A multidimensional proteomic approach to identify hypertrophy-associated proteins

Left ventricular hypertrophy (LVH) is a leading cause of congestive heart failure. The exact mechanisms that control cardiac growth and regulate the transition to failure are not fully understood, in part due to the lack of a complete inventory of proteins associated with LVH. We investigated the proteomic basis of LVH using the transverse aortic constriction model of pressure overload in mice coupled with a multidimensional approach to identify known and novel proteins that may be relevant to the development and maintenance of LVH. We identified 123 proteins that were differentially expressed during LVH, including LIM proteins, thioredoxin, myoglobin, fatty acid binding protein 3, the abnormal spindle-like microcephaly protein (ASPM), and cytoskeletal proteins such as actin and myosin. In addition, proteins with unknown functions were identified, providing new directions for future research in this area. We also discuss common pitfalls and strategies to overcome the limitations of current proteomic technologies. Together, the multidimensional approach provides insight into the proteomic changes that occur in the LV during hypertrophy.     

A proteomic approach to identify developmentally regulated proteins in Leishmania infantum

We used comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry methodologies to highlight and identify proteins that are differentially expressed in the intracellular stage of the parasite Leishmania donovani infantum, a causative agent of visceral leishmaniasis. During its digenetic life cycle, Leishmania alternates between the alimentary tract of the sandfly vector as an extracellular promastigote and the acidic phagolysosomes of macrophage cells as an intracellular amastigote. Proteins differentially expressed in the intracellular form of the parasite are thought to be important for intracellular survival and pathogenesis. We used narrow pH range strips for isoelectric focusing to resolve soluble proteins of both developmental stages of L. infantum. More than 62 proteins differentially expressed in amastigotes were detected among approximately 2000 protein spots resolved by 2-DE. A quadrupole time-of-flight analysis of few selected protein spots, specifically expressed in the amastigote stage, permitted the identification of two proteins, part of the energetic metabolism pathways, the isocitrate dehydrogenase and the glycolytic enzyme triosephosphate isomerase. The kinetic parameters of these two enzymes were measured in both developmental stages of the parasite and their activity was indeed found to be higher in amastigotes. These findings bring a new insight in our understanding of metabolic and energy requirements of the intracellular form of Leishmania. Comparative analysis of the proteome of both developmental stages of the protozoan parasite Leishmania should permit the identification of protein candidates for the development of vaccines and new drugs.     

A proteomic approach to identify substrates of matrix metalloproteinase-14 in human plasma

Matrix metalloproteinases (MMPs) are a family of zinc-containing endopeptidases that proteolyze extracellular matrix components as well as a variety of functional proteins. Here we describe a "degradomics" method that efficiently identifies substrates of MMP-14 in a complex protein mixture, such as plasma. Plasma proteins were incubated in the presence or absence of the MMP-14 catalytic domain and displayed on two-dimensional (2-D) gels. After a comparison of the gels, we selected 40 protein spots that reproducibly showed disparities. Upon in-gel digestion, mass determination, and peptide mass fingerprinting, we identified 15 different proteins from 31 spots. These proteins included six known substrates and nine potential substrates of MMP-14. Among the latter, the purified forms of apolipoprotein A-I, apolipoprotein E, and plasma gelsolin were cleaved in vitro by MMP-14, confirming that each of them is a novel substrate of MMP-14. These results demonstrate that our method rapidly and selectively identifies MMP-14 substrates from human plasma proteins. This method would thus constitute a powerful tool for identifying the substrates of MMPs and other proteases in highly complex mixtures of proteins and would enhance our understanding of the biological roles of these enzymes.     

Comparative proteomic approach to identify proteins involved in flooding combined with salinity stress in soybean

Salinity together with waterlogging or flooding, a condition that occurs frequently in the field, can cause severe damage to crops. Combined flooding and salinity decreases the growth and survival of plants more than either stress alone. We report here the first proteomic analysis to investigate the global effects of saline flooding on multiple metabolic pathways. Soybean seedlings at the emergence (VE) stage were treated with 100 mM NaCl and flooded with water or 100 mM sodium chloride solution for 2 days. Proteins were extracted from hypocotyl and root samples and analyzed by two-dimensional gel electrophoresis followed by MALDI-TOF, MALDI-TOF/TOF mass spectrometry or immunoblotting. A total of 43 reproducibly resolved, differentially expressed protein spots visualized by Coomassie brilliant blue staining were identified by MALDI-TOF MS. Identities of several proteins were also validated by MS/MS analysis or immunoblot analysis. Twenty-nine proteins were upregulated, eight proteins were downregulated and six spots were newly induced. The identified proteins include well-known salt and flooding induced proteins as well as novel proteins expressed by the salinity-flooding combined stress. The comparative analysis identified changes at the proteome level that are both specific and part of a common or shared response. The identification of such differentially expressed proteins provides new targets for future studies that will allow assessment of their physiological roles and significance in the response of glycophytes to a combination of flooding and salinity.     

