Overall protocol for 2D intact proteoform mapping by MALDI imaging
Mowei Zhou, Kevin J Zemaitis, Dusan Velickovic, Ljiljana.PasaTolic, David J Degnan
Abstract
Scope:
An overall protocol outlining several nested sub-protocols for the 2D mapping of intact proteoforms from human tissue by MALDI imaging on an UHMR Q Exactive Orbitrap MS.
Expected Outcomes:
Annotated ion images of intact proteoforms ready to be ingested and disseminated.
Steps
Sample sectioning and preparation
Tissue provided from TMCs are either shipped already cryo-sectioned, or shipped as tissue blocks and the preparation of tissues for MALDI imaging is performed similar to as described elsewhere. Once samples have been sectioned, tissue preparation has been outlined within the following protocol with several nested steps:
Instrument preparation and operation
After tissue preparation the instrument is the optimized, calibrated, and MALDI imaging is performed according to the protocol below:
Data visualization and annotation
Prior to this stage supplemental top-down proteomics by nanoPOTS/microPOTS LCMS/MS is run, once the imaging is completed the image visualization pipeline is run according to the following protocol. Proteoform annotations are then output and manually verified:
