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

Published 6/14/2017
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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.

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Banfi, C., Guarino, A., Brioschi, M., Ghilardi, S., Mastrullo, V., Tremoli, E., et al. Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve. J. Vis. Exp. (124), e55762, doi:10.3791/55762 (2017).

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 cells1,2.

Furthermore, proteins ultimately dictate the function of the cell and therefore dictate its phenotype, which can undergo dynamic changes in response to autocrine, paracrine, and endocrine factors; blood-borne mediators; temperature; drug treatment; and disease development. Thus, an expression analysis focused on the protein level is useful to characterize the proteome and to unravel the critical changes that occur to it as part of disease pathogenesis3.

Therefore, the opportunities that proteomics present to clarify health and disease conditions are formidable, despite the existing technological challenges. The particularly promising areas of research to which proteomics can contributeinclude: the identification of altered protein expression at any level (i.e., whole cells or tissue, subcellular compartments, and biological fluids); the identification, verification, and validation of novel biomarkers useful for the diagnosis and prognosis of disease; and, hopefully, the identification of new protein targets that can be used for therapeutic purposes, as well as for the assessment of drug efficacy and toxicity4.

Capturing the complexity of the proteome represents a technological challenge. The current proteomic tools offer the opportunity to perform large-scale, high-throughput analysis for the identification, quantification, and validation of altered protein levels. In addition, the introduction of fractionation and enrichment techniques, aimed at avoiding the interference caused by the most abundant proteins, has also improved protein identification by including the least abundant proteins. Finally, proteomics has been complemented by the analysis of post-translational modifications, which progressively emerge as important modulators of protein function.

However, the sample preparation and protein recovery in the biological specimens under analysis still remain the limiting steps in the proteomic workflow and increase the potential for possible pitfalls5. Indeed,in most of the molecular biology techniques that must be optimized, the first steps are tissue homogenization and cell lysis, especially during the analysis of low-abundance proteins for which amplification methods do not exist. In addition, the chemical nature of proteins can influence their own recovery. For example, the analysis of highly hydrophobic proteins is very challenging, because they easily precipitate during isoelectric focusing, while trans-membrane proteins are almost insoluble (reviewed in Reference 5). Furthermore, the tissue composition variability creates a significant barrier to developing a universal extraction method. Finally, because almost all of the clinical specimens are of limited quantity, it is essential to enable protein preparation with maximal recovery and reproducibility from minimal sample amounts6.

This work describes an optimized protocol for protein extraction from the normal human cardiac mitral valve, which represents a very challenging sample for proteomic analysis. The normal mitral valve is a complex structure lying between the left atrium and the left ventricle of the heart (Figure 1). It plays an important role in the control of blood flow from the atrium to the ventricle, preventing backflow and ensuring the proper level of oxygen supply to the whole body, thus maintaining an adequate cardiac output. However, it is often considered to be an "inactive" tissue, with a low cellularity and few components, mainly in the extracellular matrix. This is because, in normal conditions, the resident valvular interstitial cells (VICs) present a quiescent phenotype with a low protein biosynthesis rate7.

However, it has been demonstrated that, in a pathological state, the number of VICs in the spongiosa increases and their protein synthesis is activated, together with other functional and phenotypical changes8. Therefore, it is not surprising that the minimal data available in the literature focus on the analysis of pathological mitral valves9,10, in which the increased number of activated VICs might explain the relatively high number of identified proteins.

In conclusion, the present protocol may serve to develop the understanding of the pathogenic mechanisms responsible for mitral valve diseases through the study of mitral valve protein components. Indeed, a greater understanding of the underlying pathological processes could help to improve the clinical management of valve diseases, whose current indications for intervention are largely predicated on hemodynamic considerations.

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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 collected only after parental consent; on the consent sheet, they authorize (or not) the use of the cardiac tissue for research only if it is not suitable for human clinical use (i.e., microbiological, functional, and serological problems), following the guidelines of the ethics committee of Monzino Cardiologic Center.

