Identification of proteins and lipids found in varying abundances in pre-and post-operative cancer patients using a MALDI-TOF-MSMS approach

Significance of the Topic

The identification of protein and lipid markers in plasma and extracellular vesicles (EVs) offers a minimally invasive route to monitor disease progression and treatment response in cancer patients. High-throughput MALDI-TOF-MS/MS profiling can accelerate biomarker discovery for clinical diagnostics, particularly in liquid biopsy applications.

Objectives and Study Overview

This study aimed to compare the abundance of proteins and lipids in plasma and EV fractions from four patient groups: pre-operative colorectal cancer (CRC-Pre), post-operative colorectal cancer (CRC-Post), inflammatory bowel disease (IBD) and healthy controls. Using MALDI-TOF-MS/MS, multivariate statistics and database searches, the project sought to uncover differentiating molecular features linked to disease state and surgical intervention.

Methodology and Instrumentation

Workflow:

• Blood collection and plasma isolation by serial centrifugation.
• EV enrichment via ultracentrifugation; non-enriched plasma processed in parallel.
• Tryptic digestion of protein extracts and on-chip sample preparation using a MALDI-nanochip slide.
• Mass spectrometry analysis on a Shimadzu MALDI-7090 reflectron TOF-TOF instrument with high-energy CID for MS/MS.
• Data analysis using eMSTAT software for PLS-DA and peak selection; Mascot searches against SwissProt for protein identification.

Main Results and Discussion

Key findings:

• Albumin was a conserved peak across all non-enriched samples but did not differentiate groups.
• Alpha-1-antitrypsin peptide (m/z 1641.8674) identified in EV-enriched samples by PMF and MS/MS.
• Apolipoprotein A1 detected in all groups; confirmed by MS/MS (m/z 1301.64).
• Three lysophosphatidylcholines (LPC(16:0), LPC(18:1), LPC(18:0)) were annotated in non-ultracentrifuged samples.
• Transthyretin and fibrinogen alpha chain identified in EV fractions.
• EV enrichment enhanced relative intensities of low-abundance marker peaks, despite overall lower signal levels.

Benefits and Practical Applications

This MALDI-TOF-MS/MS workflow supports rapid, label-free screening of clinical plasma samples. The use of MALDI-nanochip slides simplifies sample preparation and improves detection of discriminant features. The approach holds promise for developing liquid biopsy assays to stratify cancer patients and monitor treatment efficacy.

Future Trends and Applications

Potential developments:

• Validation in larger, multi-center patient cohorts.
• Integration with complementary omics (metabolomics, transcriptomics).
• Automation and miniaturization for point-of-care diagnostics.
• Longitudinal monitoring to track disease recurrence and drug resistance.

Conclusion

The reflectron MALDI-TOF-MS/MS platform, combined with targeted statistical analysis, successfully distinguished patient groups and identified candidate protein and lipid biomarkers in plasma and EVs. This high-throughput screening pipeline lays the groundwork for future liquid biopsy diagnostics in oncology.

References

1. Serafim V. et al. Classification of cancer cell lines using MALDI-TOF mass spectrometry and statistical analysis. Int J Mol Med, 40, 1096-1104 (2017).
2. Nairn M. et al. MALDI-nanochip based Screening of Exosomal Biomarkers: Application to Cancer Diagnostics. ASMS 2020, WP-031.
3. Stübiger G. et al. MALDI-MS Protein Profiling of Chemoresistance in Extracellular Vesicles of Cancer Cells. Anal Chem, 90, 13178-13182 (2018).