Gel-Based Proteomic Identification of Suprabasin as a Potential New Candidate Biomarker in Endometrial Cancer.

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer.

Serum-Based Proteomics Profiling in Adult Patients with Cystic Fibrosis.

Cystic fibrosis (CF), the most common lethal autosomal recessive disorder among Caucasians, is caused by mutations in the CF transmembrane conductance regulator (CFTR) chloride channel gene. Despite significant advances in the management of CF patients, novel disease-related biomarkers and therapies must be identified. We performed serum proteomics profiling in CF patients (n = 28) and healthy subjects (n = 10) using the 2D-DIGE MALDI-TOF proteomic approach. Out of a total of 198 proteins identified, 134 showed a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p < 0.05), including 80 proteins with increased abundance and 54 proteins with decreased abundance in CF patients. A multiple reaction monitoring-mass spectrometry analysis of six differentially expressed proteins identified by a proteomic approach (DIGE-MALD-MS) showed a significant increase in C3 and CP proteins and a decrease in APOA1, Complement C1, Hp, and RBP4proteins compared with healthy controls. Fifteen proteins were identified as potential biomarkers for CF diagnosis. An ingenuity pathway analysis of the differentially regulated proteins indicates that the central nodes dysregulated in CF subjects involve pro-inflammatory cytokines, ERK1/2, and P38 MAPK, which are primarily involved in catalytic activities and metabolic processes. The involved canonical pathways include those related to FXR/RXR, LXR/RXR, acute phase response, IL12, nitric oxide, and reactive oxygen species in macrophages. Our data support the current efforts toward augmenting protease inhibitors in patients with CF. Perturbations in lipid and vitamin metabolism frequently observed in CF patients may be partly due to abnormalities in their transport mechanism.

A Quantitative Proteomics Study of Early Heat-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Identified OsUBP21 as a Negative Regulator of Heat Stress Responses in Rice.

To understand the early heat shock (HS)-regulated cellular responses that influence the tolerance of rice plant to high environmental temperatures, two-dimensional difference gel electrophoresis (2D-DIGE) is performed to explore the early HS-regulated proteome. Multiple proteins that show abundance changes after 1 and 5 min of HS treatment are identified. Of the early HS-regulated proteins identified, the abundance of a ubiquitin-specific protease, OsUBP21, and its Arabidopsis homolog, AtUBP13, is found to be upregulated by 5 min of HS treatment. Further, knocking the expression of OsUBP21 or AtUBP13 down or out increases the tolerance of rice and Arabidopsis plants to HS stress, suggesting that the function of these ubiquitin-specific proteases in regulating plant HS responses is conserved between monocots and dicots. 2D-DIGE showed a group of proteins are differentially regulated in wild-type and ubp21 mutant after 30 min of HS treatment. Among these proteins, 11 are found to interact directly with OsUBP21; thus, they may be targets of OsUBP21. Future analyses of the roles of these OsUBP21-interacting proteins in plant HS responses will help reveal the protein ubiquitination/deubiquitination-regulated cellular responses induced by HS in rice.

Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding gelsolin to modulate the oligomerization of VDAC complexes and cell survival.

Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used two-dimensional difference gel electrophoresis analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patients' fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes.

A 2D-DIGE-based proteomic analysis brings new insights into cellular responses of Pseudomonas putida KT2440 during polyhydroxyalkanoates synthesis.

BACKGROUND: Polyhydroxyalkanoates (PHAs) have attracted much attention in recent years as natural alternatives to petroleum-based synthetic polymers that can be broadly used in many applications. Pseudomonas putida KT2440 is a metabolically versatile microorganism that is able to synthesize medium-chain-length PHAs (mcl-PHAs). The phenomena that drive mcl-PHAs synthesis and accumulation seems to be complex and are still poorly understood. Therefore, here we determine new insights into cellular responses of Pseudomonas putida KT2440 during biopolymers production using two-dimensional difference gel-electrophoresis (2D-DIGE) followed by MALDI TOF/TOF mass spectrometry. RESULTS: The maximum mcl-PHAs content in Pseudomonas putida KT2440 cells was 24% of cell dry weight (CDW) and was triggered by nitrogen depletion. Proteomic analysis allowed the detection of 150 and 131 protein spots differentially regulated at 24 h and 48 h relative to the cell growth stage (8 h), respectively. From those, we successfully identified 84 proteins that had altered expression at 24 h and 74 proteins at 48 h of the mcl-PHAs synthesis process. The protein-protein interactions network indicated that the majority of identified proteins were functionally linkage. The abundance of proteins involved in carbon metabolism were significantly decreased at 24 h and 48 h of the cultivations. Moreover, proteins associated with ATP synthesis were up-regulated suggesting that the enhanced energy metabolism was necessary for the mcl-PHAs accumulation. Furthermore, the induction of proteins involved in nitrogen metabolism, ribosome synthesis and transport was observed. Our results indicate that mcl-PHAs accumulated in the bacterial cells changed the protein abundance involved in stress response and cellular homeostasis. CONCLUSIONS: The presented data allow us to investigate time-course proteome rearrangement in response to nitrogen limitation and biopolyesters accumulation. Our results have pointed out novel proteins that might take part in cellular responses of mcl-PHA-accumulated bacteria. The study provides an additional knowledge that could be helpful to improve the efficiency of the bioprocess and make it more economically feasible.

