Protein identification strategies in MALDI imaging mass spectrometry: a brief review

Abstract

Matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful technology used to investigate the spatial distributions of thousands of molecules throughout a tissue section from a single experiment. As proteins represent an important group of functional molecules in tissue and cells, the imaging of proteins has been an important point of focus in the development of IMS technologies and methods. Protein identification is crucial for the biological contextualization of molecular imaging data. However, gas-phase fragmentation efficiency of MALDI generated proteins presents significant challenges, making protein identification directly from tissue difficult. This review highlights methods and technologies specifically related to protein identification that have been developed to overcome these challenges in MALDI IMS experiments.

Figure 1:

An overview of the MALDI IMS workflow for protein imaging. A) A tissue sample is sectioned, washed to remove interfering salts and lipids, and coated homogenously with a MALDI matrix. After sample preparation, the sample is loaded into the instrument and the section is irradiated by a laser, moving a defined lateral distance which dictates the spatial resolution of the image. B) A mass spectrum is generated at each pixel location. C) Ion intensities for a selected mass range are then plotted in a coordinate system within the sampled tissue area, creating an ion image. D) Following, or in parallel to IMS experiments, orthogonal experiments are completed in order to generate protein identifications that can be correlated to the imaging data.

Figure 2:

A visual representation of common orthogonal proteomics experiments used to generate protein identifications in MALDI IMS workflows. The achievable spatial resolution, experimental duration, and CID/ETD compatibility is also presented.

Figure 3:

Spatial resolution and sensitivity of hydrogel-based proteomic workflows: A) Using dermal punch biopsy tools, hydrogels can be fabricated down to ~260 µm in diameter. B) Extracting peptides using a hydrogel with a diameter of 777 µm, the white matter and molecular layers of rat brain cerebellum were profiled and the proteomic profiles were compared. Figure 3 was adapted from reference [54].

Figure 4:

LESA sensitivity and applications: A) Extracting from enzymatically digested rat brain, the number of unique protein identifications was evaluated as a function of solvent extraction volume B) Intact protein extracts were gathered from select regions of whole sections of mouse pup. Top-down MS was utilized to generate protein identifications which could then be correlated to the IMS data through accurate mass matching. The ETD spectrum of thymosin B10 is presented along with its corresponding ion overlay. Figure 4 was adapted from citation [14].