Aug 31, 2022

Public workspaceOverall protocol for 2D intact proteoform mapping by MALDI imaging

DOI
https://dx.doi.org/10.17504/protocols.io.q26g74qn8gwz/v1
Overall protocol for 2D intact proteoform mapping by MALDI imaging
  • Kevin J. Zemaitis1,
  • Dusan Velickovic1,
  • David J Degnan1,
  • Mowei Zhou1,
  • Ljiljana.PasaTolic 1
  • 1Pacific Northwest National Laboratory
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
  • PNNL-TTD
Kevin J Zemaitis
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g74qn8gwz/v1
Protocol Citation: Kevin J. Zemaitis, Dusan Velickovic, David J Degnan, Mowei Zhou, Ljiljana.PasaTolic 2022. Overall protocol for 2D intact proteoform mapping by MALDI imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74qn8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 19, 2022
Last Modified: May 16, 2023
Protocol Integer ID: 68931
Keywords: intact proteoform mapping by maldi, intact proteoform mapping, annotated ion images of intact proteoform, intact proteoforms from human tissue, intact proteoform, imaging, imaging scope, 2d mapping, human tissue, annotated ion image
Funders Acknowledgements:
National Institutes of Health (NIH) Common Fund, Human Biomolecular Atlas Program (HuBMAP)
Grant ID: UG3CA256959-01
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.
Troubleshooting
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:
Citation
Kevin J. Zemaitis, Dusan Velickovic, Ljiljana.PasaTolic (2026). Tissue Preparation for Intact Proteoform MALDI-MSI on Human Tissue.
dx.doi.org/10.17504/protocols.io.6qpvr61x2vmk/v1
LINK

Instrument preparation and operation
After tissue preparation the instrument is the optimized, calibrated, and MALDI imaging is performed according to the protocol below:
Citation
Kevin J. Zemaitis, Dusan Velickovic, Mowei Zhou, Ljiljana.PasaTolic (2026). High Resolution Intact Proteoform Mass Spectrometry Imaging using UHMR HF Orbitrap.
dx.doi.org/10.17504/protocols.io.4r3l2onz4v1y/v1
LINK

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:
Citation
David J Degnan, Kevin J. Zemaitis, Dusan Velickovic, Mowei Zhou, Ljiljana.PasaTolic (2026). Image Visualization and Proteoform Assignment of MALDI-MSI from LCMS Experimental Databases.
dx.doi.org/10.17504/protocols.io.4r3l2ode3v1y/v1
LINK

Citations
Step 1
Kevin J. Zemaitis, Dusan Velickovic, Ljiljana.PasaTolic. Tissue Preparation for Intact Proteoform MALDI-MSI on Human Tissue
dx.doi.org/10.17504/protocols.io.6qpvr61x2vmk/v1
Step 2
Kevin J. Zemaitis, Dusan Velickovic, Mowei Zhou, Ljiljana.PasaTolic. High Resolution Intact Proteoform Mass Spectrometry Imaging using UHMR HF Orbitrap
dx.doi.org/10.17504/protocols.io.4r3l2onz4v1y/v1
Step 3
David J Degnan, Kevin J. Zemaitis, Dusan Velickovic, Mowei Zhou, Ljiljana.PasaTolic. Image Visualization and Proteoform Assignment of MALDI-MSI from LCMS Experimental Databases
dx.doi.org/10.17504/protocols.io.4r3l2ode3v1y/v1