ECM Remodeling in Breast Cancer with Different Grade: Contribution of 2D-DIGE Proteomics.

Tumor extracellular matrix (ECM) plays a pivotal role in outcome of breast cancer (BC) patients. Overexpression of 58 genes, encoding 43 structural ECM proteins, has been identified to determine a specific cluster of BC with accelerated metastatic potential only in the undifferentiated (grade III) phenotype. The scope of this study is to characterize protein repertoire able to predict patient outcome in BC according to ECM gene expression pattern and histological grade. The differential proteomic analysis is based on 2D-differential gel electrophoresis, MALDI-MS, bioinformatics, and immunoblotting. Results suggest a relationship among ECM remodeling, signal mechanotransduction, and metabolic rewiring in BCs characterized by a specific mRNA ECM signature and identified a set of dysregulated proteins characteristic of hormone receptors expression as fibrinogen-ß chain, collagen α-1(VI) chain, and α-1B-glycoprotein. Furthermore, in triple negative tumors with ECM signature, the FGG and α5ß1/αvß3 integrins increase whereas detyrosinated α-tubulin and mimecan decrease leading to unorganized integrin presentation involving focal adhesion kinase, activation of Rho GTPases associated to epithelial mesenchymal transition. In hormone receptors negative BCs characterized by a specific ECM gene cluster, the differentially regulated proteins, identified in the present study, can be potentially relevant to predict patient's outcome.

2D-DIGE comparative proteomic analysis of developing wheat grains under high-nitrogen fertilization revealed key differentially accumulated proteins that promote storage protein and starch biosyntheses.

Nitrogen (N) serves as a macronutrient that is essential to plant growth and development, and significantly influences storage protein and starch biosyntheses and, ultimately, grain yield and quality. In this study, we performed the first comparative proteomic analysis of developing wheat grains under high-N conditions using 2D-DIGE and tandem mass spectrometry. High-N fertilizer application caused significant increases in ear number, ear grain number, and grain yield. 2D-DIGE identified 142 differentially accumulated proteins (DAPs) during grain development in the elite Chinese bread wheat cultivar Zhongmai 175, of which 132 (93%) were identified by MALDI-TOF/TOF-MS, representing 92 unique proteins. These proteins are involved mainly in energy, N and protein metabolism, carbon metabolism, and starch biosynthesis. Subcellular localization prediction and fluorescence confocal microscopic analysis showed that the DAPs identified were localized mainly in the cytosol and chloroplast. Principal component analysis (PCA) revealed a greater proteomic difference among grain developmental periods than between the high-N and control groups. Protein-protein interaction analysis highlighted a complex network centered around enzymes involved in energy, N and protein metabolism, and starch biosynthesis. Six key DAP genes showed expression patterns consistent with their protein accumulation trends during grain development. A putative metabolic pathway was proposed, with synergistic regulatory networks of grain storage protein and starch biosyntheses in response to high-N application.

Comparative Study of Early Cold-Regulated Proteins by Two-Dimensional Difference Gel Electrophoresis Reveals a Key Role for Phospholipase Dα1 in Mediating Cold Acclimation Signaling Pathway in Rice.

