Elucidating Tissue- and Plasma-Specific Proteomic Alterations in Health and Disease

Summary

Proteomics is a high-throughput assessment of the interactions, function, composition, and structures of proteins and their cellular activities. Here, we present a protocol to assess total proteomic changes in various bio-samples, including plasma and tissues. The methodology reported herein is applicable to all bio-samples containing proteins.

Abstract

Proteomics is the large-scale study of expressed proteins, focusing on their structure, function, interactions, abundance, and post-translational modifications within a biological system. For example, phosphoproteomics, a vital subset of total proteomics, is the study of phosphorylation patterns and alterations on proteins. The methodology described herein illustrates a total proteomics approach to elucidate alterations in overall protein expression profiles, including phosphorylation patterns, in the plasma, brain, lung, spleen, and liver tissues collected from research animal models (i.e., ferrets and mice). This technique is applicable for other tissue types, as well as almost any bio-sample containing proteins (e.g., cultured cells). Following dissection, tissues of interest were flash frozen and stored at -80 °C. Tissues were then homogenized using a mortar and pestle with liquid nitrogen to preserve protein integrity and phosphorylation changes. Total protein was extracted from the tissue homogenates using a lysis buffer with a universal nuclease and protease/phosphatase inhibitors. Proteins extracted from tissue and equivalent plasma were converted to total peptides by controlled protease digestion using a commercial mass spectrometer sample preparation kit. Peptides were directly analyzed using ultra-high performance liquid chromatography/Orbitrap-tribrid tandem mass spectrometry. Identities of constituent proteins were reconstructed from the peptide mass spectral data, using proteomics bioinformatics software, as matched to a species-specific amino acid sequence library. Following this, the analysis should include strict cutoff criteria, especially using only high-confidence protein identifications. Statistically significant proteomic differences (>2-fold change; p < 0.05) can be determined between control and experimental groups.

Introduction

Proteomics, as the combination of proteome experimentation and data analysis, focuses on identifying and quantifying proteins, characterizing them based on composition, interaction, pre-translational and post-translational modifications (e.g., phosphorylation), sub-cellular localization, and structure under physiological conditions¹. Expression proteomics deals with quantitative comparisons of proteins that differ under changing biological conditions². It also holds a high importance in profiling a map of the complex, interconnected pathways, networks, and molecular systems, which directly control the major biological fu....

Protocol

Research was conducted under an IACUC-approved animal use protocol in an AAALAC International-accredited facility with a Public Health Services Animal Welfare Assurance and in compliance with the Animal Welfare Act and other federal statutes and regulations relating to laboratory animals. All supplies required for this procedure are listed in the Table of Materials. The procedure is subdivided into seven components.

CAUTION. Ensure proper safety protocols are followed when working with liquid nitrogen to homogenize tissue. The laboratory personnel wore freezer gloves, a freezer apron, a surgical face mask, and a face shield....

Results

Visualization of total proteome results by optional processing into volcano plots
The total proteome results for each sample can be readily searched by accession # or description for specific proteins of research interest and the associated relative abundances are averaged and then compared between treatment groups for significant differences (e.g., as by t-test derived p-values). Alternatively, it can be of great exploratory value to look for global changes across the entire proteome, including any .......

