancer active compounds in mushrooms has great prospects and potential.

 

In July 2023, a research team from Nankai University and The Chinese University of Hong Kong published a research achievement titled "New fungal protein from Pleurotus ferulae lanzi induces AMPK-mediated autophagy and G1-phase cell cycle arrest in A549 lung cancer cells" in the International Journal of Biological Macromolecules journal (IF=8.025). This study isolated a novel anti-NSCLC protein PFAP from Pleurotus ferulae lanzi and conducted identification and anti-tumor mechanism studies on it using proteomics technology. The analysis results of de novo sequencing showed that PFAP is a protein composed of 135 amino acid residues, with a theoretical molecular weight of 14.81 kDa. Tandem mass tag (TMT)™ quantitative proteomics analysis revealed that PFAP can induce autophagy by regulating the AMPK/mTOR pathway and inhibit the G1 phase cell cycle of A549 lung cancer cells. In addition, the study also found that PFAP can inhibit the growth of xenograft tumors in nude mice. As PFAP exhibits high resistance and low toxicity to NSCLC, it is expected to be a potential new drug for NSCLC. This study provides a solid theoretical basis for developing new strategies for treating NSCLC. The various proteomic technologies used in this study, including de novo sequencing, liquid chromatography-tandem mass spectrometry protein identification, and tandem mass tag (TMT)™ quantitative proteomics analysis, were all provided by MtoZ Biolabs.

 

Main Research Content and Results

1. Purification and Identification of the Novel Anti-Tumor Protein PFAP

Three fractions, OF1, OF2 (unadsorbed), and OF3 (adsorbed), were obtained by separating the crude protein extract of the mushroom fruiting body using hydrophobic interaction chromatography. A part of OF3 was collected and concentrated from the SDS-PAGE band, while no bands appeared for OF1 and OF2. Subsequently, the fraction OF3 was purified using a gel filtration chromatography column, resulting in a single peak with a molecular weight of 14.68 kDa, and this purified protein was named PFAP. De novo sequencing using LC-MS/MS revealed that PFAP is a protein composed of 135 amino acid residues.

 

2. PFAP Induces Cell Death in A549 Cells In Vitro

To evaluate the effect of PFAP on the viability of A549 cells, we co-cultured the cells with different concentrations (0, 50, 100, 150, and 200μg/mL) of PFAP at different time points (12, 24, and 48 hours). The results showed that with the increase of PFAP concentration and incubation time, A549 cells gradually vacuolized, and the cell growth inhibition rate was the highest after exposure to PFAP for 48 h, indicating that PFAP is a highly efficient and low toxicity anti-tumor protein.

 

3. TMT™-Based Proteomics Analysis Reveals the Impact of PFAP on A549 Cells

To further understand the mechanism of action of PFAP, we used TMT™-based quantitative proteomics analysis to analyze the changes in proteins in A549 cells. We used |log2FC|≥0.263, p<0.05 as the standard for differentially expressed proteins. A total of 226 differentially expressed proteins were identified, of which 115 were up-regulated and 111 were down-regulated.

 

4. PFAP Inhibits the Growth of Lung Adenocarcinoma Cells In Vivo

To explore the anti-tumor mechanism of PFAP in vivo, we intraperitoneally injected PFAP into nude mice with a subcutaneous implant of A549 cells. After seven injections, the tumor weight and volume were significantly reduced on the 15th day compared to the negative control group.

 

This study identified a novel anti-tumor protein PFAP through de novo sequencing technology and revealed the effect of PFAP on A549 cells through TMT™-based quantitative proteomics analysis. PFAP inhibits the growth of lung cancer A549 cells in vitro and also shows potential in inhibiting xenograft tumors in vivo. Since PFAP exhibits high resistance and low toxicity to non-small cell lung cancer, it has good prospects in cancer treatment.