To understand the early signaling steps that regulate cold responses in rice, two-dimensional difference gel electrophoresis (2-D DIGE)(1)was used to study early cold-regulated proteins in rice seedlings. Using mass spectrometry, 32 spots, which represent 26 unique proteins that showed an altered expression level within 5 min of cold treatment were identified. Among these proteins, Western blot analyses confirmed that the cellular phospholipase D α1 (OsPLDα1) protein level was increased as early as 1 min after cold treatment. Genetic studies showed that reducing the expression ofOsPLDα1makes rice plants more sensitive to chilling stress as well as cold acclimation increased freezing tolerance. Correspondingly, cold-regulated proteomic changes and the expression of the cold-responsive C repeat/dehydration-responsive element binding 1 (OsDREB1) family of transcription factors were inhibited in thepldα1mutant. We also found that the expression ofOsPLDα1is directly regulated by OsDREB1A. This transcriptional regulation ofOsPLDα1could provide positive feedback regulation of the cold signal transduction pathway in rice. OsPLDα1 hydrolyzes phosphatidylcholine to produce the signal molecule phosphatidic acid (PA). By lipid-overlay assay, we demonstrated that the rice cold signaling proteins, MAP kinase 6 (OsMPK6) and OsSIZ1, bind directly to PA. Taken together, our results suggest that OsPLDα1 plays a key role in transducing cold signaling in rice by producing PA and regulatingOsDREB1s' expression by OsMPK6, OsSIZ1, and possibly other PA-binding proteins.

Proteomic identification of rainbow trout blood plasma proteins and their relationship to seminal plasma proteins.

The characterisation of fish blood proteomes is important for comparative studies of seminal and blood proteins as well as for the analysis of fish immune mechanisms and pathways. In this study, LC-MS/MS and 2D-DIGE were applied to compare rainbow trout seminal (SP) and blood plasma (BP) proteomes. The 54 differentially abundant proteins identified in SP are involved in a variety of signalling pathways, including protein ubiquitination, liver X receptor/retinoid X receptor (LXR/RXR) and farnesoid X receptor activation, cell cycle and acute phase signalling. These findings may indicate the prevalence of acute phase signalling pathways in trout SP, and its essential role in protecting spermatozoa and reproductive tissues. Our study provides the first in-depth analysis of the trout BP proteome, with a total of 119 proteins identified. The major proteins of rainbow trout BP were recognised as acute phase proteins. Analysis of BP proteins indicated that acute phase response signalling, the complement system, liver X receptor/retinoid X receptor and farnesoid X receptor activation and the coagulation system are the top canonical pathways. This study enhances knowledge of the blood origin of trout SP proteins and understanding of fish reproductive biology. Our results provide new insight into blood proteins specifically important for fish physiology and innate immunity. The mass spectrometry data are available via ProteomeXchange with the identifier PXD005988 and https://doi.org/10.6019/PXD005988.

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is associated with several extra-pulmonary effects of which skeletal muscle wasting is one of the most common and contributes to reduced quality of life, increased morbidity and mortality. The molecular mechanisms leading to muscle wasting are not fully understood. Proteomic analysis of human skeletal muscle is a useful approach for gaining insight into the molecular basis for normal and pathophysiological conditions. METHODS: To identify proteins involved in the process of muscle wasting in COPD, we searched differentially expressed proteins in the vastus lateralis of COPD patients with low fat free mass index (FFMI), as a surrogate of muscle mass (COPDL, n = 10) (FEV1 33 ± 4.3% predicted, FFMI 15 ± 0.2 Kg.m-2), in comparison to patients with COPD and normal FFMI (COPDN, n = 8) and a group of age, smoking history, and sex matched healthy controls (C, n = 9) using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) technology, combined with mass spectrometry (MS). The effect of silencing DOT1L protein expression on markers of cell arrest was analyzed in skeletal muscle satellite cells (HSkMSCs) in vitro and assessed by qPCR and Western blotting. RESULTS: A subset of 7 proteins was differentially expressed in COPDL compared to both COPDN and C. We found an increased expression of proteins associated with muscle homeostasis and protection against oxidative stress, and a decreased expression of structural muscle proteins and proteins involved in myofibrillogenesis, cell proliferation, cell cycle arrest and energy production. Among these was a decreased expression of the histone methyltransferase DOT1L. In addition, silencing of the DOT1L gene in human skeletal muscle satellite cells in vitro was significantly related to up regulation of p21 WAF1/Cip1/CDKN1A, a marker of cell arrest and ageing. CONCLUSIONS: 2D-DIGE coupled with MS identified differences in the expression of several proteins in the wasted vastus lateralis that are relevant to the disease process. Down regulation of DOT1L in the vastus lateralis of COPDL patients may mediate the muscle wasting process through up regulation of markers of cell arrest and senescence.

