Enhanced Proteomic Coverage in Tissue Microenvironment by Immune Cell Subtype Library-Assisted DIA-MS

Area of Science:

• Proteomics
• Cancer Immunology
• Mass Spectrometry

Background:

• Tumor-infiltrating immune cells critically influence cancer progression and treatment response.
• Analyzing immune cell proteomes in small clinical samples is challenging due to low cell numbers and tissue quantity.
• Existing methods struggle with sensitivity and depth for microscale immune profiling.

Purpose of the Study:

• To develop a highly sensitive proteomic strategy for microscale tissue analysis.
• To enable comprehensive immune cell profiling within the tumor microenvironment (TME).
• To investigate the immune landscape of mesenteric lymph nodes (MLNs) in colorectal cancer (CRC) models.

Main Methods:

• Established six immune cell subtype-specific spectral libraries from murine mesenteric lymph nodes (MLNs).
• Utilized data-independent acquisition mass spectrometry (DIA-MS) with immune cell-enriched libraries.
• Applied the method to 1 μg of tumor tissue from murine colorectal cancer (CRC) models.

Main Results:

• The immune cell-enriched library approach increased protein quantification to 7419 from 6978 proteins compared to direct DIA.
• Successfully mapped 841 immune function-related proteins and identified low-abundance proteins like CD1D1 and CD244.
• Characterized MLNs in CRC models, revealing downregulated adaptive immune signaling and T cell differentiation pathways, indicating an immunosuppressive status.

Conclusions:

• The immune cell-enriched library-assisted DIA-MS strategy provides deep coverage and high sensitivity for microscale proteomic profiling.
• This method facilitates the detailed characterization of the immune landscape in challenging samples.
• The findings suggest an immunosuppressive microenvironment in MLNs of CRC models, offering insights into cancer development mechanisms.