Quantitative Imaging of Proteins in Tissue by Stable Isotope Labeled Mimetic Liquid Extraction Surface Analysis Mass Spectrometry

Absolute quantification of proteins in tissue is important for numerous fields of study. Liquid chromatography–mass spectrometry (LC–MS) methods are the norm but typically involve lengthy sample preparation including tissue homogenization, which results in the loss of information relating to spatial distribution. Here, we propose liquid extraction surface analysis (LESA) mass spectrometry (MS) of stable isotope labeled mimetic tissue models for the spatially resolved quantification of intact ubiquitin in rat and mouse brain tissue. Measured ubiquitin concentrations are in agreement with values found in the literature. Images of rat and mouse brain tissue demonstrate spatial variation in the concentration of ubiquitin and demonstrate the utility of spatially resolved quantitative measurement of proteins in tissue. Although we have focused on ubiquitin, the method has the potential for broader application to the absolute quantitation of any endogenous protein or protein-based drug in tissue.


Materials
Water, methanol and formic acid were purchased from Fisher Scientific (Leicestershire, UK) and glass slides from Thermo Scientific. Fresh frozen mouse brain tissue (extraneous tissue from culled animals) was provided by Prof. Steve Watson at the University of Birmingham, fresh frozen control rat brain (extraneous tissue from culled animals) was provided by AstraZeneca.

Preparation of mimetic tissue models
Mimetic tissue models were prepared using the method described by Groseclose and Castellino. 1 Ten whole rat brains (thawed) were homogenized using a handheld homogenizer. Approximately 2.5 mL of homogenate was portioned into individual pre-weighed tubes and final weight homogenate per tube was calculated. C 13 ,N 15 -labelled ubiquitin (enrichment 98%, Sigma-Aldrich, Gillingham, UK) solutions were prepared in methanol and water (30:70), to final concentrations such that 1% final volume of tissue was added to each sample, this prevented large volumes of solvent diluting the homogenate. Magnetic stir bars (5 mm) were added to each tube and vortexed for 1 minute. Spiked homogenates were pipetted into plastic moulds before freezing for 1 hour at -80 °C. The final concentration of labelled ubiquitin in tissue was 0, 9.9, 19.9, 67.4, 68.6, 94.7, 122.6 and 163.0 nmol/g. Rat and mouse brains were dissected and flash-frozen in liquid nitrogen before storage at -80°C. Tissue mimetics, rat and mouse brain tissue were sectioned at -20°C (Leica CM 1850 Cryostat, Milton Keynes, UK) at 14 µm thickness, and thaw-mounted onto glass slides while placed adjacent to each other.

LESA MS method
Surface sampling was performed using a TriVersa Nanomate chip-based electrospray device (Advion, Ithaca, NY). The electrospray device was coupled to a Thermo Fisher Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) instrument. The TriVersa Nanomate was controlled through LESA Points and ChipSoft 8.3.3 software (Advion, Ithaca, NY). The sample was mounted in the LESA universal adaptor plate and scanned using an Epson flatbed scanner. The scanned image was imported into LESA Points and the sampling locations were defined. Sampling locations were selected with an x, y spacing of 2 mm, covering the whole tissue section. The z coordinate was set to height 1.2 mm above the sample surface to allow the liquid microjunction to be formed. The solvent system for extraction and electrospray was methanol, water and formic acid (69.3:29.7:1). The robotic arm collected a conductive pipette tip and aspirated 2 µL of solvent from the reservoir. The arm relocated to the x, y S3 coordinates specified by LESA Points software. The pipette tip descended to the predetermined height (see above) and dispensed 1.3 µL solvent. The liquid microjunction was maintained for 10 seconds before re-aspiration. Each sample was delivered for 3 min via the Triversa Nanomate with a gas pressure of 0.3 psi and a tip voltage of 1.75 kV. Positive ion mass spectra were acquired in full scan mode (m/z 600 -2000) at a resolution of 120 000 at m/z 400. The Automatic Gain Control was turned off and the injection time fixed at 1 ms. 2 Each scan was comprised of 5 co-added microscans.

Data analysis
RAW data files were analysed in Thermo Xcalibur Software (Thermo Fisher Scientific, Bremen, Germany) and further analysed in MATLAB (MathWorks, Massachusetts, US). RAW data imaging files were converted to mzML files using ProteoWizard's MSConvert software. 3 These files were converted into imzML files using imzML converter. ImzML files were analysed in MATLAB with SpectralAnalysis software. 4

Assessment of homogenate mixing
Two sections were taken from the top and one from the bottom of the mimetic tissue model (see Figure S3) and all three were subject to LESA MS under the same sampling conditions. Ion images of endogenous and labelled ubiquitin were produced which reveal that a similar level of variation is seen for both species. This suggests that the labelled ubiquitin was assumed to be distributed uniformly throughout the homogenate prior to freezing. It also suggests that the variation in intensity is due to variation in LESA sampling and not due to inhomogeneity in the sample.

S8
The initial tissue mimetics experiment The whole experiment was performed and completed 1 year after the preliminary results. The Figure   S5 summarizes the LESA MS preliminary experiment.