A Two-Dimensional Difference Gel Electrophoresis (2D-DIGE) Protocol for Studies of Neural Precursor Cells.

This chapter describes the basics of two-dimensional difference gel electrophoresis (2D-DIGE) for multiplex analysis of up to distinct proteomes. The example given describes the analysis of undifferentiated and differentiated neural precursor cells labelled with fluorescent Cy3 and Cy5 dyes in comparison to a pooled standard labelled with Cy2. After labelling, the proteomes are mixed together and electrophoresed on the same 2D gels. Scanning the gels at wavelengths specific for each dye allows direct overlay of the two different proteomes and the differences in abundance of specific protein spots can be determined through comparison to the pooled standard.

Short communication: Comparative proteomic analysis of the lactating and nonlactating bovine mammary gland.

The objective of this study was to determine how bovine mammary protein profiles vary during lactation and the dry period. Three lactating and 3 nonlactating cows were selected for mammary gland tissue sampling. Compared with the mammary proteins in nonlactating cows, a total of 60 differentially expressed proteins (DEP, including 57 upregulated and 3 downregulated) were identified in lactating cows using 2-dimensional difference gel electrophoresis combined with mass spectrometry. These DEP included enzymes and proteins associated with various macromolecular metabolic processes, and appeared to promote the increased metabolic activity associated with milk synthesis and secretion. The increased DEP were primarily related to initiation, maintenance, and involution of lactation, and included proteins involved in glycolysis/gluconeogenesis, the tricarboxylic acid cycle, the pentose phosphate pathway, oxidative phosphorylation, aminoacyl-transfer RNA biosynthesis, and fatty acid biosynthesis. Identified DEP were further validated by real-time, reverse-transcription PCR and Western blot. Five new DEP associated with lactation were uniquely identified. This work provided some protein-associated insights to facilitate further investigation of the mechanisms underlying lactation in dairy cows.

Revealing oxidative damage to enzymes of carbohydrate metabolism in yeast: An integration of 2D DIGE, quantitative proteomics, and bioinformatics.

Clinical usage of lidocaine, a pro-oxidant has been linked with severe, mostly neurological complications. The mechanism(s) causing these complications is independent of the blockade of voltage-gated sodium channels. The budding yeast Saccharomyces cerevisiae lacks voltage-gated sodium channels, thus provides an ideal system to investigate lidocaine-induced protein and pathway alterations. Whole-proteome alterations leading to these complications have not been identified. To address this, S. cerevisiae was grown to stationary phase and exposed to an LC50 dose of lidocaine. The differential proteomes of lidocaine treatment and control were resolved 6 h post exposure using 2D DIGE. Amine reactive dyes and carbonyl reactive dyes were used to assess protein abundance and protein oxidation, respectively. Quantitative analysis of these dyes (⩾ 1.5-fold alteration, p ⩽ 0.05) revealed a total of 33 proteoforms identified by MS differing in abundance and/or oxidation upon lidocaine exposure. Network analysis showed enrichment of apoptotic proteins and cell wall maintenance proteins, while the abundance of proteins central to carbohydrate metabolism, such as triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase, and redox proteins superoxide dismutase and peroxiredoxin were significantly decreased. Enzymes of carbohydrate metabolism, such as phosphoglycerate kinase and enolase, the TCA cycle enzyme aconitase, and multiple ATP synthase subunits were found to be oxidatively modified. Also, the activity of aconitase was found to be decreased. Overall, these data suggest that toxic doses of lidocaine induce significant disruption of glycolytic pathways, energy production, and redox balance, potentially leading to cell malfunction and death.

Phosphoproteomic analysis of wild-type and antimony-resistant Leishmania braziliensis lines by 2D-DIGE technology.

Protein phosphorylation is one of the most studied post-translational modifications that is involved in different cellular events in Leishmania. In this study, we performed a comparative phosphoproteomics analysis of potassium antimonyl tartrate (SbIII)-resistant and -susceptible lines of Leishmania braziliensis using a 2D-DIGE approach followed by MS. In order to investigate the differential phosphoprotein abundance associated with the drug-induced stress response and SbIII-resistance mechanisms, we compared nontreated and SbIII-treated samples of each line. Pair wise comparisons revealed a total of 116 spots that showed a statistically significant difference in phosphoprotein abundance, including 11 and 34 spots specifically correlated with drug treatment and resistance, respectively. We identified 48 different proteins distributed into seven biological process categories. The category "protein folding/chaperones and stress response" is mainly implicated in response to SbIII treatment, while the categories "antioxidant/detoxification," "metabolic process," "RNA/DNA processing," and "protein biosynthesis" are modulated in the case of antimony resistance. Multiple sequence alignments were performed to validate the conservation of phosphorylated residues in nine proteins identified here. Western blot assays were carried out to validate the quantitative phosphoproteome analysis. The results revealed differential expression level of three phosphoproteins in the lines analyzed. This novel study allowed us to profile the L. braziliensis phosphoproteome, identifying several potential candidates for biochemical or signaling networks associated with antimony resistance phenotype in this parasite.

