(620af) Fabricating Surfaces for High-Throughput Sample Preparation and to Quantify Spatial Resolution in MALDI Imaging Mass Spectrometry (Rapid Fire)

Matrix assisted laser desorption/ ionization imaging mass spectrometry (MALDI – IMS) is powerful technology in medical, biological and material sciences. In a typical experiment, the tissue surface is coated with matrix, a small organic acid. Consequently, a laser pulse is directed on the tissue surface ablating and ionizing biomolecules. These molecules are separated based on their mass-to-charge ratio.  The laser is rastered across the tissue surface to create molecular heat maps. Current techniques for studying molecular imaging such as immunohistochemistry and histology rely on specific targets requiring apriori knowledge of the biological specimen and monitor one molecular species at a time. In contrast, MALDI can detect hundreds of molecular species in a single experiment requiring no apriori knowledge of the tissue making it particularly useful for discovery.

Current instruments are limited to mass range of up to 20,000 m/z under typical operating conditions. Additionally, a MALDI spectrum consists of intact masses that tell us little about the identity of proteins. To address these challenges, the analytes within the tissue can be enzymatically digested and the resulting peptides can be identified using tandem mass spectrometry. Current methodology employs robotic spotters to deposit trypsin in spatially discrete regions followed by matrix deposition. This process requires many hours for a single tissue specimen and the spatial resolution is limited to 200 µm. We have developed MALDI targets pre-coated with trypsin and matrix, where tissue is mounted onto these coated surfaces.

The pre-coated targets are developed for both fresh-frozen tissues and formalin-fixed paraffin-embedded (FFPE) tissues. Gold slides are uniformly coated with alpha-cyano-4-hydroxycinnamic acid by solvent casting and subsequently spray coated with trypsin. Three micrometer tissue sections are sliced and thaw mounted on top of the pre-coated targets. The slide is then placed in a chamber saturated with N,N-diisopropylethylamine (DIEA) isopropylethylamine and water that converts the matrix into an ionic form with neutral pH of 7-8. After digestion for 2 hours at 50 °C, the sample is washed with 10% tri-fluoroacetic acid and analyzed with MALDI IMS. For FFPE tissue sections, the wax is first removed using a xylene wash and the rest of the process is similar.

We have successfully used pre-coated methods for protease digestion of fresh frozen tissues, reducing sample preparation time and improving spatial resolution 2-fold down to 100 µm. Thin tissue sections from rat brain were analyzed. Several proteins were identified including Myelin Basic Protein, PEP-19, Neurogranin and others. For each parent protein, several peptides were observed in the MALDI FT-ICR spectrum and matched to their theoretical mass to less than 1 ppm. Furthermore, each of the matched peptides for a specific protein was co-localized enabling high confidence in the identifications.

In addition to pre-coated surfaces, we are also fabricating synthetic patterns to evaluate instrument performance particularly spatial resolution. The standard reticle slides with a synthetic pattern of a laser desorbable compound can accurately measure laser spot sizes at multiple laser fluences, assess the positional accuracy of ablation spots on a target, and measure the reproducibility and accuracy of scans across the targets.  This work also includes production of standard protocols and user friendly software tools to accomplish these tasks in the biological and clinical laboratory. Lines ranging from 10 – 100 µm are fabricated. These patterns are designed in computer-aided software and printed as masks that are used to fabricate photoresist masters using photolithographic techniques. PDMS molds are produced using soft-lithography.

The PDMS stamp is inked with a 2 mM solution of hexadecanethiol, and placed on a gold slide to selectively functionalize the gold surface yielding hydrophilic and hydrophobic regions. The substrate is immersed in a solution of crystal violet causing crystal deposition in pre-defined hydrophilic areas. The patterned surfaces are imaged using an Ultraflextreme MALDI TOF/TOF and FT-ICR instruments. The data are analyzed using MATLAB tools to quantify the spatial resolution as measured from the isolated spots on the reticle. Overall, the objective is to develop of a standard reticle to quantify MALDI imaging spatial resolution in the range of 10-100 um ± 10%.

We have also tested the patterned organic material to evaluate delocalization by different matrix application methods, comparing an excessively wet spray with a dry application process (sublimation). One slide was sprayed with sinapinic acid in 50% acetonitrile using a solvent sprayer. A second slide was coated with the same matrix by sublimation. The two slides were imaged using a Bruker Ultraflextreme MALDI TOF instrument with a raster step size of 50 µm. The ion images show that the solvent based spray application caused extensive delocalization while the sublimated sample preserved the pattern. This shows the importance of carefully controlling the surface wetness with solvent spray devices and the need for patterned slide to evaluate the quality of the matrix coating.

In summary, the pre-coated slides standardize the sample preparation for tissue specimen removing the burden of sample preparation from the end user. The reticle slides can evaluate the instrumentation performance particularly its spatial resolution. Combined these new methods can ensure standardized and reproducible performance so that MALDI IMS can be readily adapted in the clinical environment.