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Updated: May 20, 2026
Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells
Putty-Reddy Sudhir1, Chein-Hung Chen, Madireddy Pavana Kumari
1Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Abstract:
Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.
Insights
Activated KRAS (Kirsten rat sarcoma viral oncogene homolog) drives lung cancer. This study identifies novel KRAS target proteins and N-glycoproteins in human bronchial epithelial cells, offering new insights for lung adenocarcinoma diagnosis and therapy.
Area of Science:
- Proteomics and Glycoproteomics
- Cancer Biology
- Molecular Oncology
Background:
- Mutational activation of KRAS is a key driver in various cancers, notably lung adenocarcinoma.
- Understanding the downstream molecular targets of activated KRAS is crucial for developing effective therapies.
- Limited knowledge exists regarding the specific proteins and glycoproteins regulated by activated KRAS.
Purpose of the Study:
- To identify and characterize KRAS target proteins and N-glycoproteins in human bronchial epithelial cells.
- To discover novel molecular targets that mediate the downstream effects of activated KRAS.
- To explore potential diagnostic biomarkers and therapeutic targets for KRAS-driven lung adenocarcinoma.
Main Methods:
- Utilized OFFGEL peptide fractionation and hydrazide chemistry coupled with LTQ-Orbitrap mass spectrometry for proteomic and N-glycoproteomic analysis.
- Employed label-free quantitation to identify differentially regulated proteins and N-glycoproteins in cells with and without activated KRAS (KRAS(V12)).
- Integrated bioinformatics analysis, including IPA-Biomarker® filtering and analysis of lung adenocarcinoma microarray data, along with shRNA knockdown experiments for validation.
Main Results:
- Identified 5713 proteins and 608 N-glycosites, revealing differential regulation of 23 proteins and 14 N-glycoproteins by activated KRAS, with 84% being novel findings.
- Prioritized potential cancer biomarkers, including proteins (e.g., CA2, CTSD) and N-glycoproteins (e.g., ALCAM, TIMP-1).
- Validated down-regulation of FABP5 and PDCD4, and confirmed PDCD4 as a KRAS target. Observed structural N-glycan alterations in SEMA4B and functional role of TIMP-1 N-glycosylation.
Conclusions:
- This study presents the largest proteome and N-glycoproteome datasets for human bronchial epithelial cells, identifying numerous novel KRAS targets.
- The identified targets and biomarkers hold significant potential for advancing the understanding, diagnosis, and therapeutic strategies for KRAS-induced lung adenocarcinoma.
- Findings highlight the importance of N-glycosylation in KRAS-mediated oncogenesis and suggest new avenues for targeted cancer therapies.