Candidate prioritization for low-abundant differentially expressed proteins in 2D-DIGE datasets.

BACKGROUND: Two-dimensional differential gel electrophoresis (2D-DIGE) provides a powerful technique to separate proteins on their isoelectric point and apparent molecular mass and quantify changes in protein expression. Abundantly available proteins in spots can be identified using mass spectrometry-based approaches. However, identification is often not possible for low-abundant proteins. RESULTS: We present a novel computational approach to prioritize candidate proteins for unidentified spots. Our approach exploits noisy information on the isoelectric point and apparent molecular mass of a protein spot in combination with functional similarities of candidate proteins to already identified proteins to select and rank candidates. We evaluated our method on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. Using leave-one-out cross-validation, we show that the true-positive rate for the top-5 ranked proteins is 43.8%. CONCLUSIONS: Our approach shows good performance on a 2D-DIGE dataset comparing protein expression in uninfected and HIV-1 infected T-cells. We expect our method to be highly useful in (re-)mining other 2D-DIGE experiments in which especially the low-abundant protein spots remain to be identified.

Proteomics analysis of malignant and benign prostate tissue by 2D DIGE/MS reveals new insights into proteins involved in prostate cancer.

BACKGROUND: The key to a more effective diagnosis, prognosis, and therapeutic management of prostate cancer (PCa) could lie in the direct analysis of cancer tissue. In this study, by comparative proteomics analysis of PCa and benign prostate hyperplasia (BPH) tissues we attempted to elucidate the proteins and regulatory pathways involved in this disease. METHODS: The samples used in this study were fresh surgical tissues with clinically and histologically confirmed PCa (n = 19) and BPH (n = 33). We used two dimensional difference in gel electrophoresis (2D DIGE) coupled with mass spectrometry (MS) and bioinformatics analysis. RESULTS: Thirty-nine spots with statistically significant 1.8-fold variation or more in abundance, corresponding to 28 proteins were identified. The IPA analysis pointed out to 3 possible networks regulated within MAPK, ERK, TGFB1, and ubiquitin pathways. Thirteen of the identified proteins, namely, constituents of the intermediate filaments (KRT8, KRT18, DES), potential tumor suppressors (ARHGAP1, AZGP1, GSTM2, and MFAP4), transport and membrane organization proteins (FABP5, GC, and EHD2), chaperons (FKBP4 and HSPD1) and known cancer marker (NME1) have been associated with prostate and other cancers by numerous proteomics, genomics or functional studies. We evidenced for the first time the dysregulation of 9 proteins (CSNK1A1, ARID5B, LYPLA1, PSMB6, RABEP1, TALDO1, UBE2N, PPP1CB, and SERPINB1) that may have role in PCa. The UBE2N, PSMB6, and PPP1CB, involved in cell cycle regulation and progression were evaluated by Western blot analysis which confirmed significantly higher abundances of UBE2N and PSMB6 and significantly lower abundance of PPP1CB in PCa. CONCLUSION: In addition to the identification of substantial number of proteins with known association with PCa, the proteomic approach in this study revealed proteins not previously clearly related to PCa, providing a starting point for further elucidation of their function in disease initiation and progression.

Potential predictive plasma biomarkers for cervical cancer by 2D-DIGE proteomics and Ingenuity Pathway Analysis.

