A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging

Abstract

MALDI imaging requires careful sample preparation to obtain reliable, high-quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good-quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses.

Figure 1

MALDI spectra from (a) methanol soft landed/washed rat lens tissue and (b) carbon tape mounted and lyophilized rat lens tissue showing major signals due to αA-crystallin products.

Figure 2

MALDI IMS comparison of a methanol soft landed/washed 100 day old rat lens tissue sample (left panel), a methanol soft landed/unwashed 21 day old rat lens (center panel), and replicates of carbon tape mounted/unwashed 100 day old rat lens tissue (right panel) displaying the distribution of (a) m/z 6,413 αA crystallin 1–53, ((b) m/z 9,844 αA crystallin 1–83, (c) m/z 19,835 αA 1–173 and (d) overlay of the previous m/z values. Scale bars represent 2 mm. Tissue margins are indicated by dashed white lines.

Figure 3

3D optical profilometry image (4x magnification) of an unwashed 11.5 month old mouse lens methanol soft landed onto a gold coated MALDI plate displaying the cortex and nuclear regions where the tissue can be seen to be lifting from the surface.

Figure 4

MALDI IMS images displaying α-crystallin products from mouse and rat lens tissue located in (a) nuclear regions, (b) cortex region, (c) outer cortex region and (d) overlay of multiple signals. Scale bars represent 1 mm.

Figure 5

Slit lamp image of a normal mouse eye in vivo.

Figure 6

Optical and MALDI IMS images from a carbon tape mounted adult zebrafish lens (a) Optical image of washed (top) and unwashed (bottom) zebrafish lens sections and spotted matrix array. (b) Optical image of regions of interest where data were acquired. (c) MALDI images generated from putative γ-crystallin at m/z 20,569. (d) Multiple MALDI images displaying concentric rings observed in the zebrafish lens.