Laser capture microdissection-capillary zone electrophoresis-tandem mass spectrometry (LCM-CZE-MS/MS) for spatially resolved top-down proteomics: a pilot study of zebrafish brain

Abstract

Mass spectrometry (MS)-based spatially resolved top-down proteomics (TDP) of tissues is crucial for understanding the roles played by microenvironmental heterogeneity in the biological functions of organs and for discovering new proteoform biomarkers of diseases. There are few published spatially resolved TDP studies. One of the challenges relates to the limited performance of TDP for the analysis of spatially isolated samples using, for example, laser capture microdissection (LCM) because those samples are usually mass-limited. We present the first pilot study of LCM-capillary zone electrophoresis (CZE)-MS/MS for spatially resolved TDP and used zebrafish brain as the sample. The LCM-CZE-MS/MS platform employed a non-ionic detergent and a freeze-thaw method for efficient proteoform extraction from LCM isolated brain sections followed by CZE-MS/MS without any sample cleanup step, ensuring high sensitivity. Over 400 proteoforms were identified in a CZE-MS/MS analysis of one LCM brain section via consuming the protein content of roughly 250 cells. We observed drastic differences in proteoform profiles between two LCM brain sections isolated from the optic tectum (Teo) and telencephalon (Tel) regions. Proteoforms of three proteins (npy, penkb, and pyya) having neuropeptide hormone activity were exclusively identified in the isolated Tel section. Proteoforms of reticulon, myosin, and troponin were almost exclusively identified in the isolated Teo section, and those proteins play essential roles in visual and motor activities. The proteoform profiles accurately reflected the main biological functions of the Teo and Tel regions of the brain. Additionally, hundreds of post-translationally modified proteoforms were identified.

Fig. 1

Schematic overview of the LCM-CZE-MS/MS workflow.

Fig. 2

(A) An image of one 20 μm-thick zebrafish brain slice used in the study. The collected square regions of the zebrafish brain Optic Tectum (Teo) and Telencephalon (Tel) are highlighted. Each region has an area of 500 μm². (B) The microscopic images of two square regions of Teo and Tel after the microdissection. (C) The corresponding tissue sections collected in (B) on the caps of Eppendorf tubes that were used for sample collection in the LCM experiment.

Fig. 3

The number of proteoform and protein identifications from the LCM-CZE-MS/MS experiments using the OG-sonication sample preparation method (A) and OG-freeze–thaw method (B). The error bars represent the standard deviations of the number of protein and proteoform identifications from duplicate CZE-MS/MS runs. (C) Total ion current (TIC) electropherograms of the Teo2 sample prepared by the OG-freeze–thaw method and analyzed by CZE-MS/MS in duplicate. One example proteoform mass spectrum is shown in the inserted figure. (D) Overlaps of identified proteoforms between duplicate CZE-MS/MS analyses of Teo2 and Tel2 samples prepared by the OG-freeze–thaw method. Correlations of proteoform intensity between duplicate analyses of Teo2 (E) and Tel2 (F) samples. (G) Sequence and fragmentation pattern of one identified histone H4 proteoform from the Teo2 sample.

Fig. 4

(A) Overlaps of identified proteoforms and proteins from Tel2 and Teo2 samples. The data was from the duplicate CZE-MS/MS analyses (technical replicates). (B) Base peak electropherograms of the Tel2 and Teo2 samples with the mass spectrum of the most abundant peak highlighted in the inserted figure. (C) The number of identified proteoforms of several genes from the Tel2 and Teo2 samples. For those genes, the Tel2 sample had much more proteoforms than the Teo2 sample. (D) The number of identified proteoforms of several genes from the Tel2 and Teo2 samples. For those genes, the Teo2 sample had much more proteoforms than the Tel2 sample.

Fig. 5

Data about proteoforms with PTMs. (A) The number of proteoforms with various PTMs identified from the Tel2 and Teo2 samples. (B–D) Sequences and fragmentation patterns of three proteoforms with combinations of various PTMs.