A proteomic approach to identify phosphoproteins encoded by cDNA libraries

We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.     

A mass spectrometric approach to identify arbuscular mycorrhiza-related proteins in root plasma membrane fractions

One of the most important morphological changes occurring in arbuscular mycorrhizal (AM) roots takes place when the plant plasma membrane (PM) invaginates around the fungal arbuscular structures resulting in the periarbuscular membrane formation. To investigate whether AM symbiosis-specific proteins accumulate at this stage, two complementary MS approaches targeting the root PM from the model legume Medicago truncatula were designed. Membrane extracts were first enriched in PM using a discontinuous sucrose gradient method. The resulting PM fractions were further analysed with (i) an automated 2-D LC-MS/MS using a strong cation exchange and RP chromatography, and (ii) SDS-PAGE combined with a systematic LC-MS/MS analysis. Seventy-eight proteins, including hydrophobic ones, were reproducibly identified in the PM fraction from non-inoculated roots, representing the first survey of the M. truncatula root PM proteome. Comparison between non-inoculated and Glomus intraradices-inoculated roots revealed two proteins that differed in the mycorrhizal root PM fraction. They corresponded to an H(+)-ATPase (Mtha1) and a predicted glycosylphosphatidylinositol-anchored blue copper-binding protein (MtBcp1), both potentially located on the periarbuscular membrane. The exact role of MtBcp1 in AM symbiosis remains to be investigated.     

Using proteomic approach to identify tumor-associated proteins as biomarkers in human esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers in China. The lower survival rate of ESCC is attributed to late diagnosis and poor therapeutic efficacy; therefore, the identification of tumor-associated proteins as biomarkers for early diagnosis, and the discovery of novel targets for therapeutic intervention, seems very important for increasing the survival rate of ESCC. To identify tumor-associated proteins as biomarkers in ESCC, we have analyzed ESCC tissues and adjacent normal tissues by two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analysis. The results showed that a total of 104 protein spots with different expression levels were found on 2DE, and 47 proteins were eventually identified by MALDI-TOF MS. Among these identified proteins, 33 proteins including keratin 17 (KRT17), biliverdin reductase B (BLVRB), proteasome activator subunit 1 (PSME1), manganese superoxide dismutase (MnSOD), high-mobility group box-1(HMGB1), heat shock protein 70 (HSP70), peroxiredoxin (PRDX1), keratin 13 (KRT13), and so on were overexpressed, and 14 proteins including cystatin B (CSTB), tropomyosin 2 (TPM2), annexin 1 (ANX1), transgelin (TAGLN), keratin 19 (KRT19), stratifin (SFN), and so on were down-expressed in ESCC. Biological functions of these proteins are associated with cell proliferation, cell motility, protein folding, oxidative stress, and signal transduction. In the subsequent study using immunoassay on ESCC serum samples and tissue-array slides, two representative proteins, HSP70 and HMGB1, were selected as examples for the purpose of validation. The results showed that both HSP70 and HMGB1 can induce autoantibody response in ESCC sera and have higher expression in ESCC tissues. Especially, the frequency of antibodies to HSP70 in ESCC sera was significantly higher than that in normal human sera. The preliminary results suggest that some of these identified proteins might contribute to esophageal cell differentiation and carcinogenesis, certain proteins could be used as tumor-associated antigen (TAA) biomarkers in cancer diagnosis, and further studies on these identified proteins should provide more evidence of how these proteins are involved in carcinogenesis of ESCC.     

Inflammatory markers associated with abdominal aortic aneurysm

Purpose

Inflammation with leukocytic infiltration, degradation of extracellular matrix (ECM), and depletion of vascular smooth muscle cells (VSMC) are pathological hallmarks of abdominal aortic aneurysm (AAA). The aim of this study was to further evaluate relationships betweenAAAand inflammatory biomarkers, interleukin- 6 (IL-6), tumour necrosis factor-α (TNF-α), endothelin-1 (ET-1) and soluble urokinase-type plasminogen activator receptor (suPAR), by comparing levels in 65-year-old men with and without AAA at ultrasound screening.We also evaluated whether any biomarker can independently predict AAA at screening, and clarified potential correlations between aortic diameter and blood levels of these biomarkers.

