MALDI in-source decay of high mass protein isoforms: application to alpha- and beta-tubulin variants

Area of Science:

• Biochemistry
• Proteomics
• Cancer Research

Background:

• Tubulin is a primary target in cancer chemotherapy, with over 20% of agents targeting it.
• Tubulin isoform variations, particularly in the C-terminus, are linked to treatment resistance.
• Accurate characterization of tubulin C-terminal domains is critical for understanding drug resistance mechanisms.

Purpose of the Study:

• To develop and validate a novel mass spectrometry-based method for characterizing tubulin C-terminal isotypes.
• To investigate the potential of reflectron in-source decay (reISD) for top-down analysis of tubulin C-termini.
• To establish new avenues for biomarker discovery related to cancer drug resistance and progression.

Main Methods:

• Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) fragmentation of synthetic tubulin C-terminal peptides.
• In-source decay (ISD) fragmentation of C-termini from HeLa cell isotypes, coupled with a pseudo MS(3) technique (T(3)-sequencing).
• Guanidination of peptides to facilitate characterization of the beta-III tubulin C-terminus.

Main Results:

• Successful characterization of tubulin isotypes from HeLa cells using reISD coupled with T(3)-sequencing.
• Demonstration of preferential y(n)-series ion generation during in-source fragmentation.
• First reported instance of reISD for the C-terminus of a 50 kDa protein, potentially via a CID-like mechanism in the MALDI plume.

Conclusions:

• The developed reISD approach provides a powerful new tool for the top-down characterization of clinically relevant protein biomarkers like beta-III tubulin.
• This method offers significant potential for identifying markers of drug resistance and tumor progression.
• The study highlights the importance of protein isotype characterization for early cancer detection and treatment monitoring.