Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Summary

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Abstract

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

Introduction

Recent evidence has altered the understanding of the roles of the many regulatory mechanisms that occur after mRNA synthesis. Indeed, translational, post-transcriptional, and proteolytic processes can regulate protein abundance and function. The dogma – which says that mRNA concentrations are proxies to those of the corresponding proteins, assuming that transcript levels are the main determinant of protein abundance – has been partially revised.Indeed, transcript levels only partially predict protein abundance, suggesting that post-transcriptional events occur to regulate the proteins within cells^1,2

Protocol

In this protocol, the human hearts are collected during multiorgan explantation (cold ischemia time of 4-12 h, mean 6 ± 2 h) from multi-organ donors excluded from organ transplantation for technical or functional reasons, despite normal echocardiographic parameters. They are sent to the Cardiovascular Tissue Bank of Milan, Monzino Cardiologic Center (Milan, Italy) for the banking of the aortic and pulmonary valves. The mitral posterior leaflets are not used for clinical purposes, so they are collected during the aortic and pulmonary valve isolation after informed consent is obtained from the donors' relatives. The tissue for transplantation and research is co....

Results

The extraction and dissolution of proteins in the urea buffer is directly compatible with proteomic methods based on isoelectrofocusing (two-dimensional electrophoresis (2-DE)¹¹ and liquid-phase isoelectric focusing (IEF)¹²) and with immunoblotting after dilution in Laemmli buffer¹³ containing a protease inhibitor cocktail¹⁴.

For gel-.......

Discussion

One critical step of this protocol is the use of liquid nitrogen to freeze the sample and to chill the grinder system. The use of liquid nitrogen prevents biological degradation and allows for efficient powdering, but it requires specific training for safe handling.

In this protocol features a grinder system for sample grinding because small samples are difficult to recover from standard mortar and pestles. In this case, small samples spread as a fine powder over the mortar surface, rendering .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Saline solution			0.9 % NaCl
Eurocollins A	SALF	30874046	Balanced organ's transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins
Eurocollins B	SALF	30874022	Balanced organ's transport medium. Combine 400 mL of Eurocollins A with 100 mL Eurocollins B to obtain balanced medium Eurocollins
Wisconsin	Bridge life	RM/N 4081	Balanced organ's transport medium
Biohazard vertical flow air	Burdinola		Class A GMP classification
Dewar Flask	Thermo Scientific	Nalgene 4150-1000
Cryogrinder system	OPS diagnostics	CG 08-01	Grinder system containing mortars, pestles and screwdriver
Stainless steel forceps
Stainless steel spatula
Disposable sterile scalpel	Medisafe	MS-10
Stainless steel scissors			Autoclavable
Stainless steel picks			Autoclavable
Disposable sterile drap	Mon&Tex	3.307.08
Sterilizing solution with isopropyl alcohol			70% isopropyl alcohol
Sterilizing solution with hydrogen peroxide			6% hydrogen peroxide
Micropipette, 1 mL, with tips
15 mL centrifuge tubes	VWR international	9278
1.7 mL centrifuge tubes	VWR international	PIER90410
Urea buffer			8 M urea, 2 M thiourea, 4 % w/v CHAPS, 20 mM Trizma, 55 mM Dithiotreitol
Urea	Sigma aldrich	U6504-1KG	To be used for Urea buffer
Thiourea	Sigma aldrich	T8656	To be used for Urea buffer
CHAPS	Sigma aldrich	C3023-5GR	To be used for Urea buffer
Dithiotreitol	Sigma aldrich	D0632-5G	To be used for Urea buffer
Syringe 50 mL	PIC		To be used to filter Urea buffer
0.22 µm filter	Millipore	SLGP033RB	To be used to filter Urea buffer
PFTE Pestle, 2 mL	Kartell	6302	Part of Potter-Elvehjem homogenizer
Borosilicate glass mortar	Kartell	6102	Part of Potter-Elvehjem homogenizer
Stirrer	VELP scientifica	Stirrer DLH	To be used for homogenization by Potter-Elvehjem
Bradford Protein assay	Bio-Rad laboratories	5000006
Tube rotator	Pbi International	F205
Liquid nitrogen
Aluminum foil
Ice
Polystyrene box
Dry ice
Centrifuge			For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g
Freezer -80°C
Precision balance
Autoclave			For sterilization
Cryogenic gloves for liquid nitrogen
Gloves
Professional forced ventilation and natural air convection oven			For sterilization
Protease inhibitor cocktail	Sigma aldrich	P8340-5ML	100X solution
ProteoExtract Protein Precipitation Kit	Calbiochem	539180
RapiGest	Waters	186001861
Cytoscape	www.cytoscape.org	version 2.7	Software platform for Gene Ontology analysis
BiNGO	http://apps.cytoscape.org/apps/bingo	version 3.0.3	Plugin for Gene ontology analysis
AlphaB Crystallin/CRYAB Antibody	Novus Biologicals	NBP1-97494	Mouse monoclonal antibody against CryAB
Septin-11 Antibody	Novus Biologicals	NBP1-83824	Rabbit polyclonal antibody against septin-11
FHL1 Antibody	Novus Biologicals	NBP-188745	Rabbit polyclonal antibody against FHL-1
Dermatopontin Antibody	Novus Biologicals	NB110-68135	Rabbit polyclonal antibody against dermatopontin
Goat Anti mouse IgG HRP	Sigma aldrich	A4416-0.5ML	Secondary antibody for immunoblotting
Goat Anti rabbit IgG HRP	Bio-Rad laboratories	170-5046	Secondary antibody for immunoblotting