1. Mitral valve preparation

  1. Harvest the human mitral valve as soon as possible after organ explantation (cold ischemia time of 4-12 h).
  2. In a clean room, remove the heart from the transport bag containing a cold (4 °C) solution (i.e., saline solution or balanced medium Eurocollins or Wisconsin). Put it into a bucket and place it in a biosafety cabinet (biohazard vertical air flow, class A, Good Manufacturing Practices (GMP) classification) to proceed with the valve preparation.
  3. Place the heart on a sterile disposable drape in the cabinet. Using a sterile disposable scalpel, cut the heart completely, perpendicularly to its major axis, on the level of the left and right ventricles, about 4 cm away from the apex.
  4. Move the ascending aorta and pulmonary artery to display the left atrial roof.
  5. With sterile autoclavable forceps and picks, cut around the left auricle on the left atrial roof, making the mitral valve visible and allowing for the great mitral leaflet (anterior) and the small mitral leaflet (posterior) to be identified.
    NOTE: Antero-lateral and the posterior medial commissures define the border of the anterior leaflet and the posterior area.
  6. Using sterile autoclavable scissors and non-traumatic forceps, dissect the left atrium and the ventricle wall thickness around the circumference of the whole mitral valve.
  7. Identify the mitro-aortic valve continuity.
    NOTE: The left ventricle contains the whole mitral valve and chords.
  8. Separate the anterior mitral valve leaflet from the posterior mitral valve leaflet, cutting the posterior leaflet along the insertion with the ventricle (commissure).
  9. Wash the posterior leaflet in the saline solution. Cut the leaflet into small pieces (<1 cm2) and individually wrap them in aluminum foil. Snap-freeze them with liquid nitrogen.
    Caution: Follow organizational safety procedures when using liquid nitrogen.
    1. Sanitize the table of the cabinet with a 70% isopropyl alcohol solution and a 6% hydrogen peroxide solution at the end of the procedure.

2. Protein extraction

  1. Use forceps to pick up the sample stored in liquid nitrogen and immediately place it on dry ice while still wrapped in the aluminum foil. Do not leave the sample to thaw during any transfers.
  2. Prior to grinding, chill the porcelain/zirconium mortar and pestles of a grinder system (e.g., CryoGrinder), together with the sample, by putting them in a Dewar flask containing liquid nitrogen (~500 mL).
    Caution: Follow organizational safety procedures when using liquid nitrogen.
  3. Put the mortar and pestles in a polystyrene box containing dry ice. Remove the sample from the aluminum foil and put it into the mortar.
  4. Grind the sample with the big pestle against the mortar 15-20 times, using the screwdriver to rotate the pestle. Mix the sample with the tip of a pre-chilled spatula during the grinding process.
    1. Repeat with the small pestle.
  5. Transfer the ground sample to a previously weighed tube (e.g., 15 mL centrifuge tube) by inverting the tube, placing it over the mortar, and inverting them together to move the sample to the tube. Use a pre-chilled spatula to recover all material from the mortar.
    1. Keep the tube with the sample on dry ice to avoid sample thawing during the transfer.
  6. Calculate the net weight of the sample.
  7. Clean the mortar and the pestles after each sample and decontaminate them by autoclaving or heating them at 200 °C for 2 h.
  8. Transfer the powdered sample from the centrifuge tube to the glass tube of a homogenizer by inversion.
  9. Add filtered urea buffer (8 M urea, 2 M thiourea, 4% w/v CHAPS, 20 mM Tris, and 55 mM dithiotreitol) to the glass tube, 200 µL of urea buffer for every 10 mg of powdered tissue.
    NOTE: Residual powdered sample left in the centrifuge tube can be recovered using part of the calculated volume of urea buffer.
  10. Homogenize the sample using a stirrer equipped with a borosilicate glass mortar and a polytetrafluoroethylene (PTFE) pestle. Slowly press the pestle onto the sample with a twisting motion (1,500 rpm) 10 times.
  11. Recover the supernatant and transfer it to a clean 1.7 mL centrifuge tube. Again extract the remaining sample with fresh urea buffer, adding half of the volume used during the first extraction.
  12. Repeat step 2.10.
  13. Recover the supernatant and combine it with the supernatant from step 2.11. Place the combined supernatant on a tube rotator for 30 min.
  14. Centrifuge the tube for 30 min at 13,000 x g and 4 °C.
  15. Recover the supernatant and measure the protein concentration using the Bradford protein assay, per the manufacturer's instructions. Store the sample at -80 °C until use.

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Representative 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-free mass spectrometry-based methods (i.e., liquid chromatography coupled to data-independent mass spectrometry analysis (LC/MSE) and two-dimensional LC/MSE (2D-LC/MSE))15, the samples extracted in the described urea buffer need to be further treated to eliminate the urea and thiourea, which could interfere with subsequent protein digestion and liquid chromatography separation. This desalting step can be accomplished using the commercial protein precipitation kits, following the manufacturer's instructions to precipitate the proteins. The sample can be then dissolved in 25 mM NH4HCO3 containing 0.1% cleavable detergents for protein digestion16. The precipitation of the proteins can eliminate buffer components, minimally affecting the protein content (protein recovery: >85%), thus rendering the sample suitable for every kind of analysis.

The application of this protocol for protein extraction from human mitral valves allowed for the identification of a total of 422 proteins, combining four different proteomics approaches previously described in detail11,15. Specifically, 169 proteins were identified by 2-DE, 330 proteins by liquid-phase IEF, 96 proteins by LC/MSE, and 148 proteins by 2D-LC/MSE (Table 1).

To classify the 422 identified proteins in terms of subcellular localization, a software was used for the gene ontology (GO) analysis (e.g., Cytoscape). The network created with the software and its corresponding plug-in showed that, besides the expected proteins localized in the extracellular region (see the upper-right portion of Figure 2), most of the proteins identified by the proteomic approaches were from the intracellular region (i.e., cytoplasm, organelles, vesicles, and cytoskeleton). Cell-surface proteins were also identified (Figure 2).

