This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.
The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) has been employed as an image based technique for two decades1,2, analyzing a range of biomolecules including: lipids3, peptides2,4, proteins2,5, metabolites6,7, N-glycans8, and synthetic molecules such as therapeutic drugs9,10. The number of publications demonstrating the utility of this technique have grown significantly over the last decade6,11,12,13. Certain molecules, such as lipids, are relatively easy to analyze via MALDI MSI, as they ionize readily due to their chemical nature and thus require little prior preparation3. However, for more difficult targets such as peptides, the steps required to effectively ionize these molecules are extensive and generally complicated14. There are currently very few publications that aim to address or demonstrate reproducibility in the methodologies that are employed to prepare tissue for this unique visual technique15. For this reason, we have compiled observations and implemented optimizations into a single, easy to implement, methodology that should require little to no modification, for the analysis of peptides from a formaldehyde cross-linked tissue source14.
In this manuscript, we have described a validated, low cost reproducible methodology for the detection and spatial mapping of peptides, generated from formalin-fixed frozen (FFF) and formalin-fixed paraffin-embedded (FFPE) tissue sections. This methodology does not require or rely on any specialized instrumentation3. Specifically, we address the many aspects of specialized sample preparation necessary to analyze peptides; steps such as antigen retrieval16 and matrix coating. Our protocol also utilizes inexpensive equipment and reagents, thereby making this methodology accessible to a wider community who would otherwise be unable to afford the alternate robotic apparatus17.
The reasoning behind developing a manual sample preparation method was two-fold: Firstly, the use of a sublimator creates a consistent and homogenous coating of matrix crystals that are ~1 µm in length18, something unachievable with more common spraying techniques. Secondly, the relatively small set up costs: the total cost of the custom apparatus was <$1500 AUD. We note, in terms of cost effectiveness, the price per sample is far cheaper when there is no robotic machinery involved. The use of sublimation has been reported previously, however, to the best of our knowledge, step-by-step methodologies that describe this process and sample preparation have not been reported nor described in the literature.
This protocol is intended to assist researchers that have access to a MALDI mass spectrometer and who are intent on generating spatial information in relation to a bio-molecule of interest19. In essence, MALDI MSI is a form of histological screening that does not rely on antibodies or stains2.
CAUTION: All applicable safety precautions should be followed when performing this procedure, including the use of appropriate personal protective equipment (PPE) (e.g., lab coats, nitrile gloves, safety glasses, etc.)
1. Preparation of Reagents and Equipment
2. Preparation of Tissue Sections
3. Methylene Crosslink Hydrolysis
4. Tissue Digestion
5. Matrix Coating via Sublimation
6. Recrystallization
7. Instrumentation
If followed correctly, this protocol produces images that clearly represent the gross morphology of the tissue without any scratches or other deformations (Figure 1). The ideal validation for a correctly performed sample preparation, is the ability to distinguish between different physical structures by changing the molecule being viewed (Figure 2).
A good guide for determining if samples have been incorrectly prepared is to check for the presence of delocalization or matrix aggregation; peptide signatures that extend beyond the borders of the physical tissue are perfect indicators that molecules have moved during sample preparation. This is explained in detail in O'Rourke and Padula (2017)15.
The produced peptide spectrum should contain a high abundance of discrete molecules that are well distributed across the chosen mass range1,23 (this is largely sample dependent, however, it is not unusual to see in excess of 50 discrete peptide signatures from FFPE tissue) (Figure 3)18.
Figure 1: A correctly prepared sample of FFF human brain tissue. This processed tissue specimen has been imaged at 50 µm and shows good macro structure and a clear difference between white matter (WM) and grey matter (GM). Successfully prepared samples will show clear differentiation of different tissue locations. Here, there is a clear region of up-regulation of the peptide (represented by mass-to-charge ratio, M/Z) 1085 in the WM region of panel A. Differentiation is further demonstrated by the absence of that defined shape in panel B followed by its return in panel C. By assaying the distribution of three different molecules on the same tissue section and showing that some are uniformly distributed, while others are limited to discrete physical locations, we can see that sample preparation has been performed correctly. Please click here to view a larger version of this figure.
