TY - JOUR
T1 - MALDI-IHC-Guided In-Depth Spatial Proteomics: Targeted and Untargeted MSI Combined
AU - Claes, B.S.R.
AU - Krestensen, K.K.
AU - Yagnik, G.
AU - Grgic, A.
AU - Kuik, C.
AU - Lim, M.J.
AU - Rothschild, K.J.
AU - Vandenbosch, M.
AU - Heeren, R.M.A.
N1 - Funding Information:
This work was supported by the Dutch province of Limburg through the LINK program. Portions of this work were funded by the SBIR grant CA236097 from the NIH (National Cancer Institute) to AmberGen, Inc. R.M.A.H., the M4i team and AmberGen, Inc. acknowledges the continuous technical support of Bruker Daltonics during this project. B.S.R.C. and R.M.A.H. acknowledge financial support from the NIH of the United States of America─i.e., R01 CA213492. The authors thank Annet A.M. Duivenvoorden (NUTRIM, department of surgery, Maastricht University) for help with the immunohistochemical interpretation of the data.
Publisher Copyright:
© 2023 The Authors. Published by American Chemical Society.
PY - 2023/1/13
Y1 - 2023/1/13
N2 - Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDIIHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDIIHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-alpha SM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.
AB - Recently, a novel technology was published, utilizing the strengths of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) and immunohistochemistry (IHC), achieving highly multiplexed, targeted imaging of biomolecules in tissue. This new technique, called MALDIIHC, opened up workflows to target molecules of interest using MALDI-MSI that are usually targeted by standard IHC. In this paper, the utility of targeted MALDIIHC and its complementarity with untargeted on-tissue bottom-up spatial proteomics is explored using breast cancer tissue. Furthermore, the MALDI-2 effect was investigated and demonstrated to improve MALDI-IHC. Formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections were stained for multiplex MALDI-IHC with six photocleavable mass-tagged (PC-MT) antibodies constituting a breast cancer antibody panel (CD20, actin-alpha SM, HER2, CD68, vimentin, and panCK). K-means spatial clusters were created based on the MALDI-IHC images and cut out using laser-capture microdissection (LMD) for further untargeted LC-MS-based bottom-up proteomics analyses. Numerous peptides could be tentatively assigned to multiple proteins, of which three proteins were also part of the antibody panel (vimentin, keratins, and actin). Post-ionization with MALDI-2 showed an increased intensity of the PC-MTs and suggests options for the development of new mass-tags. Although the on-tissue digestion covered a wider range of proteins, the MALDI-IHC allowed for easy and straightforward identification of proteins that were not detected in untargeted approaches. The combination of the multiplexed MALDI-IHC with image-guided proteomics showed great potential to further investigate diseases by providing complementary information from the same tissue section and without the need for customized instrumentation.
KW - BREAST-CANCER
KW - MASS
KW - IDENTIFICATION
KW - IMMUNOHISTOCHEMISTRY
KW - BIOMARKERS
KW - PROTEINS
KW - PATHWAY
KW - TISSUE
U2 - 10.1021/acs.analchem.2c04220
DO - 10.1021/acs.analchem.2c04220
M3 - Article
C2 - 36638208
SN - 0003-2700
VL - 95
SP - 2329
EP - 2338
JO - Analytical Chemistry
JF - Analytical Chemistry
IS - 4
ER -