Results were further confirmed in three independent mitral valve samples. Immunoblotting was used to analyze a group of four proteins (i.e., septin-11, four and a half LIM domains protein 1 (FHL-1), dermatopontin, and alpha-crystallin B (CryAB)) that have never been identified in the normal mitral valve (Figure 3).

Figure 1
Figure 1: Mitral valve structure. Top view of the human heart showing the closed (A) or open (B) human mitral valve. Front view of the left ventricle of a human heart (C). Please click here to view a larger version of this figure.

Figure 2
Figure 2: Analysis of the identified mitral valve proteins in term of cellular distribution. Cytoscape and the plugin BiNGO were used to obtain the distribution of gene ontology (GO) terms from the cellular component categories. The circle size is proportional to the number of protein components associated with the selected GO terms, and the color scale for the p-value of over-representation is reported. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Immunoblotting analysis of septin-11, FHL-1, dermatopontin, and CryAB in whole extract from three human normal mitral valve leaflets. Immunoblotting was performed using mouse monoclonal antibody against CryAB and rabbit polyclonal antibodies against the septin-11, FHL-1, and dermatopontin antibodies. Please click here to view a larger version of this figure.

Accession Description 2-DE 2D-LC LC-MSE liquid phase IEF
A6NMZ7 Collagen alpha VI x
O00151 PDZ and LIM domain protein 1 x
O00299 Chloride intracellular channel protein 1 x
O00764 Pyridoxal kinase x
O14558 Heat shock protein beta 6 x
O43399 Tumor protein D54 x
O43488 Aflatoxin B1 aldehyde reductase member 2 x
O43707 Alpha actinin 4 x x
O43866 CD5 antigen like precursor x
O60493 Sorting nexin 3 x
O60701 UDP glucose 6 dehydrogenase x
O75223 Uncharacterized protein x
O75368 SH3 domain binding glutamic acid rich like protein x
O75390 Citrate synthase mitochondrial precursor x
O75489 NADH dehydrogenase ubiquinone iron sulfur protein 3 mitochondrial precursor x x
O75608 Acyl protein thioesterase 1 x
O75828 Carbonyl reductase NADPH 3 x
O75874 Isocitrate dehydrogenase NADP cytoplasmic x
O75955 Flotillin x
O94760 NG NG dimethylarginine dimethylaminohydrolase 1 x
O94788 Retinal dehydrogenase 2 x
O95865 NG NG dimethylarginine dimethylaminohydrolase 2 x x
P00325 Alcohol dehydrogenase 1B x x
P00338 L lactate dehydrogenase A chain x
P00352 Retinal dehydrogenase 1 x
P00441 Superoxide dismutase Cu Zn x x
P00450 Ceruloplasmin precursor x x
P00488 Coagulation factor XIII A chain precursor x
P00491 Purine nucleoside phosphorylase x
P00492 Hypoxanthine guanine phosphoribosyltransferase x
P00558 Phosphoglycerate kinase 1 x x x
P00568 Adenylate kinase isoenzyme 1 x
P00734 Prothrombin x x
P00738 Haptoglobin x x x x
P00739 Haptoglobin related protein precursor x
P00751 Complement factor B x x x
P00915 Carbonic anhydrase 1 x
P00918 Carbonic anhydrase 2 x
P01008 Antithrombin III precursor x x x
P01009 Alpha 1 antitrypsin x x x x
P01011 Alpha 1 antichymotrypsin x x x x
P01019 Angiotensinogen precursor x x
P01023 Alpha 2 macroglobulin x x
P01024 Complement C3 x x x x
P01033 Metalloproteinase inhibitor 1 precursor x
P01042 Kininogen 1 precursor x
P01593 Ig kappa chain V I region AG x
P01598 Ig kappa chain V I region EU x
P01600 Ig kappa chain V I region Hau x x
P01611 Ig kappa chain V I region Wes x
P01620 Ig kappa chain V III region SIE x
P01625 Ig kappa chain V IV region Len x
P01766 Ig heavy chain V III region BRO x x
P01781 Ig heavy chain V III region GAL x
P01834 Ig kappa chain C region x x x x
P01842 Ig lambda chain C regions x x x
P01857 Ig gamma 1 chain C region x x x x
P01859 Ig gamma 2 chain C region x x x x
P01860 Ig gamma 3 chain C region x x x
P01861 Ig gamma 4 chain C region x x x
P01871 Ig mu chain C region x x x
P01876 Ig alpha 1 chain C region x x x x
P01877 Ig alpha 2 chain C region x
P02144 Myoglobin x x
P02452 Collagen alpha 1 I chain x x x x
P02511 Alpha crystallin B chain x
P02545 Lamin A C 70 kDa lamin x x x x
P02647 Apolipoprotein A I x x x x
P02649 Apolipoprotein E x x x x
P02671 Fibrinogen alpha chain x x x x
P02675 Fibrinogen beta chain x x x x
P02679 Fibrinogen gamma chain x x x x
P02689 Myelin P2 protein x
P02735 Serum amyloid A protein precursor x
P02741 C reactive protein precursor x
P02743 Serum amyloid P component x x x x
P02746 Complement C1q subcomponent subunit B x x
P02747 Complement C1q subcomponent subunit C precursor x
P02748 Complement component C9 x x x
P02749 Beta 2 glycoprotein 1 x x x x
P02750 Leucine rich alpha 2 glycoprotein precursor x
P02751 Fibronectin x x
P02760 AMBP protein precursor x x x x