Results

There were significant (p ? 0.05) differences between subjects with and without AAA for the following variables: p-leukocyte count (TLC) (p<0.001), p-homocysteine (p<0.001), p-TNF-α (p = 0.023), p-IL-6 (p<0.001), p-ET-1 (p = 0.002), p-suPAR (p<0.001), ankle brachial index (ABI) (p<0.001), plasma (p)-creatinine (p = 0.049), p-total cholesterol (p<0.001), p-high density lipoprotein (HDL) (p<0.001) and low density lipoprotein (LDL) cholesterol (p = 0.001), smoking habits (p<0.001), and use of antihypertensive (p<0.001) and lipid-lowering (p = 0.001) drugs. When the above variables were stepwise excluded in a logistic regression model, only p-IL-6 (p = 0.002), p-homocysteine (p = 0.015), p-HDL (p = 0.004), ABI in the right (p = 0.005) and left (p = 0.094) leg, smoking habits (p = 0.003), and antihypertensive drug use (p = 0.045), differed between groups. Significant correlations with aortic diameter existed for p-TNF-α (p = 0.028), p-IL-6 (p<0.001), p-ET-1 (p = 0.002) and p-suPAR (p<0.001) in the entire study population, and for p-TNF-α (p = 0.023), p-ET-1 (p = 0.009) and p-suPAR (p = 0.001) among men with AAA.

Conclusions

Several inflammatory biomarkers were significantly elevated and correlated with aortic diameter among 65-year old men with AAA at ultrasound screening. IL-6, homocysteine and use of antihypertensive medication remained elevated in the logistic regression model, together with known risk markers for AAA such as smoking and signs of atherosclerosis.

     

Biomechanics of abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a condition whereby the terminal aorta permanently dilates to dangerous proportions, risking rupture. The biomechanics of AAA has been studied with great interest since aneurysm rupture is a mechanical failure of the degenerated aortic wall and is a significant cause of death in developed countries. In this review article, the importance of considering the biomechanics of AAA is discussed, and then the history and the state-of-the-art of this field is reviewed--including investigations into the biomechanical behavior of AAA tissues, modeling AAA wall stress and factors which influence it, and the potential clinical utility of these estimates in predicting AAA rupture.     

A proteomic approach to identification of plutonium-binding proteins in mammalian cells

Plutonium can enter the body through different routes and remains there for decades; however its specific biochemical interactions are poorly defined. We, for the first time, have studied plutonium-binding proteins using a metalloproteomic approach with rat PC12 cells. A combination of immobilized metal ion chromatography, 2D gel electrophoresis, and mass spectrometry was employed to analyze potential plutonium-binding proteins. Our results show that several proteins from PC12 cells show affinity towards Pu⁴⁺-NTA (plutonium bound to nitrilotriacetic acid). Proteins from seven different spots in the 2D gel were identified. In contrast to the previously known plutonium-binding proteins transferrin and ferritin, which bind ferric ions, most identified proteins in our experiment are known to bind calcium, magnesium, or divalent transition metal ions. The identified plutonium interacting proteins also have functional roles in downregulation of apoptosis and other pro-proliferative processes. MetaCore™ analysis based on this group of proteins produced a pathway with a statistically significant association with development of neoplastic diseases.     

A proteomic approach to analyze salt-responsive proteins in rice leaf sheath

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to 3-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY363) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (SOD). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, SOD maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of SOD was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY363 and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY363 was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.     

Genomic and proteomic determinants of outcome in patients undergoing thoracoabdominal aortic aneurysm repair

Thoracoabdominal aortic aneurysm repair, with its requisite intraoperative mesenteric ischemia-reperfusion, often results in the development of systemic inflammatory response syndrome, multiorgan dysfunction syndrome (MODS), and death. In the present study, an adverse clinical outcome following thoracoabdominal aortic aneurysm repair was identified by blood leukocyte genomic and plasma proteomic responses. Time-dependent changes in the expression of 146 genes from blood leukocytes were observed (p < 0.001). Expression of 138 genes (p < 0.001) and the concentration of seven plasma proteins discriminated between patients who developed MODS and those who did not, and many of these differences were evident even before surgery. These findings suggest that changes in blood leukocyte gene expression and plasma protein concentrations can illuminate pathophysiological processes that are subsequently associated with the clinical sequelae of systemic inflammatory response syndrome and MODS. These changes in gene expression and plasma protein concentrations are often observed before surgery, consistent with either a genetic predisposition or pre-existing inflammatory state.     

A proteomics approach to identify the ubiquitinated proteins in mouse heart

There is a growing need for the large-scale identification of the ubiquitinated proteins and ubiquitin attachment sites. As part of this effort, we generated a transgenic mouse expressing a tagged ubiquitin in the heart. We found that the majority of ubiquitinated proteins in mouse heart are insoluble in detergent-free buffer and were chemically cleaved after methionine with CNBr. CNBr cleaved the proteins into smaller polypeptides while preserving the ubiquitin chains. Ubiquitin-conjugated polypeptides were then purified under denaturing conditions, digested with Lys-C and trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. We identified 121 proteins that were ubiquitinated in mouse heart, and we detected 33 ubiquitination sites in 21 of the proteins. Components of cardiac muscle and many mitochondrial proteins were identified as substrates for ubiquitination, strongly suggesting that proteins related to major heart functions such as contraction and energy production are under continuous quality control by the ubiquitin system.     