The current methods available for screening and detecting cervical squamous cell carcinoma (CSCC) have insufficient sensitivity and specificity. As a result, many patients suffered from erroneous and missed diagnosis. Because CSCC is usually asymptomatic at potentially curative stages, identification of biomarkers is an urgent need for the early detection of CSCC. Comparative proteomics based on two-dimensional differential in-gel electrophoresis (2D-DIGE) was employed to quantitatively analyze plasma proteins of healthy Uyghur women and with early stage cervical carcinoma. The 2D-DIGE image were analyzed statistically using DeCyder™ 2D software. The statistical analysis of proteomic data revealed that 43 protein spots showed significantly different expression (ratio > 1.5, P < 0.01). A further identification of these protein spots by MALDI-TOF-MS found out 16 different proteins. Bioinformatic analysis within the framework of Ingenuity Pathway Analysis (IPA(@)) showed that 10 plasma proteins as candidate biomarker were screened, mainly including lipid metabolism-related proteins (APOA4, APOA1, APOE), complement (EPPK1, CFHR1), metabolic enzymes (CP, F2, MASP2), glycoprotein (CLU), and immune function-related proteins (IGK@). Networks involved in lipid metabolism, molecular transport, and small molecule biochemistry were dysfunctional in CSCC. Acute phase response signaling and JAK/Stat signaling and IL-4 signaling, etc., were identified as the canonical pathways that are overrepresented in CSCC. Furthermore, the expression of three proteins (APOA1, APOE, CLU) were validated using ELISA in plasma of patients with different stage cervical lesion. With the combined proteomic and bioinformatic approach, this study was successful in identifying biomarker signatures for cervical cancer and might provide new insights into the mechanism of CSCC progression, potentially leading to the design of novel diagnostic and therapeutic strategies.

Proteome-wide search for PP2A substrates in fission yeast.

PP2A (protein phosphatase 2A) is a major phosphatase in eukaryotic cells that plays an essential role in many processes. PP2A mutations in Schizosaccharomyces pombe result in defects of cell cycle control, cytokinesis and morphogenesis. Which PP2A substrates are responsible for these changes is not known. In this work, we searched for PP2A substrates in S. pombe using two approaches, 2D-DIGE analysis of PP2A complex mutants and identification of PP2A interacting proteins. In both cases, we used MS to identify proteins of interest. In the DIGE experiment, we compared proteomes of wild-type S. pombe, deletion of pta2, the phosphoactivator of the PP2A catalytic subunit, and pab1-4, a mutant of B-type PP2A regulatory subunit. A total of 1742 protein spots were reproducibly resolved by 2D-DIGE and 51 spots demonstrated significant changes between PP2A mutants and the wild-type control. MS analysis of these spots identified 27 proteins that include key regulators of glycerol synthesis, carbon metabolism, amino acid biosyntesis, vitamin production, and protein folding. Importantly, we independently identified a subset of these proteins as PP2A binding partners by affinity precipitation, suggesting they may be direct targets of PP2A. We have validated our approach by demonstrating that phosphorylation of Gpd1, a key enzyme in glycerol biogenesis, is regulated by PP2A and that ability of cells to respond to osmotic stress by synthesizing glycerol is compromised in the PP2A mutants. Our work contributes to a better understanding of PP2A function and identifies potential PP2A substrates.

Protein profiles of peripheral blood mononuclear cells as a candidate biomarker for Behçet's disease.

OBJECTIVES: To investigate the pathophysiology of Behçet's disease (BD) and find biomarkers for the disease, we analysed protein profiles of peripheral blood mononuclear cells (PBMCs). METHODS: Proteins, extracted from PBMCs, were comprehensively analysed in 16 patients with BD, 16 patients with rheumatoid arthritis (RA), 12 patients with Crohn's disease (CD), and 16 healthy control subjects (HC) by 2-dimensional differential gel electrophoResis (2D-DIGE). Differently expressed proteins were identified by mass spectrometry. RESULTS: 563 protein spots were detected. We completely discriminated between the BD and HC groups, between the BD and RA groups, and between the BD and CD groups by multivariate analysis of intensity of 23, 35, and 1 spots, respectively. The spots contributing to the differences included proteins related to cytoskeleton, transcription/translation, T cell activation, bone turnover, regulating apoptosis, and microbial infection. Intensity of 3 spots (tyrosine-protein phosphatase non-receptor type 4, threonine synthase-like 2, and ß-actin) provided area under the receiver operating characteristic curves (AUROC) of 0.889 for discrimination between the BD group and the non-BD groups. Informatively, intensity of the above 1 spot completely discriminated the CD group from the other groups (AUROC 1.000). This spot, identified as ß-actin, had different pI from the above ß-actin-spot probably due to different post-translational modification. CONCLUSIONS: PBMC protein profiles, especially the profile of the 3 spots, would be candidate biomarkers for BD. The latter ß-actin subtype would be useful for discriminating inflammatory bowel diseases from BD and other diseases. The identified proteins may play important roles in the pathophysiology of BD.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eß thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.