P02763 Alpha 1 acid glycoprotein 1 x x x
P02765 Alpha 2 HS glycoprotein precursor x
P02766 Transthyretin precursor x x x
P02768 Serum albumin x x x x
P02774 Vitamin D binding protein precursor x
P02787 Serotransferrin x x x x
P02788 Lactotransferrin precursor x
P02790 Hemopexin x x x x
P02792 Ferritin light chain x
P04004 Vitronectin x x x x
P04075 Fructose bisphosphate aldolase A x
P04083 Annexin A1 x x x x
P04179 Superoxide dismutase Mn mitochondrial precursor x
P04196 Histidine rich glycoprotein precursor x
P04217 Alpha 1B glycoprotein precursor x x
P04350 Tubulin beta 4 chain x
P04406 Glyceraldehyde 3 phosphate dehydrogenase x x x x
P04792 Heat shock protein beta 1 x x x x
P05091 Aldehyde dehydrogenase mitochondrial precursor x x
P05155 Plasma protease C1 inhibitor precursor x
P05156 Complement factor I precursor x
P05413 Fatty acid binding protein heart x
P05452 Tetranectin precursor TN x x
P05787 Keratin type II cytoskeletal 8 x
P06396 Gelsolin x x x x
P06576 ATP synthase subunit beta mitochondrial precursor x x
P06732 Creatine kinase M type x x
P06733 Alpha enolase x x x x
P06753 Tropomyosin alpha 3 chain x x
P07108 Acyl CoA binding protein x
P07195 L lactate dehydrogenase B chain x x x
P07196 Neurofilament light polypeptide x
P07197 Neurofilament medium polypeptide x x
P07237 Protein disulfide isomerase precursor x x
P07339 Cathepsin D precursor x x
P07355 Annexin A2 x x x x
P07360 Complement component C8 gamma chain precursor x
P07437 Tubulin beta chain x x x x
P07585 Decorin x x x x
P07737 Profilin 1 x
P07858 Cathepsin B precursor x
P07900 Heat shock protein HSP 90 alpha x x
P07951 Tropomyosin beta chain x
P07954 Fumarate hydratase mitochondrial precursor x
P07996 Thrombospondin 1 x x x
P08107 Heat shock 70 kDa protein 1A 1B x x x x
P08123 Collagen alpha 2 I chain x x x x
P08133 Annexin A6 x x
P08238 Heat shock protein HSP 90 beta x x
P08253 72 kDa type IV collagenase precursor x
P08294 Extracellular superoxide dismutase Cu Zn precursor x x x
P08590 Myosin light polypeptide 3 x x
P08603 Complement factor H x x x x
P08670 Vimentin x x x x
P08729 Keratin type II cytoskeletal 7 x
P08758 Annexin A5 x x x x
P09211 Glutathione S transferase P x x x
P09382 Galectin 1 x x x
P09417 Dihydropteridine reductase x
P09493 Tropomyosin 1 alpha chain x x
P09525 Annexin A4 x x
P09651 Heterogeneous nuclear ribonucleoprotein A1 x
P09871 Complement C1s subcomponent precursor x
P09936 Ubiquitin carboxyl terminal hydrolase isozyme L1 x
P09972 Fructose bisphosphate aldolase C x
P0C0L4 Complement C4 A precursor x
P0CG05 Ig lambda 2 chain C regions x
P0CG38 POTE ankyrin domain family member I x
P10515 Dihydrolipoyllysine residue acetyltransferase component of pyruvate dehydrogenase complex x
P10768 S formylglutathione hydrolase x
P10809 60 kDa heat shock protein mitochondrial precursor x
P10909 Clusterin x x x x
P10915 Hyaluronan and proteoglycan link protein 1 precursor x x
P11021 78 kDa glucose regulated protein x x x
P11047 Laminin subunit gamma 1 precursor x
P11142 Heat shock cognate 71 kDa protein x x x x
P11177 Pyruvate dehydrogenase E1 component subunit beta mitochondrial precursor x
P11217 Glycogen phosphorylase muscle form x
P11310 Medium chain specific acyl CoA dehydrogenase mitochondrial precursor x
P11413 Glucose 6 phosphate 1 dehydrogenase x
P11766 Alcohol dehydrogenase class 3 chi chain x
P12036 Neurofilament heavy polypeptide x
P12109 Collagen alpha 1 VI chain x x x x
P12110 Collagen alpha 2 VI chain x x x x
P12111 Collagen alpha 3 VI chain x x x
P12277 Creatine kinase B type x
P12429 Annexin A3 x x
P12814 Alpha actinin 1 x x
P12829 Myosin light polypeptide 4 x
P12882 Myosin 1 x
P12883 Myosin 7 x x
P12955 Xaa Pro dipeptidase x
P13489 Ribonuclease inhibitor x
P13533 Myosin 6 x x
P13611 Versican core protein precursor x x
P13639 Elongation factor 2 x
P13716 Delta aminolevulinic acid dehydratase x
P13796 Plastin 2 x
P13804 Electron transfer flavoprotein subunit alpha mitochondrial precursor x
P13929 Beta enolase x x
P14136 Glial fibrillary acidic protein astrocyte x
P14314 Glucosidase 2 subunit beta precursor x
P14550 Alcohol dehydrogenase NADP x
P14618 Pyruvate kinase isozymes M1 M2 x x x
P14625 Endoplasmin precursor x x
P15121 Aldose reductase x
P15259 Phosphoglycerate mutase 2 x
P16152 Carbonyl reductase NADPH 1 x
P17066 Heat shock 70 kDa protein 6 x x x
P17174 Aspartate aminotransferase cytoplasmic x
P17540 Creatine kinase sarcomeric mitochondrial precursor x
P17661 Desmin x x x x
P17980 26S protease regulatory subunit 6A x
P17987 T complex protein 1 subunit alpha x
P18206 Vinculin x x
P18428 Lipopolysaccharide binding protein precursor x
