An Integrated Approach to Proteome Analysis: Identification of Proteins Associated with Cardiac Hypertrophy

doi:10.1006/abio.1998.2566

Analytical Biochemistry

Volume 258, Issue 1, 10 April 1998, Pages 1-18

https://doi.org/10.1006/abio.1998.2566 Get rights and content

Abstract

Hypertrophy of cardiac myocytes is a primary response of the heart to overload, and is an independent predictor of heart failure and death. Distinct cellular phenotypes are associated with hypertrophy resulting from different causes. These phenotypes have been described by others at the molecular level by analysis of gene transcription patterns. An alternative approach is the analysis of large-scale protein expression patterns (the proteome) by two-dimensional polyacrylamide gel electrophoresis. Realization of this goal requires the ability to rigorously analyze complex 2D gel images, efficiently digest individual gel isolated proteins (especially those expressed at low levels), and analyze the resulting peptides with high sensitivity for rapid database searches. We have undertaken to improve the technology and experimental approaches to these challenges in order to effectively study a cell culture model for cardiac hypertrophy. The 2D gel patterns for cell lysates from multiple samples of cardiac myocytes with or without phenylephrine-induced hypertrophy were analyzed and spots which changed in abundance with statistical significance were located. Eleven such spots were identified usingimproved procedures for in-gel digestion of silver-stained proteins and high-sensitivity mass spectrometry. The incorporation of low levels of sodium dodecyl sulfate into the digestion buffer improved peptide recovery. The combination of matrix-assisted laser desorption mass spectrometry for initial measurements and capillary liquid chromatography–ion trap mass spectrometry for peptide sequence determination yielded efficient protein identification. The integration of 2D gel image analysis and routine identification of proteins present in gels at the subpicomole level represents a general model for proteome studies relating genomic sequence with protein expression patterns.

References (91)

K. Iwaki et al.
J. Biol. Chem.
(1990)
P.H. O'Farrell
J. Biol. Chem.
(1975)
W.F. Patton
J. Chromatogr. A
(1995)
J.I. Garrels
J. Biol. Chem.
(1989)
H. Ji et al.
J. Biol. Chem.
(1993)
J.D. Lin et al.
J. Chromatogr. B Biomed. Appl.
(1995)
J.R. Yates et al.
Anal. Biochem.
(1993)
D.J.C. Pappin et al.
Curr. Biol.
(1993)
P. James et al.
Biochem. Biophys. Res. Commun.
(1993)
J.K. Eng et al.
J. Am. Soc. Mass Spectrom.
(1994)

Electrophoresis

(1995)

G.J. Hughes et al.

Electrophoresis

(1993)

J.M. Corbett et al.

Electrophoresis

(1994)

Cited by (124)

