Pre-fabricated Surfaces for Assessment of Spatial Resolution and Proteomic Analysis of Tissue Samples using Imaging Mass Spectrometry

Abstract

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is an enabling tool in medical and biological research. From a single experiment, hundreds of biomolecules can be spatially mapped on a tissue section. A key bottleneck remains the time-consuming sample preparation that requires several hours for each tissue specimen. Current methods employ robotic spotters to serially deposit enzyme and matrix in spatially discrete regions on the tissue. This work develops an alternative using targets pre-coated with trypsin and a MALDI matrix, where the tissue is mounted onto these coated surfaces and then undergoes chemical transformation and incubation to allow digestion of its proteins. Tryptic fragments from proteins including myelin basic protein, PEP-19, neurogranin, and brain acid soluble protein 1 have been successfully imaged by this approach. Further, this pre-coated method provides a standardized target offering reduced sample preparation time and improved spatial resolution down to 75 µm.

A second goal was to fabricate synthetic patterns as imaging standard for evaluating instrument performance and spatial resolution. A standard reticle slide was prepared using lithographic and self-assembly techniques. A PDMS stamp functionalized a gold surface with hexadecanethiol to drive the self-assembly of crystal violet into a specified pattern. The developed reticle slide allowed accurate measurement of laser spot sizes under operating conditions, determination of the positional accuracy of laser on a target, and a comparison of the reproducibility and accuracy of scans across the targets. Together with developed criteria for analysis, an objective assessment of spatial resolution for MALDI-IMS is provided.

These two efforts provide greater reliability for MALDI-IMS by reducing variability. The pre-coated slides streamline the sample preparation for tissue specimen and reduce the burden of sample preparation on the user. The reticle slides allow routine evaluation of instrumentation performance and spatial resolution. Together, these methods can ensure standardized and reproducible performance to aid adoption of MALDI IMS in the clinical environment.