P18669 Phosphoglycerate mutase 1 x
P19105 Myosin regulatory light chain 2 x x
P19623 Spermidine synthase x
P19652 Alpha 1 acid glycoprotein 2 precursor x x
P19823 Inter alpha trypsin inhibitor heavy chain H2 x
P19827 Inter alpha trypsin inhibitor heavy chain H1 x x
P20073 Annexin A7 x
P20618 Proteasome subunit beta type 1 precursor x
P20774 Mimecan x x x x
P21266 Glutathione S transferase Mu 3 x
P21333 Filamin A x x
P21796 Voltage dependent anion selective channel protein 1 x
P21810 Biglycan x x x x
P21980 Protein glutamine gamma glutamyltransferase 2 x x
P22105 Tenascin X x x
P22314 Ubiquitin activating enzyme E1 x
P22352 Glutathione peroxidase 3 precursor x x
P22626 Heterogeneous nuclear ribonucleoproteins A2 B1 x
P22695 Ubiquinol cytochrome c reductase complex core protein 2 mitochondrial precursor x
P23141 Liver carboxylesterase 1 precursor x
P23142 Fibulin 1 x x x x
P23284 Peptidyl prolyl cis trans isomerase B precursor x
P23381 Tryptophanyl tRNA synthetase cytoplasmic x
P23526 Adenosylhomocysteinase x x
P23528 Cofilin 1 x
P24752 Acetyl CoA acetyltransferase mitochondrial precursor x
P25311 Zinc alpha 2 glycoprotein precursor x
P25705 ATP synthase subunit alpha mitochondrial x
P25788 Proteasome subunit alpha type 3 x x
P25789 Proteasome subunit alpha type 4 x
P26447 Protein S100 A4 x
P27348 14 3 3 protein theta x x
P27797 Calreticulin precursor x
P28066 Proteasome subunit alpha type 5 x
P28070 Proteasome subunit beta type 4 precursor x x
P28072 Proteasome subunit beta type 6 precursor x
P28074 Proteasome subunit beta type 5 precursor x
P28331 NADH ubiquinone oxidoreductase 75 kDa subunit mitochondrial precursor x
P28838 Cytosol aminopeptidase x
P29218 Inositol monophosphatase x
P29401 Transketolase x
P29692 Elongation factor 1 delta x
P29966 Myristoylated alanine rich C kinase substrate x
P30040 Endoplasmic reticulum protein ERp29 precursor x
P30041 Peroxiredoxin 6 x x
P30043 Flavin reductase x
P30044 Peroxiredoxin 5 mitochondrial precursor x
P30085 UMP CMP kinase x
P30086 Phosphatidylethanolamine binding protein 1 x
P30101 Protein disulfide isomerase A3 precursor x x x
P30613 Pyruvate kinase isozymes R L x
P30740 Leukocyte elastase inhibitor x
P31025 Lipocalin 1 precursor x
P31937 3 hydroxyisobutyrate dehydrogenase mitochondrial precursor x
P31942 Heterogeneous nuclear ribonucleoprotein H3 x
P31943 Heterogeneous nuclear ribonucleoprotein H x
P31946 14 3 3 protein beta alpha x x
P31948 Stress induced phosphoprotein 1 x
P31949 Protein S100 A11 x
P32119 Peroxiredoxin 2 x x
P34931 Heat shock 70 kDa protein 1 like x x
P34932 Heat shock 70 kDa protein 4 x
P35232 Prohibitin x
P35237 Serpin B6 x
P35443 Thrombospondin 4 x
P35555 Fibrillin 1 x x
P35579 Myosin 9 x x x
P35580 Myosin 10 x x
P35609 Alpha actinin 2 x
P35625 Metalloproteinase inhibitor 3 x x
P35998 26S protease regulatory subunit 7 x
P36871 Phosphoglucomutase 1 x x
P36955 Pigment epithelium derived factor x x
P37802 Transgelin 2 x x
P37837 Transaldolase x
P38117 Electron transfer flavoprotein subunit beta x
P38646 Stress 70 protein mitochondrial precursor x x
P39687 Acidic leucine rich nuclear phosphoprotein 32 family member A x
P40121 Macrophage capping protein x
P40925 Malate dehydrogenase cytoplasmic x
P40926 Malate dehydrogenase mitochondrial precursor x
P41219 Peripherin x
P42330 Aldo keto reductase family 1 member C3 x
P45880 Voltage dependent anion selective channel protein 2 x
P47755 F actin capping protein subunit alpha 2 x x
P47756 F actin capping protein subunit beta x x
P47985 Ubiquinol cytochrome c reductase iron sulfur subunit mitochondrial precursor x
P48047 ATP synthase O subunit mitochondrial precursor x
P48637 Glutathione synthetase x
P48741 Heat shock 70 kDa protein 7 x x
P49189 4 trimethylaminobutyraldehyde dehydrogenase x
P49368 T complex protein 1 subunit gamma x
P49747 Cartilage oligomeric matrix protein x x x x
P49748 Very long chain specific acyl CoA dehydrogenase mitochondrial precursor x
P50395 Rab GDP dissociation inhibitor beta x x
P50454 Serpin H1 precursor x
P50995 Annexin A11 x
P51452 Dual specificity protein phosphatase 3 x
P51884 Lumican x x x x
P51888 Prolargin precursor x x x x
P52565 Rho GDP dissociation inhibitor 1 x
P52566 Rho GDP dissociation inhibitor 2 x
P54652 Heat shock related 70 kDa protein 2 x x x x
P55072 Transitional endoplasmic reticulum ATPase x
P55083 Microfibril associated glycoprotein 4 precursor x x
P57053 Histone H2B type F x
P60174 Triosephosphate isomerase x x x
P60660 Myosin light polypeptide 6 x x
P60709 Actin cytoplasmic 1 x x x x
P60981 Destrin x
P61086 Ubiquitin conjugating enzyme E2 25 kDa x
P61088 Ubiquitin conjugating enzyme E2 x