John T. Stults
2015, The Encyclopedia of Mass Spectrometry: Volume 9: Historical Perspectives, Part B: Notable People in Mass Spectrometry
A crayfish insulin-like-binding protein: Another piece in the androgenic gland insulin-like hormone puzzle is revealed
2013, Journal of Biological Chemistry
Citation Excerpt :
Proteins were separated on 4–12% NuPAGE Bis-Tris mini gels (Invitrogen) using MES running buffer according to the manufacturer's instructions. Bands were visualized by either Coomassie Brilliant Blue or silver staining (26). For immunoblot analyses, separated protein bands were transferred to a nitrocellulose membrane.
Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development.
Background: In Crustacea, a male-specific insulin-like androgenic hormone governs sexual differentiation.
Results: A crayfish insulin-like growth factor-binding protein (Cq-IGFBP) was expressed and found to bind the androgenic hormone.
Conclusion: Cq-IGFBP is part of the scheme modulating the crustacean androgenic hormone.
Significance: Cq-IGFBP is the first IGFBP found that interacts with an insulin-like gender-specific ligand linking early evolution of insulin pathways with gender.
Differential protein expression profiling of myocardial tissue in a mouse model of hypertrophic cardiomyopathy
2010, Journal of Molecular and Cellular Cardiology
Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in genes encoding sarcomere proteins. The mechanisms involved in the development of cardiac hypertrophy and heart failure remain poorly understood. Global proteomic profiling was used to study the cardiac proteome of mice predisposed to developing HCM. Hearts from three groups of mice (n = 3 hearts per group) were studied: non-transgenic (NTG) and cardiac-specific transgenic models over-expressing either the normal (TnI^WT) or a mutant cardiac troponin I gene (Gly203Ser; TnI^G203S). Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was used to identify proteins. Image analysis was performed using Progenesis SameSpots. A total of 34 proteins with at least a twofold change in the TnI^G203S mouse model were identified. Alterations were detected in components involved in energy production, Ca²⁺ handling, and cardiomyocyte structure. Expression level changes in cytoskeletal and contractile proteins were well represented in the study, including the intermediate filament protein desmin, which was further investigated in two additional physiological and pathological settings, i.e., exercise treatment, and severe heart failure in a novel double-mutant TnI-203/MHC-403 model of HCM. This study highlights the potential role of tissue proteomic profiling for mapping proteins, which may be critical in cardiac dysfunction and progression to heart failure in HCM.
A protein involved in the assembly of an extracellular calcium storage matrix
2010, Journal of Biological Chemistry
Gastroliths, the calcium storage organs of crustaceans, consist of chitin-protein-mineral complexes in which the mineral component is stabilized amorphous calcium carbonate. To date, only three proteins, GAP 65, gastrolith matrix protein (GAMP), and orchestin, have been identified in gastroliths. Here, we report a novel protein, GAP 10, isolated from the gastrolith of the crayfish Cherax quadricarinatus and specifically expressed in its gastrolith disc. The encoding gene was cloned by partial sequencing of the protein extracted from the gastrolith matrix. Based on an assembled microarray cDNA chip, GAP 10 transcripts were found to be highly (12-fold) up-regulated in premolt gastrolith disc and significantly down-regulated in the hypodermis at the same molt stage. The deduced protein sequence of GAP 10 lacks chitin-binding domains and does not show homology to known proteins in the GenBank^TM data base. It does, however, have an amino acid composition that has similarity to proteins extracted from invertebrate and ascidian-calcified extracellular matrices. The GAP 10 sequence contains a predicted signal peptide and predicted phosphorylation sites. In addition, the protein is phosphorylated and exhibits calcium-binding ability. Repeated daily injections of GAP 10 double strand RNA to premolt C. quadricarinatus resulted in a prolonged premolt stage and in the development of gastroliths with irregularly rough surfaces. These findings suggest that GAP 10 may be involved in the assembly of the gastrolith chitin-protein-mineral complex, particularly in the deposition of amorphous calcium carbonate.
Guanine deaminase functions as dihydropterin deaminase in the biosynthesis of aurodrosopterin, a minor red eye pigment of Drosophila
2009, Journal of Biological Chemistry
Dihydropterin deaminase, which catalyzes the conversion of 7,8-dihydropterin to 7,8-dihydrolumazine, was purified 5850-fold to apparent homogeneity from Drosophila melanogaster. Its molecular mass was estimated to be 48 kDa by gel filtration and SDS-PAGE, indicating that it is a monomer under native conditions. The pI value, temperature, and optimal pH of the enzyme were 5.5, 40 °C, and 7.5, respectively. Interestingly the enzyme had much higher activity for guanine than for 7,8-dihydropterin. The specificity constant (k_cat/K_m) for guanine (8.6 × 10⁶m⁻¹·s⁻¹) was 860-fold higher than that for 7,8-dihydropterin (1.0 × 10⁴m⁻¹·s⁻¹). The structural gene of the enzyme was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis as CG18143, located at region 82A1 on chromosome 3R. The cloned and expressed CG18143 exhibited both 7,8-dihydropterin and guanine deaminase activities. Flies with mutations in CG18143, SUPor-P/Df(3R)A321R1 transheterozygotes, had severely decreased activities in both deaminases compared with the wild type. Among several red eye pigments, the level of aurodrosopterin was specifically decreased in the mutant, and the amount of xanthine and uric acid also decreased considerably to 76 and 59% of the amounts in the wild type, respectively. In conclusion, dihydropterin deaminase encoded by CG18143 plays a role in the biosynthesis of aurodrosopterin by providing one of its precursors, 7,8-dihydrolumazine, from 7,8-dihydropterin. Dihydropterin deaminase also functions as guanine deaminase, an important enzyme for purine metabolism.
Application of proteomics in cardiovascular medicine
2006, International Journal of Cardiology
Proteomics is an emerging field that has the potential to uncover new therapeutic targets for the treatment and prevention of cardiovascular disease, as well as new diagnostic biomarkers for early disease detection. The basic strategy when carrying out proteomic analysis of cardiovascular disease is to compare the protein complements of diseased hearts or sera with controls. Any proteins that have altered expression between the two groups can be studied further for their involvement in disease pathogenesis. A number of steps need to be taken to identify changes in protein expression, including sample preparation, protein separation, imaging, and protein identification. Such studies are already underway in some cardiovascular conditions including dilated cardiomyopathy and atrial fibrillation. This review provides a summary of the techniques used in proteomic analysis and their application to cardiovascular research.

View all citing articles on Scopus

^☆: Snyder, A. P.

View full text

Regular ArticleAn Integrated Approach to Proteome Analysis: Identification of Proteins Associated with Cardiac Hypertrophy☆

Abstract

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. A

J. Biol. Chem.

J. Biol. Chem.

J. Chromatogr. B Biomed. Appl.

Anal. Biochem.

Curr. Biol.

Biochem. Biophys. Res. Commun.

J. Am. Soc. Mass Spectrom.

J. Biol. Chem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.

J. Chromatogr. B Biomed. Appl.

Trends Cardiovasc. Med.

J. Biol. Chem.

Curr. Opin. Cardiol.

Br. Heart J.

Circulation

Endocrine

Circ. Res.

Annu. Rev. Med.

Humangenetik

Electrophoresis

Eur. Heart J. (Suppl. D)

Electrophoresis

Electrophoresis

Circulation

Electrophoresis

Electrophoresis

Electrophoresis

Biotech. Gen. Eng. Rev.

Electrophoresis

Electrophoresis

Nat. Med.

Electrophoresis

Electrophoresis

Electrophoresis

Electrophoresis

Electrophoresis

Regular Article
An Integrated Approach to Proteome Analysis: Identification of Proteins Associated with Cardiac Hypertrophy☆