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. 2022 Jul 18;61(29):e202200721.
doi: 10.1002/anie.202200721. Epub 2022 May 17.

Proteoform-Selective Imaging of Tissues Using Mass Spectrometry

Manxi Yang  1 , Hang Hu  1 , Pei Su  1   2 , Paul M Thomas  2 , Jeannie M Camarillo  2 , Joseph B Greer  2 , Bryan P Early  2 , Ryan T Fellers  2 , Neil L Kelleher  2 , Julia Laskin  1
Affiliations

Proteoform-Selective Imaging of Tissues Using Mass Spectrometry

Manxi Yang et al. Angew Chem Int Ed Engl. .

Abstract

Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform-specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) for the proteoform-selective imaging of biological tissues. Nano-DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof-of-concept experiments demonstrate that nano-DESI MSI combined with on-tissue top-down proteomics is ideally suited for the proteoform-selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.

Keywords: Mass Spectrometry Imaging; On-Tissue Top-down Proteomics; Post-Translational Modifications; Proteoforms.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Average mass spectra of the rat brain tissue showing protein signals in the a) white matter and b) blood vessel regions highlighted with red circles in the corresponding optical images. c) A table summarizing the features labeled in (a) and (b). d) MS/MS spectrum of the unmodified hemoglobin subunit alpha (HBA1) and its fragmentation map obtained using the normalized collision energy (NCE) of 23 and isolation window of 3 m/z.
Figure 2
Figure 2
a) An optical image of an axial rat brain tissue section after nano‐DESI MSI analysis. Ion images of the intact proteins normalized to TIC: b) m/z 894.938417+, 15 197 Da, hemoglobin subunit alpha (HBA1), unmodified (level 1), c) m/z 992.28116+, 15 861 Da, hemoglobin subunit beta‐1 (HBB‐1), R104 di‐methylation (level 1), d) m/z 765.406713+, 9 937 Da, acyl‐CoA‐binding protein (ACBP), acetylation (level 2A), e) m/z 784.5647+, 5 485 Da, cytochrome c oxidase subunit 7c, mitochondrial (COX7c), unmodified (level 1), f) m/z 998.794710+, 9 978 Da, cytochrome c oxidase subunit 6B1 (COX6B1), N‐terminal acetylation (level 1), g) m/z 709.93757+, 4 963 Da, thymosin beta‐4, acetylation (level 2A), h) m/z 741.809419+, 14 075 Da, myelin basic protein (MBP), unmodified (level 1), i) m/z 682.357227+, 18 397 Da, myelin basic protein (MBP), N‐terminal acetylation (level 1). Scale bar: 3 mm.
Figure 3
Figure 3
Ion images of the +19 charge state of the 14.1 kDa MBP proteoforms. Top panels show ion images normalized to the TIC. Bottom panels show images generated by plotting the ratio of the individual proteoform signal to the sum of signals of all the MBP proteoforms: a) m/z 744.233519+, 14 121 Da, N‐terminal acetylation (level 1), b) m/z 745.023519+, 14 136 Da, N‐terminal acetylation, methionine sulfoxide (level 1), c) m/z 745.813219+, 14 151 Da, phosphorylation (level 3), d) m/z 747.706319+, 14 187 Da, di‐methylation, phosphorylation (level 2A), e) m/z 748.390119+, 14 200 Da, N‐terminal acetylation, phosphorylation (level 2A), f) m/z 752.492319+, 14 278 Da, N‐terminal acetylation, di‐phosphorylation (level 2A). Scale bar: 3 mm.
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