MALDI-nanochip based Screening of Exosomal Biomarkers: Application to Cancer Diagnostics

Importance of the Topic

The analysis of exosomal proteins offers a non-invasive window into disease processes, particularly for cancer diagnostics. Exosomes circulating in blood carry molecular signatures reflective of their tissue of origin. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) profiling of these vesicles enables rapid screening and differentiation of patient groups, supporting liquid biopsy approaches and improving clinical decision making.

Objectives and Study Overview

This work extends earlier MALDI-MS profiling of extracellular vesicles to identify specific protein biomarkers in plasma exosomes from four cohorts: colorectal cancer patients (pre- and post-surgery), inflammatory bowel disease (IBD) patients and healthy controls. The primary goals were to compare proteomic fingerprints, discover differentially expressed proteins across groups and validate the use of a MALDI-nanochip workflow for high-throughput screening.

Methodology

Blood specimens were processed by sequential centrifugation to isolate exosomes. Protein extracts were split into two workflows: direct on-chip tryptic digestion on MALDI-nanochips and in-solution digestion followed by C18 ZipTip cleanup. Samples were mixed with α-cyano-4-hydroxycinnamic acid matrix and analyzed by reflectron MALDI-TOF on both benchtop and high-resolution MS/MS platforms. Multivariate statistical tools (partial least squares discriminant analysis) and bioinformatics searches (Mascot against SwissProt/Uniprot) were applied to detect and identify discriminant peptide peaks.

Used Instrumentation

• Shimadzu AXIMA Performance MALDI-TOF
• Shimadzu MALDI-7090 TOF/TOF for MS/MS analyses
• Tethis MALDI-nanochip targets
• FlexiMass-DS target plates
• Data analysis software: Clover MS Data Analysis and eMSTAT
• Mascot protein database search against SwissProt/Uniprot

Results and Discussion

MALDI-nanochip processing revealed enhanced signal intensity for several m/z peaks (>8000) compared to conventional ZipTip cleanup. PLS-DA clustering clearly separated the four study groups, demonstrating reproducible proteomic patterns. MS/MS identification pinpointed Immunoglobulin heavy constant alpha 1 (IGHA1) and other candidate biomarkers that varied significantly between colorectal cancer, IBD and healthy donors. On-chip cleanup effectively removed high-abundance plasma proteins and unveiled lower-abundance discriminant peaks that were otherwise masked.

Benefits and Practical Applications of the Method

• Rapid, label-free screening of exosomal protein profiles
• High throughput potential for clinical cohorts
• Non-invasive liquid biopsy supporting early cancer detection
• Capability to uncover low-abundance biomarkers through selective cleanup

Future Trends and Potential Applications

Further refinement of marker panels and workflow automation may enhance diagnostic accuracy. Integration with complementary omics and expansion to other cancer types or inflammatory diseases could broaden clinical utility. Advances in MALDI-nanochip design and data-driven bioinformatics promise more sensitive, multiplexed assays for routine liquid biopsy applications.

Conclusion

The MALDI-nanochip-based proteomic workflow demonstrated clear discrimination of exosomal protein signatures among colorectal cancer, IBD and healthy subjects. It offers a promising platform for rapid, high-throughput biomarker discovery and potential translation into clinical liquid biopsy diagnostics.

References

1. Serafim V, et al. Classification of cancer cell lines using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and statistical analysis. Int J Mol Med. 2017;40:1096–1104.
2. Stübiger G, et al. MALDI-MS Protein Profiling of Chemoresistance in Extracellular Vesicles of Cancer Cells. Anal Chem. 2018;90:13178–13182.