Figure 2: An incorrectly prepared section of FFF human brain tissue. This specimen has been imaged at 50 µm but shows a large-scale level of delocalization. The patterns of the molecules present across panels 1, 2 and 3 clearly demonstrate that, unlike Figure 1, there is no clear definition between biological regions or differences in the abundance of the molecules displayed. Since the images are the same, irrespective of the molecules selected for display, we can conclude that it has been delocalized due to being incorrectly prepared. Since this is brain tissue, this result does not make logical, biological sense. Please click here to view a larger version of this figure.
Figure 3: The global mass spectrum of the image displayed in Figure 1. The spectrum contains an abundance of peptide signatures across the lower end of the mass range, with several high mass peptide peaks present as well. Please click here to view a larger version of this figure.
Supplementary Figure 1: Schematic for cutting blotting paper used in vapor chamber. Thick blotting paper is cut into a circular shape with a diameter of 94 mm. A rectangular tab is also cut out to correspond with the size of a standard glass microscope slide. Please click here to view a larger version of this figure.
Supplementary Figure 2: Assembly schematic of vapor chamber. This schematic shows how the vapor chamber should be assembled with specific note of how the sample slides, blotting paper and adhesive tape all correspond with each other. Please click here to view a larger version of this figure.
Supplementary Figure 3: Schematic of sublimation chamber assembly: This figure shows how the sample slide, sublimator, and heating apparatus are arranged to allow reproducible sublimation. Please click here to view a larger version of this figure.
This protocol was designed to maximize the generation of ionizable molecular species while eliminating delocalization of analytes. The key factors involve using the same overriding principle when applying matrix, digesting the sample, or recrystallizing after sublimation24; namely, that an even deposition of vapor, matrix or otherwise, needs to be created and maintained. Pipetting solvent washes for recrystallization and digestion, evenly underneath the sample, prevents any individual area receiving more solvent vapor than anywhere else. This is important for preventing the formation of visible condensation and overall inhibits delocalization of the surface molecules as well as ensuring that the sample is evenly digested and recrystallized. It is imperative to achieve an even deposition of matrix, as too little will show a relatively low intensity and diversity of species, and too much will suppress the ions from the sample, also lowering intensity and diversity of species25. To achieve an even deposition of matrix, ensure that an even, thin layer of matrix crystals of a homogenous size, are placed directly below the footprint of the suspended microscope slide. If the layer is uneven or too thin, then sublimation of the matrix will proceed too slowly or will be uneven26. The potential issues with manual application of solvent and enzyme comes down to the individual experience of the user. Some level of "practice" is needed in order to ensure a repeatable result, and there may be some initial accidental incorrect preparation. However, if care is taken to ensure that the final image is in accordance with the properties of the "good" sample we have provided, then any improperly prepared samples will not lead to false biological conclusions.
In order to effectively digest a tissue sample preserved in formaldehyde (FFPE or FFF), it is essential to remove as many of the methylene crosslinks as possible prior to trypsin deposition16. This is easily achieved by heating the sample in a pressure cooker at >100 °C, for a short duration, without actually causing bubbles that can mechanically dissociate the sample. Tissue can easily be lost when treated in such a way, so in order to combat this, NC slides are employed. While not critical, it does ensure the physical integrity of the sample when subjected to heat, pressure, and organic solvent. Digestion of the sample is then achieved by applying the enzyme in a state that prevents its activation and then allowing it to dry, i.e. suspended in ultra-pure water. The vapor chamber then follows the same principle as recrystallization, providing enough of a humid environment to allow localized movement of the enzyme to encounter and cleave the protein backbone, but not enough "wetness" to allow the resulting peptides to drift significantly from their initial location26. Pipetting directly on the slide will not delocalize any surface analytes, due to the effects of residual crosslinking and the general insolubility of large proteins in water.