P61224 Ras related protein Rap 1b precursor x
P61978 Heterogeneous nuclear ribonucleoprotein K x x
P61981 14 3 3 protein gamma x x x
P62258 14 3 3 protein epsilon 14 3 3E x
P62491 Ras related protein x
P62714 Serine threonine protein phosphatase 2A catalytic subunit beta isoform x
P62736 Actin aortic smooth muscle x x x
P62805 Histone H4 x
P62826 GTP binding nuclear protein Ran x
P62873 Guanine nucleotide binding protein G I G S G T subunit beta 1 x
P62879 Guanine nucleotide binding protein G I G S G T subunit beta 2 x
P62937 Peptidyl prolyl cis trans isomerase A x x
P62987 Ubiquitin 60S ribosomal protein L40 x
P63104 14 3 3 protein zeta delta x x x x
P63241 Eukaryotic translation initiation factor 5A 1 x
P63244 Guanine nucleotide binding protein subunit beta 2 like 1 x
P63267 Actin gamma enteric smooth muscle x x
P67936 Tropomyosin alpha 4 chain x
P68032 Actin alpha cardiac muscle 1 x
P68104 Elongation factor 1 alpha 1 x x x
P68133 Actin alpha skeletal muscle x
P68363 Tubulin alpha 1B chain x x x
P68371 Tubulin beta 2C chain x x x
P68402 Platelet activating factor acetylhydrolase IB subunit beta x
P68871 Hemoglobin subunit beta x x x
P69905 Hemoglobin subunit alpha x x
P78371 T complex protein 1 subunit beta x
P78417 Glutathione transferase omega 1 x x
P80748 Ig lambda chain V III region LOI x
P98095 Fibulin 2 x x
Q01082 Spectrin beta chain brain 1 x
Q01449 Myosin regulatory light chain 2 atrial isoform x
Q01518 Adenylyl cyclase associated protein 1 x
Q01995 Transgelin Smooth muscle protein 22 alpha x
Q03252 Lamin B2 x x
Q03591 Complement factor H related protein 1 precursor x
Q04917 14 3 3 protein eta x x
Q06323 Proteasome activator complex subunit 1 x
Q06828 Fibromodulin x x x x
Q06830 Peroxiredoxin 1 x x
Q07507 Dermatopontin x x x x
Q07960 Rho GTPase activating protein 1 x
Q08257 Quinone oxidoreductase x
Q08431 Lactadherin x x
Q12765 Secernin 1 x
Q13011 Delta 3 5 Delta 2 4 dienoyl CoA isomerase mitochondrial precursor x x
Q13228 Selenium binding protein 1 x x
Q13404 Ubiquitin conjugating enzyme E2 variant 1 x
Q13409 Cytoplasmic dynein 1 intermediate chain 2 x
Q13509 Tubulin beta 3 chain x x x
Q13642 Four and a half LIM domains protein 1 x
Q13765 Nascent polypeptide associated complex subunit alpha x
Q13885 N Tubulin beta 2A chain x x
Q14194 Dihydropyrimidinase related protein 1 x
Q14195 Dihydropyrimidinase related protein 3 x x x x
Q14624 Inter alpha trypsin inhibitor heavy chain H4 precursor x
Q14697 Neutral alpha glucosidase AB precursor x
Q14764 Major vault protein x
Q14767 Latent transforming growth factor beta binding protein 2 x x
Q14894 Mu crystallin homolog NADP regulated thyroid hormone binding protein x
Q15063 Periostin precursor x x x x
Q15084 Protein disulfide isomerase A6 precursor x
Q15113 Procollagen C endopeptidase enhancer 1 precursor x
Q15181 Inorganic pyrophosphatase x
Q15365 Poly rC binding protein 1 x
Q15366 Poly rC binding protein 2 x
Q15582 Transforming growth factor beta induced protein x x x
Q15819 Ubiquitin conjugating enzyme E2 variant 2 x
Q16352 Alpha internexin x
Q16473 Putative tenascin XA x
Q16555 Dihydropyrimidinase related protein 2 x x x
Q16698 2 4 dienoyl CoA reductase mitochondrial precursor x
Q16891 Mitochondrial inner membrane x
Q562R1 Beta actin like protein 2 x
Q6S8J3 POTE ankyrin domain family member E x
Q6UWY5 Olfactomedin like protein 1 precursor x x
Q71U36 Tubulin alpha 1A chain x x x
Q7Z7G0 Target of Nesh SH3 precursor x x x
Q8WUM4 Programmed cell death 6 interacting protein x
Q8WWX9 Thioredoxin like selenoprotein M precursor x
Q92597 Protein NDRG1 x
Q92945 Far upstream element binding protein 2 x
Q96CN7 Isochorismatase domain containing protein 1 x
Q96CX2 BTB POZ domain containing protein x
Q96KK5 Histone H2A type 1 H x x
Q96KP4 Cytosolic nonspecific dipeptidase x
Q99426 Tubulin specific chaperone B x
Q99497 Protein DJ 1 x x
Q99536 Synaptic vesicle membrane protein x x x
Q99714 3 hydroxyacyl CoA dehydrogenase type 2 x
Q99715 Collagen alpha 1 XII chain x x
Q99798 Aconitate hydratase mitochondrial precursor x
Q9BQE3 Tubulin alpha 6 chain x
Q9BUF5 Tubulin beta 6 chain x x
Q9BUT1 3 hydroxybutyrate dehydrogenase type 2 x
Q9BVA1 Tubulin beta 2B chain x x x
Q9BXN1 Asporin x x x x
Q9H0W9 Ester hydrolase C11orf54 x
Q9H4B7 Tubulin beta 1 chain x
Q9NRN5 Olfactomedin like protein 3 precursor x x
Q9NRV9 Heme binding protein 1 x
Q9NSB2 Keratin type II cuticular Hb4 x x
Q9NVA2 Septin 11 x
Q9UBR2 Cathepsin Z precursor x
Q9UBX5 Fibulin 5 x x
Q9UK22 F box only protein 2 x
Q9Y277 Voltage dependent anion selective channel protein 3 x
Q9Y281 Cofilin 2 x
Q9Y490 Talin 1 x
Q9Y696 Chloride intracellular channel protein 4 x
Q9Y6C2 EMILIN 1 x x

