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

Abstract

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 multiple reaction monitoring (MRM) mass spectrometry (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 time-of-flight (TOF) MS data. This external calibration curve was then used to determine 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 show the feasibility for the precise and accurate quantitation of tissue protein concentrations over 2 orders of magnitude, while maintaining the spatial localization information for the proteins.

Quantification in MALDI-MS imaging: what can we learn from MALDI-selected reaction monitoring and what can we expect for imaging?

“…Among the different normalization methods, the use of labeled internal standards (ISTDs) showed the best linear regression fit [24,25]. ISTD can be a labeled version of the analyte [26][27][28]23], a structural analogue [29], or an endogenous signal (e.g., a peptide in the same mass range as the targeted analyte [30] or a lipid species [31]). In Q-MSI, ISTD normalization compensates for signal variations resulting in difference in analyte extraction, co-crystallization, and ionization [32], assuming that the ISTD is homogenously distributed over the region of interest.…”

Novel mass spectrometry imaging software assisting labeled normalization and quantitation of drugs and neuropeptides directly in tissue sections

“…Efforts have been made for relative quantitation of amyloid beta peptides using insulin as an internal standard [6]. Measurement of endogenous peptides using multiple reaction monitoring (MRM) has been reported directly on tissue sections of the rat brain [7].…”

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

“…Because the MALDI MS process is only able to provide relative abundance information based on the local ionization efficiency within each pixel, care must be taken when interpreting MALDI MSI data without stable isotope labeled internal standards 49 . Furthermore, for confidence in the localization results, confirmation of any distribution patterns detected must be done using alternative methods.…”