A Streamlined Approach for Mass Spectrometry-Based Proteomics Using Selected Tissue Regions

We have developed a streamlined method for studying the proteome in specific regions of higher tissue samples using mass spectrometry. Our goal is to enhance proteomic technique, including sample preparation and mass spectrometry analysis, to achieve high accuracy and reliability. One challenge in FFPE-based spatial proteomics is incomplete protein digestion, which reduces overall protein coverage.

Overcoming these requires optimized digestion protocols and enhanced mass spectrometry strategies to improve proteome depths and accuracy. We identified distinct protein alterations across different type of pancreatic disease, providing in insight into the differences between benign and precancerous conditions. These findings could aid in the early diagnosis of pancreatic cancer.

We aim to advance patient proteinase method to achieve high resolution protein mapping within tissue regions. Utilizing mass spectrometry-based proteomics, we seek to identify region-specific molecular signatures that contribute to biomarker discovery. Our approach integrates those patient trappings and pre-preparation with data independent occasion mass spectrometry to rapidly generate high-quality proteome data from selected FFPE tissue regions.

This method enables great quantitation of the spatial proteome. To begin, prepare a hematoxylin and eosin-stained or immunohistochemical-stained tissue slide with the region of interest indicated by a pathologist. Place the unstained tissue slide and the stained tissue slide back to back, ensuring proper alignment.

Using a scalpel, remove tissue regions that are not of interest and scrape the tissue regions of interest towards the center of the slide. Transfer the collected tissue into a clean 1.5 milliliter low protein binding tube and add 180 microliters of SDS lysis buffer to each tube. Perform probe sonication at 20%amplitude for 10 cycles of five seconds on and five seconds off.

Now, incubate the samples at 100 degrees Celsius for 3.5 hours while maintaining a speed of 1, 000 G.After incubation, allow the sample to cool at room temperature for 10 minutes. Then, centrifuge at 16, 000 G for 10 minutes at room temperature to separate tissue debris from the supernatant. Collect the supernatant into a clean labeled tube and store at minus 80 degrees Celsius until further use.

For acetone precipitation, place the protein sample corresponding to 100 to 300 micrograms into an acetone-compatible tube. Then, add ice cold acetone, pre-chilled to minus 20 degrees Celsius, in a volume five times the sample volume. Incubate the mixture at minus 20 degrees Celsius for 18 hours.

After incubation, centrifuge the tube at 16, 000 G for 15 minutes. Carefully dispose of the supernatant without disturbing the protein pellet. Add 500 microliters of acetone tempered at minus 20 degrees Celsius and centrifuge.

After decanting the supernatant, air dry the sample. Prepare the suspension trapping lysis buffer in deionized water. Then, add 40 microliters of the prepared buffer to the sample tube and vortex thoroughly to dissolve the air-dried protein pellet.

Place the sample in a shaking incubator at 100 degrees Celsius at 1, 000 G for 35 minutes. Next, add 10 microliters of the alkylating reagent to the sample and incubate the sample at room temperature at 300 G for one hour. Inside a chemical fume hood, add five microliters of the 10%trichloroacetic acid to the sample, making the total volume 55 microliters.

Check the pH of the sample using pH paper to ensure it is less than one. Next, add 350 microliters of binding buffer one to the sample to trap proteins. Place the suspension trapping column into a two milliliter tube and transfer the entire sample, including any insoluble material, into the column.

Centrifuge the column at 4, 000 G for 50 seconds to trap proteins. Then, add 400 microliters of wash buffer two. After centrifuging, discard the flowthrough.

Following the final wash, centrifuge the column at 4, 000 G for 1.25 minutes to ensure complete passage of buffer one. Add 125 microliters of digestion buffer to the suspension trapping column and cap it to prevent evaporation. Incubate the sample at 37 degrees Celsius for 18 hours without shaking.

Now, add 80 microliters of elution buffer one to the suspension trapping column and centrifuge at 4, 000 G for 1.25 minutes. Pull the eluted peptides and transfer them to a clean new tube. After peptide quantification, lyophilize 20 micrograms of peptides.

Re-dissolve the lyophilized peptides in 40 microliters of aqueous buffer containing 3%acetonitrile in 0.1%formic acid water and sonicate for 10 minutes in a sonication bath. Centrifuge the sample at 16, 000 G for 60 minutes and transfer the supernatant into vials for mass spectrometry analysis. Inject two microliters of each sample using the autosampler of the nano-liquid chromatography mass spectrometry system.

Separate peptides on a reversed phase column packed with three micrometers C18 material using a 127 minute gradient of five to 35%acetonitrile at 100 nanoliters per minute. Ionize the peptides via a nanospray ion source and transfer them into an Orbitrap-based mass spectrometer. Analyze peptides using a narrow-range data-independent acquisition method.

Precise region of interest isolation during FFPE tissue processing was achieved across different pancreatic cystic formalin-fixed paraffin-embedded tissues. Reproducible total ion chromatograms were obtained between biological trireplicates for each type of pancreatic cystic neoplasm. Liquid chromatography mass spectrometry analysis identified 9, 703 proteins.

The abundances of all identified proteins spanned 6.25 orders of magnitude, demonstrating comprehensive proteome coverage, and known pancreatic cancer protein markers were quantified. A total of 933 differentially expressed proteins were identified, with 457 upregulated and 476 downregulated in intraductal papillary mucinous neoplasms. Bioinformatics analysis revealed that differentially expressed proteins were associated with several pancreatic cancer-related pathways.