Table 1: List of the proteins identified in the mitral valve tissue extract when four different proteomic approaches were applied: two-dimensional electrophoresis (2-DE), two-dimensional LC-MSE (2D-LC/MSE), LC/MSE, and liquid-phase IEF. The method by which each protein was identified is reported.

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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 collection difficult. Another advantage is that the grinder is motorized, which allows for a greater number of samples to be processed in a reproducible manner and without added fatigue. One limitation on the use of a grinder is that the size of the sample which must be small (100 mg or less) for the pestle to be pressed effectively against the mortar. Furthermore, the grinder components must be warmed to room temperature between uses for cleaning. Consequently, the procedure is time consuming and, if many samples are processed daily, many sets are needed.

An additional critical step is the preparation of the extraction buffer. The salt concentrations, especially for the urea (8 M) and thiourea (2 M), are quite high; thus, the volume of salt is almost half the total volume of the solution. Furthermore, the dissolution is not easy considering that heat must be avoided because, at more than 37 °C, urea can lead to protein carbamylation at the N termini of proteins/peptides and at the side-chain amino groups of lysine and arginine residues17. Once dissolved, the urea buffer must be filtered with 0.22-µm filters and can be stored at -80 °C for 4 weeks without affecting its extraction efficacy, but it must be warmed to more than 15 °C before use to allow for complete dissolution.

