Abstract
Purpose: :
MALDI imaging provides an alternative multiplex imaging modality that depends on the molecular weight of the species present in tissue sections. Thus, intact and modified proteins can be imaged in a single experiment. The purpose of this study is to develop sample preparation methods that allow distributions of beta- and gamma-crystallins and their modified forms to be imaged from bovine and human lens tissue sections.
Methods: :
Frozen bovine and human lens sections were obtained at 20 um thickness. Lens tissue was washed with 50% acetonitrile (ACN) and sinapinic acid matrix (50:49.9:0.1 ACN:H2O:TFA) was applied using the Labcyte Portrait 630 spotter. Images were acquired using a Bruker Autoflex III in linear mode with a spatial resolution of 300 µm. Proteins were identified by top down tandem mass spectrometry (both collision induced dissociation and electron transfer dissociation) on-line with HPLC separation of lens homogenates or directly from purified lens crystallins.
Results: :
A modified sample preparation protocol allowed images to be produced of beta-and gamma- crystallins from bovine and human lens sections. Gamma-S crystallin showed an outer cortical localization while other gamma crystallins were observed in the nucleus. Partially truncated beta crystallins were distributed in an outer nuclear ring.
Conclusions: :
MALDI imaging methods have been expanded to include not only alpha-crystallin, but now beta- and gamma-crystallins in lens tissues. Images can be used to detect age-related modifications and their spatial localization.
Keywords: crystallins • imaging/image analysis: non-clinical • proteomics