Discussion

The above method utilized tissues that were snap frozen in liquid nitrogen to immediately halt all biological activity before transferring to -80 °C for long-term storage. Once all samples were collected, tissue homogenization occurred. During homogenization, it is critical to keep all tissues and tools cold to prevent reactivation or degradation of the proteins. Also, during the initial extraction of protein from homogenized tissue, samples are kept cold to prevent loss of protein activity or protein degradation. W.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
660 nm Protein Assay Kit	Pierce / Thermo Scientific	22662	For BCA to determine protein concentrations of samples, contains Pierce 660nm Protein Assay Reagent and Pre-Diluted Protein Assay Standards
Acetonitrile, Optima LC/MS grade	Thermo Fisher Scientific	047138.K2	Ultra-pure acetonitrile used for liquid chromatography/mass spectrometry
Aluminum Foil Plate-Seal	Beckman Coulter	538619	Seal used during development of protein assay plate
BioTek Synergy Neo2 Multi-mode Reader / UV-Vis Spectrometer	Agilent	https://www.agilent.com/en/product/microplate-instrumentation/microplate-readers/multimode-microplate-readers/biotek-synergy-neo2-hybrid-multimode-reader-1623195	Spectrophotometer/assay plate reader instrument used to complete 660nm absorbance readings to determine protein concentrations
ChromaCare LC-MS Instrument Flush Solution	Thermo Fisher Scientific	T111101000	Solution containing 25% each of acetonitrile, isopropranol, methanol, and water use to help clean out the LC system after running samples
CK28 Precellys tube	Bertin Corp / Technologies	P000911LYSK0A.0	Homogenized tissues are placed in these tubes for tissue lysis and protein isolation using the Precellyis instrument. These are the tubes utilized by our lab referred to in the protocol as "homogenizing tubes."
Cryopod	Brooks / Azenta Life Sciences	https://www.azenta.com/products/cryopod-carrier	Container used to hold liquid nitrogen for chilling mortar during tisuse homogenization
EasyPep Mini MS Sample Prep Kit	Thermo Fisher Scientific	A40006	One kit can process 20 samples, contents include the lysis solution for protein extraction, universal nuclease supplemented in the extraction solution, and regeants needed for Steps 1.4 and 1.5
EASY-Spray C18 LC column (15 cm L, 50 µm Dia, and 2 µm particle size)	Thermo Fisher Scientific	ES901	Electrospray LC column used for the seperation of peptides prior to MS detection
FlexMix calibration solution	Pierce / Thermo Scientific	A39239	Mixture of 16 standard molecules used to calibrate the detector accuracy and sensitivity of MS instrument prior to sample runs
Formic Acid, LC/MS grade	Thermo Fisher Scientific	85178	Ultra-pure formic acid used to acifidy all LC solutions and samples at a final concentration of 0.1% (v/v)
Halt Protease & Phosphotase Single-Use Inhibitor Cocktail (100x)	Thermo Fisher Scientific	78442	Supplemented in the extraction buffer to prevent degradation of proteins and phosphoproteins in sample
Methanol, Optima LC/MS grade	Thermo Fisher Scientific	047192.K2	Ultra-pure methanol used for liquid chromatography/mass spectrometry
Porcelain Mortar, 150 mL	Thermo Fisher Scientific	FB961C	150 mL capacity, used for homogenizing smaller ferret tissues (i.e., brain)
Porcelain Mortar, 275 mL	Thermo Fisher Scientific	FB961D	275 mL capacity, used for homogenizing large ferret tissues (i.e., liver)
Porcelain Mortar, 50 mL	Thermo Fisher Scientific	FB961A	50 mL capacity, used for homogenizing mouse tissues
Porcelain Pestl, 50 mL	Thermo Fisher Scientific	FB961K	For use with 50 mL mortar
Porcelain Pestles, 150 mL	Thermo Fisher Scientific	FB961M	For use with 150 mL mortar
Porcelain Pestles, 275 mL	Thermo Fisher Scientific	FB961N	For use with 275 mL mortar
Precellys	Bertin Corp / Technologies	https://www.bertin-technologies.com/product/sample-preparation-homogenizers/precellys-evolution-homogenizer/	Instrument used to homogenize tissue samples. The following settings were used: 4500 RPM, 2 x 30 s per cycle, 30 s pause.
Protein Assay Reagent	Pierce / Thermo Scientific	22662	Protein Assay Reagent used in steps 3.4–3.8
Proteome Discoverer	Thermo Fisher Scientific	CSW0064764	Bioinformatics software for analysis of proteomics/phosphoproteomics data
SureSTART MSCERT screw cap LC injection vials (0.02–1.5 mL capacity)	Thermo Fisher Scientific	6PMCK39TR	High preformance application certified, screw cap (PTFE, pre-slit septa) glass vials used to contain samples for injection on the LC/MS instrument
Ultimate 3000 UHPLC / Orbitrap Fusion Lumos Tribrid MS (LC/MS) system	Thermo Fisher Scientific	LC: discountinued / MS: FETD2-10002, by special request	Ultra-high preformance liquid chromatography/mass spectrometery (LC/MS) instrument used to separate out and then detect by molecular weight the peptides in the samples
Universal Nuclease for Cell Lysis, 100kU, 400 µL	Pierce / Thermo Scientific	88702	Additional universal nuclease was purchased
Vacufuge Plus	Eppendorf	https://www.eppendorf.com/us-en/Products/Centrifugation/Concentrator/Vacufuge-plus-p-PF-25748	Vacufuge used to dry peptide samples before reconstitution.
Water, Optima LC/MS grade	Thermo Fisher Scientific	047146.K2	Ultra-pure water used for liquid chromatography/mass spectrometry