Modifications and troubleshooting

In this protocol, protein extraction is performed using the described urea buffer because it is one of the most commonly used protein extraction solutions for proteomic studies due to its compatibility with isoelectrofocusing and its efficiency in solubilizing sparingly soluble proteins18. It has been demonstrated that this buffer can very efficiently solubilize sparingly soluble proteins, such as integral membrane proteins19, or proteins that are highly prone to aggregation, such as tubulin18. Furthermore, this buffer is fully compatible with the Bradford assay to determine protein concentration, and it can be used directly in 2-DE and liquid-phase IEF analyses.

However, this buffer is not ideal for the solubilization of all proteins present in a sample. It is possible that different extraction buffers could reveal proteins undetectable by this protocol. For example, it is well known that ribosomal and nuclear proteins could be better extracted with acid extraction or trichloroacetic acid/acetone precipitation20, while alkaline pH levels are more suitable for membrane proteins21,22. Therefore, the use of alternative buffers might require additional steps for protein precipitation to eliminate salts that interfere with 2-DE or liquid-phase IEF.

Limitations of the technique

The number of proteins identified by this protocol is relatively low, but the number of identifications and the coverage of the proteomic analysis can be further increased through the use of more modern instruments, whose mass accuracy and sequencing speed have increased dramatically in the last few years23.It is possible to cover a large part of the proteome, without any prefractionation steps, by employing a long gradient for liquid chromatography separations coupled with a high-resolution MS instrument that has a fast sequencing speed24.

Significance of the technique with respect to existing/alternative methods

The ability to identify and quantify proteins in the human cardiac valves, such as the mitral valve, is an important and challenging task that will help to elucidate the mechanisms of physiological/pathological processes in valve diseases. Defining the changes of the mitral valve proteome will greatly increase the understanding of the nature of the biological processes that are associated with the disease state of the tissue.

The current knowledge on the physiopathology of the mitral valve is limited and generally obtained through the analysis of individual proteins involved in specific processes, such as extracellular matrix remodeling, hemostasis, inflammation, or oxidative stress25, mainly studied at the tissue level by immunohistochemistry As anticipated in the Introduction, the minimal proteomic data available in the literature focuses on the analysis of pathological mitral valves9,10.

The lack of comprehensive proteomic studies can be ascribed to the complexity of this low-cellularity tissue that is highly rich in extracellular matrix proteins (i.e., proteoglycans, collagen, and elastin). These proteins make up around 80% of the total amount, hampering the analysis of low-abundance proteins26.

Thus, it was necessary to establish an efficient extraction protocol to maximize protein solubilization in order to describe the proteome of this tissue. This protocol allowed for the extraction of ~50 µg of protein from 1 mg of tissue. This is is a relatively low yield in comparison with "soft" tissue, such as liver (~135 µg from 1 mg of tissue), but it is sufficient to perform a protein analysis on individual samples. This is particularly relevant when defining the intra-individual variability of a phenomenon.

Furthermore, this method has the advantage of being compatible with many analytical applications. The mitral valve proteins dissolved in the proposed extraction buffer can be directly used for immunoblotting and proteomic analysis based on two-dimensional electrophoresis and liquid-phase isoelectrophoresis11,12 or, after protein precipitation to eliminate buffer component interference, for other assays, such as gel-free mass spectrometry15.

With the application of this extraction protocol, a more exhaustive characterization of the protein components of normal mitral valve tissue has been obtained through the identification of many intracellular proteins. These proteins are localized in the cytosol or in organelles, not only in the extracellular matrix, and have different molecular and biological functions. Other interesting proteins (i.e., CryAB, septin-11, FHL-1, and dermatopontin) were also identified. These proteins have unknown functions in the mitral valve, but their biological properties suggest a possible role in valve diseases.

Future applications or directions after mastering this technique

With this protocol, it is possible to correlate data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analyses. Indeed, when used together, these approaches will lead to a more comprehensive understanding of the molecular mechanisms underlying disease, from mRNA to post-translational protein modification. Thus, this method can be of interest to researchers focused on cardiac valve physiopathology. Finally, this protocol can also be applied to the porcine mitral valve, which bears a close resemblance to the human valve27 and is used as an experimental model for valve function evaluation.

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The Italian Ministry of Health supported this study (RC 2013-BIO 15). We thank Barbara Micheli for her excellent technical assistance.

Materials

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

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References

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