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.
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.
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.
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
- Harvest the human mitral valve as soon as possible after organ explantation (cold ischemia time of 4-12 h).
- 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.
- 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.
- Move the ascending aorta and pulmonary artery to display the left atrial roof.
- 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.
- 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.
- Identify the mitro-aortic valve continuity.
NOTE: The left ventricle contains the whole mitral valve and chords.
- Separate the anterior mitral valve leaflet from the posterior mitral valve leaflet, cutting the posterior leaflet along the insertion with the ventricle (commissure).
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- Repeat with the small pestle.
- 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.
- Keep the tube with the sample on dry ice to avoid sample thawing during the transfer.
- Calculate the net weight of the sample.
- Clean the mortar and the pestles after each sample and decontaminate them by autoclaving or heating them at 200 °C for 2 h.
- Transfer the powdered sample from the centrifuge tube to the glass tube of a homogenizer by inversion.
- 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.
- 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.
- 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.
- Repeat step 2.10.
- Recover the supernatant and combine it with the supernatant from step 2.11. Place the combined supernatant on a tube rotator for 30 min.
- Centrifuge the tube for 30 min at 13,000 x g and 4 °C.
- 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.
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: 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: 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: 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|
|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|
|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|
|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|
|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|
|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|
|P01023||Alpha 2 macroglobulin||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|
|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|
|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|
|P02760||AMBP protein precursor||x||x||x||x|
|P02763||Alpha 1 acid glycoprotein 1||x||x||x|
|P02765||Alpha 2 HS glycoprotein precursor||x|
|P02774||Vitamin D binding protein precursor||x|
|P02792||Ferritin light chain||x|
|P04075||Fructose bisphosphate aldolase A||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|
|P06576||ATP synthase subunit beta mitochondrial precursor||x||x|
|P06732||Creatine kinase M type||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|
|P07360||Complement component C8 gamma chain precursor||x|
|P07437||Tubulin beta chain||x||x||x||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|
|P08107||Heat shock 70 kDa protein 1A 1B||x||x||x||x|
|P08123||Collagen alpha 2 I chain||x||x||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|
|P08729||Keratin type II cytoskeletal 7||x|
|P09211||Glutathione S transferase P||x||x||x|
|P09493||Tropomyosin 1 alpha chain||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|
|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|
|P12814||Alpha actinin 1||x||x|
|P12829||Myosin light polypeptide 4||x|
|P12955||Xaa Pro dipeptidase||x|
|P13611||Versican core protein precursor||x||x|
|P13639||Elongation factor 2||x|
|P13716||Delta aminolevulinic acid dehydratase||x|
|P13804||Electron transfer flavoprotein subunit alpha mitochondrial precursor||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|
|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|
|P17980||26S protease regulatory subunit 6A||x|
|P17987||T complex protein 1 subunit alpha||x|
|P18428||Lipopolysaccharide binding protein precursor||x|
|P18669||Phosphoglycerate mutase 1||x|
|P19105||Myosin regulatory light chain 2||x||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|
|P20618||Proteasome subunit beta type 1 precursor||x|
|P21266||Glutathione S transferase Mu 3||x|
|P21796||Voltage dependent anion selective channel protein 1||x|
|P21980||Protein glutamine gamma glutamyltransferase 2||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|
|P23284||Peptidyl prolyl cis trans isomerase B precursor||x|
|P23381||Tryptophanyl tRNA synthetase cytoplasmic||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|
|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|
|P29692||Elongation factor 1 delta||x|
|P29966||Myristoylated alanine rich C kinase substrate||x|
|P30040||Endoplasmic reticulum protein ERp29 precursor||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|
|P34931||Heat shock 70 kDa protein 1 like||x||x|
|P34932||Heat shock 70 kDa protein 4||x|
|P35609||Alpha actinin 2||x|
|P35625||Metalloproteinase inhibitor 3||x||x|
|P35998||26S protease regulatory subunit 7||x|
|P36955||Pigment epithelium derived factor||x||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|
|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|
|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|
|P51452||Dual specificity protein phosphatase 3||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|
|P60660||Myosin light polypeptide 6||x||x|
|P60709||Actin cytoplasmic 1||x||x||x||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|
|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|
|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|
|Q03591||Complement factor H related protein 1 precursor||x|
|Q04917||14 3 3 protein eta||x||x|
|Q06323||Proteasome activator complex subunit 1||x|
|Q07960||Rho GTPase activating protein 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|
|Q15084||Protein disulfide isomerase A6 precursor||x|
|Q15113||Procollagen C endopeptidase enhancer 1 precursor||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|
|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|
|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|
|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|
|Q9UBR2||Cathepsin Z precursor||x|
|Q9UK22||F box only protein 2||x|
|Q9Y277||Voltage dependent anion selective channel protein 3||x|
|Q9Y696||Chloride intracellular channel protein 4||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.
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.
The authors have nothing to disclose.
The Italian Ministry of Health supported this study (RC 2013-BIO 15). We thank Barbara Micheli for her excellent technical assistance.
|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|
|Centrifuge||For centrifugation of 1.7 mL centrifuge tubes at 13,000 x g|
|Cryogenic gloves for liquid nitrogen|
|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|
|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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