1. A Repeatable Cost Effective Method For The Analysis Of Fresh Frozen Tissue Sections Via Matrix Assisted Laser Desorption Ionisation Imaging Mass Spectrometry (MALDI-IMS)
Matthew O’Rourke, Steven Djordjevic, Matthew Padula.
The ithree Institute, University of Technology, Sydney.
Introduction
Matrix Assisted Laser Desorption Ionisation Imaging Mass Spectrometry (MALDI-IMS) is a robust technique that has seen great improvement in recent time and has the potential to further the field of diagnostic imaging [1]. However with automated sample preparation approaches becoming more and more prevalent [2], little work is being done to understand and standardize the critical matrix application and co-crystallisation techniques in use or investigate more economical non-machine based approaches. In this work we have attempted to dissect and define the conditions responsible for the successful implementation of a sublimation based approach for sample preparation, that is able to maintain homogeneity of matrix coverage as well as prevent delocalisation. By quantifying each condition and empirically determining their ideal conditions, we hope to provide a repeatable and cost effective method for the universal MS imaging of fresh frozen tissue.
Aims
To create a cost effective method of Imaging fresh frozen tissue via MALDI IMS and characterise and define the conditions responsible for an effective repeatable methodology.
Methodology
Methodology
Preparation Of Tissue
Tissue is prepared in 3 stages:
Freezing, Sectioning and Washing:
Freezing is performed in -20C isopentane to exclude water and prevent tissue cracking [3].
Sectioning is performed at 12 µm and sections are thaw-mounted onto indium tin oxide (ITO) slides coated in Liquid Nitrocellulose (fig1).
Washing of sections is performed in six stages with organic solvent to remove lipids and salts.
Sublimation & Recrystalisation
Following sectioning and washing, the tissue is mounted in the sublimator. Matrix is placed in the bottom petri dish, the sublimator is evacuated to 25 mTorr and ice is packed into the cooling finger.
The chamber is then heated in an oil bath at 175oC for 20 minutes (fig4) to achieve matrix deposition of 0.2 mg/cm2.The chamber is vented and the ice removed.
Once deposition is complete, the slide is mounted in the vapour chamber and recrystallised for 1 hour at 37oC with 650 µl of 50% ACN: 0.1%TFA.
Figure 4: Assembled sublimation apparatus and oil bath. Chamber is suspended in bath via a steel ring and retort stand
Figure 1: Thaw mounted rat liver tissue on an ITO slide (slide is mounted to apparatus with copper tape)
Figure 5: Point spectra and associated images for 4 tissue types. 1) Rat kidney, 2) Rat liver, 3) Rat brain, 4) Rat Heart. Note the differential spectra of proteins and consistent signal to noise across all tissue. Mass range is between 3 and 20 kDa and all images are at 60 µm spatial resolution.
Modification Of Apparatus
It was necessary to modify the sublimator in order to predictably control the pattern of matrix deposition. The top of chamber was modified to “flatten” the cooling finger via copper tape and a petri dish (fig2a). The bottom was modified with another petri dish and river sand (fig2b). When assembled the two internal surfaces were flat relative to each other (fig2c).
To control recrystallisations the vapour chamber was constructed from a plastic agar dish and thick blotting paper. A microscope slide shape was cut into the blotting paper and slide shapes drawn on the bottom of the agar dish (fig3a). Another shape is drawn on the top half corresponding to the intended side position (fig 3b). An ITO slide containing a sample is placed on the drawn shape and affixed with a metal slide via a magnet (fig3c). The chamber is assembled to align the paper tab and the slide (fig3d). A C
Results
We found that with the aforementioned modifications, matrix application and recrystallisation is robust and repeatable, therefore the images generated are consistent.
We have successfully demonstrated the universality of this method by applying it to rat Brain, Kidney, Liver, Heart, and Spinal cord (data not shown) with no variation in results regarding quality of spectra or image (fig5). The method used for each tissue type was not altered in any way. We are thereby confident that with minimal intuitive adjustment this method could be easily adopted by other institutions. A B B C D
Conclusions
Through the implementation of this standardised protocol the UTS proteomics core is now able to image any type of fresh frozen tissue reliably and consistently. This technique is now being adapted for use for several discreet projects.
Future Directions
While imaging intact protein on fresh frozen tissue is now repeatable, there are limitations with this; most tissue is preserved or cross-linked in some way. The method to combat this, is peptide imaging via on tissue enzymatic digestion [4]. Our future work will be to adapt this protocol to include digestion by using a method whereby a section is coated with an enzyme such as trypsin in such a way as to maintain crystal homogeneity and prevent delocalisation of the analyte.
References
Grey, A.C., et al., MALDI imaging mass spectrometry of integral membrane proteins from ocular lens and retinal tissue. J Proteome Res, (7): p
Nimesh, S., et al., Current status and future perspectives of mass spectrometry imaging. Int J Mol Sci, (6): p
Kaletas, B.K., et al., Sample preparation issues for tissue imaging by imaging MS. Proteomics, (10): p
Gustafsson, O.J., et al., Matrix-assisted laser desorption/ionization imaging protocol for in situ characterization of tryptic peptide identity and distribution in formalin-fixed tissue. Rapid Commun Mass Spectrom, (6): p