Quantitation of spatially-localized proteins in tissue samples using MALDI-MRM imaging

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Imaging

Background:

• Matrix-assisted laser desorption/ionization (MALDI) imaging creates molecular images of tissues by correlating ion abundances with histology.
• MALDI imaging faces challenges with ion suppression, hindering accurate concentration measurements and comparisons.
• Accurate protein quantitation is crucial for screening applications and precise sample comparisons.

Purpose of the Study:

• To develop a novel MALDI imaging method for the localization and accurate quantitation of proteins in tissues.
• To overcome the limitations of ion suppression in traditional MALDI imaging for quantitative analysis.
• To enable precise comparisons between samples and support screening applications.

Main Methods:

• Optimization of in situ tryptic digestion for tissue samples.
• Reproducible deposition of isotopically labeled standard peptides using an aerosol-generating device.
• Data acquisition via MALDI multiple reaction monitoring (MRM) mass spectrometry (MS).
• Quantitation based on the ratio of endogenous unlabeled peptides to applied labeled peptides.
• External calibration using MALDI time-of-flight (TOF) MS for labeled peptide quantification.

Main Results:

• Demonstrated feasibility of precise and accurate protein quantitation in tissues using MALDI MRM-based imaging.
• Achieved accurate quantitation of proteins over 2 orders of magnitude.
• Maintained spatial localization information of proteins throughout the analysis.
• Standard curves generated showed coefficients of determination greater than 0.97.

Conclusions:

• The developed MALDI MRM imaging method enables accurate protein quantitation in tissues.
• This approach overcomes ion suppression issues, allowing for reliable concentration measurements.
• The method preserves spatial information, facilitating biomarker discovery and tissue analysis.