Although we have focused on the application of this methodology to peptides, it can be just as easily applied to workflows for the analysis of metabolites, lipids, and intact proteins. Some steps would need to be removed or augmented; intact protein imaging does not require any proteolytic cleavage steps, but the fundamental workflow of sample sectioning and mounting followed by matrix sublimation, recrystallization, and analysis can be applied to almost any sample type. Our intention for developing this protocol and ensuring its reliability and robustness through extensive empirical investigations, was to create a method that requires little or no modification, that uses inexpensive equipment, and is amenable to a wide community with varying degrees of instrumentation and biochemical skill. We hope that this opens new avenues of investigation to laboratories that would have otherwise dismissed this technique as too complicated or expensive. We are also confident that we have provided the first definitive sample preparation method for peptide MSI. At the time of writing, most methodologies have been largely instrumentation-based, with a particular emphasis on the ease of use of robotic spraying based methodologies27,28,29. There has also been little in the way of reliable methylene hydrolysis methodologies with conjecture as to the correct procedure and apparatus to be used.
In conclusion, we consider that the application of the above methodology to FFPE and FFF tissues, will result in greater confidence in the efficacy of MSI for the analysis of peptides.
The authors have nothing to disclose.
The authors would like to acknowledge the Sydney Medical School Foundation and Blues and Foundation for funding part of this work through their PhD scholarship program for Alzheimer’s Disease Research and an ARC Discovery grant (DP160102063) awarded to PKW.
Cryo Microtome | Leica | CM3050 | For preparation and section of tissue. |
Indium Tin Oxide Microscope slides | Bruker | 8237001 | For preparation and section of tissue. |
Coplin Jars | Sigma Aldrich | S5516 | For preparation and section of tissue. |
Pressure Cooker | Kambrook | KPR620BSS | For preparation and section of tissue. |
Sublimator | Chem Glass | NA | For sublimation procedure. Similar in design to the CG-3038 however it was custom made |
Sand bath | NA | NA | For sublimation procedure. Fine grade river sand held in folded aluminium foil sourced from outside not from any specific company |
Glass Petri Dish | Sigma Aldrich | CLS70165100 | For sublimation procedure. |
Vacuum Pump | NA | NA | For sublimation procedure. Sourced as a spare part from an old mass spectrometer |
Cold trap | Chem Glass | CG-4510-02 | For sublimation procedure. |
Hot Plate | John Morris | EW-15956-32. | For sublimation procedure. |
Plastic petri dish | Sigma Aldrich | Z717223 | For sublimation procedure. |
37 °C incubator | NA | NA | For sublimation procedure. Not applicable, incubator is non sterile and over 30 years old |
Blotting paper | Sigma Aldrich | P7796 | For sublimation procedure. |
Nitrocellulose | Sigma Aldrich | N8395 | For washing of slides. |
Acetone | Sigma Aldrich | 650501 | For washing of slides. |
Xylene | Sigma Aldrich | 214736 | For washing of slides. |
100% EtOH | Sigma Aldrich | 1.02428 | For washing of slides. |
70% EtOH | Sigma Aldrich | NA | For washing of slides. Made in lab from 95% stock ethanol |
Chloroform | Sigma Aldrich | C2432 | For washing of slides. |
Glacial Acetic Acid | Sigma Aldrich | ARK2183 | For washing of slides. |
Tris HCL pH 8.8 | Sigma Aldrich | TRIS-RO | For proteolytic cleavage. Powder made to 1M followed by equilibration with 32% HCl to PH 8.8 |
Milli Q Ultra-Pure Water | Sigma Aldrich | NA | For proteolytic cleavage. Purification performed in house by sartorious water purification system |
Ammonium Bircarbonate | Sigma Aldrich | A6141 | For proteolytic cleavage. |
Trypsin | Sigma Aldrich | T0303 | For proteolytic cleavage. |
CHCA Matrix | Sigma Aldrich | C2020 | For recrystallisation. |
Acetonitrile | Sigma Aldrich | 1.00029 | For recrystallisation. |
Trifluoroacetic Acid (TFA) | Sigma Aldrich | 302031 | For recrystallisation. |