Quantitation of Spatially-Localized Proteins in Tissue Samples using MALDI-MRM Imaging.

Anal Chem. 2012 Apr 17;84(8):3514-22. Epub 2012 Mar 26.
Clemis EJ, Smith DS, Camenzind AG, Danell RM, Borchers CH.

MALDI Imaging allows the creation of a "molecular image" of a tissue slice. This image is reconstructed from the ion abundances in spectra obtained while rastering the laser over the tissue. These images can then be correlated with tissue histology to detect potential biomarkers of, for example, aberrant cell types. MALDI, however, is known to have problems with ion suppression, making it difficult to correlate measured ion abundance with concentration. It would be advantageous to have a method which could provide more accurate protein concentration measurements, particularly for screening applications or for precise comparisons between samples. In this paper, we report the development of a novel MALDI Imaging method for the localization and accurate quantitation of proteins in tissues. This method involves optimization of in-situ tryptic digestion, followed by reproducible and uniform deposition of an isotopically-labeled standard peptide from a target protein onto the tissue, using an aerosol-generating device. Data is acquired by MALDI-MRM-MS and accurate peptide quantitation is determined from the ratio of MRM transitions for the endogenous unlabeled proteolytic peptides to the corresponding transitions from the applied isotopically-labeled standard peptides. In a parallel experiment, the quantity of the labeled peptide applied to the tissue was determined using a standard curve generated from MALDI-TOF-MS data. This external calibration curve was then used to extrapolate the quantity of endogenous peptide in a given area. All standard curves generate by this method had coefficients of determination greater than 0.97. These proof-of-concept experiments using MALDI MRM-based Imaging shows the feasibility for the precise and accurate quantitation of tissue protein concentrations over two orders of magnitude, while maintaining the spatial localization information for the proteins.