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. 2018 Mar 15;32(5):442-450.
doi: 10.1002/rcm.8042.

Protein identification in imaging mass spectrometry through spatially targeted liquid micro-extractions

Daniel J Ryan  1   2 , David Nei  2   3 , Boone M Prentice  2   3 , Kristie L Rose  2   3 , Richard M Caprioli  1   2   3   4   5 , Jeffrey M Spraggins  1   2   3
Affiliations

Protein identification in imaging mass spectrometry through spatially targeted liquid micro-extractions

Daniel J Ryan et al. Rapid Commun Mass Spectrom. .

Abstract

Rationale: Liquid extraction surface analysis (LESA) can be used to generate spatially directed protein identifications in an imaging mass spectrometry (IMS) workflow. This approach involves the use of robotic micro-extractions coupled to online liquid chromatography (LC). We have characterized the extraction efficiency of this method as well as its ability to identify proteins from a matrix assisted laser/desorption ionization (MALDI) IMS experiment.

Methods: Proteins and peptides were extracted from transverse sections of a rat brain and sagittal sections of a mouse pup using liquid surface extractions. Extracts were either analyzed by online LC coupled to a high mass resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometer or collected offline and analyzed by traditional LC/MS methods. Identifications were made using both top-down and bottom-up methodologies. MALDI images were acquired on a 15T FTICR mass spectrometer at 125 μm spatial resolution.

Results: Robotic liquid surface extractions are reproducible across various tissue types, providing significantly improved spatial resolution, with respect to extractions, while still allowing for a robust number of protein identifications. A single 2-μL extract can provide identification of over 14,000 peptides with little sample preparation, increasing throughput for spatially targeted workflows. Surface extractions from tissue were coupled directly to LC to gather spatially relevant proteomics data.

Conclusions: Robotic liquid surface extractions can be used to interrogate discrete regions of tissue to provide protein identifications with high throughput, accuracy, and robustness. The direct coupling of tissue surface extractions and LC offers a new and effective approach to provide spatial proteomics data in an imaging experiment.

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Figures

Figure 1
Figure 1
A) The measured droplet resolution of the LESAplus system on water-sensitive paper decreases as the volume dispensed increases and as the relative organic fraction of the solvent increases. B) The measured droplet resolution of the LESAplus system on rat brain tissue (cerebellum) using hematoxylin stain to visualize the solvent diffusion during extraction. For both experiments, droplet diameter measurements were made using bright field microscopy.
Figure 2
Figure 2
Five LESA extractions gathered sequentially from the same spot on trypsin-digested rat brain that show a decrease in unique protein identifications and peptides identified through sequential extractions.
Figure 3
Figure 3
Proteins identified from 3 regions of a mouse pup tissue section by combining 2 liquid surface extractions from the same spot. In total, 2879 proteins were identified from the cerebral cortex, 2290 proteins were identified from the cerebellum, and 602 proteins were identified from the kidney. The 3 regions tested displayed overlaps in the proteins identified as highlighted in the Venn diagram.
Figure 4
Figure 4
A & B) Spectral and imaging data from a 125 μm spatial resolution MALDI protein image from a sagittal section of a mouse pup acquired on a 15T FTICR MS. Four ions were chosen to exemplify the many tissue substructures present as highlighted in the image overlay (B). The proteins sequenced below are highlighted by a star in the average mass spectrum of the protein image. C & D) The high resolution, top-down mass spectrum of m/z 4,898.57 allows for its identification as N-acetylated Thymosin β10. E & F) The high resolution, top-down mass spectrum of m/z 5,675.21 allows for its identification as ATP synthase subunit ε. G & H) The high resolution, top-down mass spectrum of m/z 6,628.37 allows for its identification as N-acetylated histone H2A Type 1. I & J) The high resolution, top-down mass spectrum of m/z 7,513.88 allows for its identification as dimethylated hemoglobin subunit A.
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