A machine learning approach to identify hydrogenosomal proteins in Trichomonas vaginalis

The protozoan parasite Trichomonas vaginalis is the causative agent of trichomoniasis, the most widespread nonviral sexually transmitted disease in humans. It possesses hydrogenosomes-anaerobic mitochondria that generate H(2), CO(2), and acetate from pyruvate while converting ADP to ATP via substrate-level phosphorylation. T. vaginalis hydrogenosomes lack a genome and translation machinery; hence, they import all their proteins from the cytosol. To date, however, only 30 imported proteins have been shown to localize to the organelle. A total of 226 nuclear-encoded proteins inferred from the genome sequence harbor a characteristic short N-terminal presequence, reminiscent of mitochondrial targeting peptides, which is thought to mediate hydrogenosomal targeting. Recent studies suggest, however, that the presequences might be less important than previously thought. We sought to identify new hydrogenosomal proteins within the 59,672 annotated open reading frames (ORFs) of T. vaginalis, independent of the N-terminal targeting signal, using a machine learning approach. Our training set included 57 gene and protein features determined for all 30 known hydrogenosomal proteins and 576 nonhydrogenosomal proteins. Several classifiers were trained on this set to yield an import score for all proteins encoded by T. vaginalis ORFs, predicting the likelihood of hydrogenosomal localization. The machine learning results were tested through immunofluorescence assay and immunodetection in isolated cell fractions of 14 protein predictions using hemagglutinin constructs expressed under the homologous SCSα promoter in transiently transformed T. vaginalis cells. Localization of 6 of the 10 top predicted hydrogenosome-localized proteins was confirmed, and two of these were found to lack an obvious N-terminal targeting signal.     

A proteomic approach to analyze nitrogen- and cytokinin-responsive proteins in rice roots

Nitrogen plays a central role in rice growth and development because it modulates a wide variety of processes, including cytokinin (CK) metabolism. CK-mediated signaling is also related to nitrogen metabolism. The functional relation between nitrogen and CK are extremely complex and unclear. In this study, a comparative proteomic analysis was carried out to analyze proteins regulated by nitrogen and CK in rice roots. Proteins extracted from rice roots are separated by two-dimensional polyacrylamide gel electrophoresis. Thirty-two protein spots that expressed similarly by nitrogen and CK treatments are selected for identification by mass spectrometry. Of these spots, 28 are successfully identified. These proteins were categorized into classes related to energy, metabolism, disease/defense, protein degradation, signal transduction, transposons, and unclear classification. Energy gives the largest functional category, suggesting that the glycolysis (two enzymes detected) and tricarboxylic acid cycle (six enzymes detected) are accurately regulated by nitrogen and CK, thus promoting the synthesis of amino acid. The identification of novel proteins provides new insights into the coordination of nitrogen and CK in rice. The possible role of these proteins is discussed.     

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa

Background

Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0.

Result

We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction.

Conclusion

The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.     

Using proteomic approach to identify tumor-associated antigens as markers in hepatocellular carcinoma

Many studies have demonstrated that intracellular proteins, which are involved in carcinogenesis, can provoke autoantibody responses. Therefore, autoantibodies can be used clinically for cancer detection and for proteomic analysis in identification of tumor-associated antigens (TAAs) that are potentially involved in malignant transformation. Liver cancer, especially hepatocellular carcinoma (HCC), is one of the most common tumors in the world. The majority of people with HCC will die within 1 year of its detection. This high case fatality rate can partially be attributed to a lack of diagnostic methods that allow early detection. In the present study, sera from 20 patients with HCC, 30 patients with chronic hepatitis (CH), and 30 patients with liver cirrhosis (LC) as well as sera from 10 normal individuals were used in a proteomic approach to identify HCC-related TAAs. Thirty-four immunoreactive protein spots were excised from the two-dimensional gel electrophoresis (2DE), digested with trypsin, and subsequently analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Of 34 immunoreactive protein spots, 28 were identified. Seventeen of them were not only reactive with serum antibodies in HCC but also with antibodies in pre-HCC conditions, and 11 were only reactive with serum antibodies in HCC but not with antibodies in pre-HCC conditions. In the subsequent analysis, two representative proteins, HSP60 and HSP70, were selected as examples for the validation purpose. The results from immunoassay were consistent with the data from proteomic analysis, supporting our hypothesis that proteins identified with autoantibodies that have been present in precancer conditions may be not appropriate to use as TAA markers in cancer detection.