Two-dimensional Difference Gel Electrophoresis Research Articles

A novel molecular target, superoxide dismutase 1, in ALK inhibitor-resistant lung cancer cells, detected through proteomic analysis

BackgroundsTo the best of our knowledge, there are no reports of proteomic analysis for the identification of unknown proteins involved in resistance to anaplastic lymphoma kinase (ALK) inhibitors. In this study, we investigated the proteins involved in resistance to alectinib, a representative ALK inhibitor, through proteomic analysis and the possibility of overcoming resistance. MethodsAn ALK-positive lung adenocarcinoma cell line (ABC-11) and the corresponding alectinib-resistant cell line (ABC-11/CHR2) were used. Two-dimensional difference gel electrophoresis (2D DIGE) was performed; the stained gel was scanned and the spots were analyzed using DeCyder TM2D 7.0. Mass spectrometry (MS) with the UltrafleXtreme matrix-assisted laser desorption ionization-tandem time-of-flight (MALDI-TOF/TOF) MS system was performed. For the MS/MS analysis, the samples were spotted on an AnchorChipTM 600 TF plate. The peptide masses obtained in the reflector positive mode were acquired at m/z of 400−6,000. MS/MS data were searched against the NCBI protein databases. Growth inhibition was measured using an MTT assay. The isobologram and combination index were calculated based on the median-effect analysis. Western blotting was performed using antibodies, including superoxide dismutase (SOD) 1, MET, ERK, PARP, AKT, and BRCA1. ResultsThe 2D DIGE for ABC-11 and ABC-11/CHR2 showed different expression levels in about 2,000 spots. SOD was identified from spots highly expressed in resistant strains. Western blotting also confirmed SOD1 overexpression in ABC-11/CHR2. siSOD1 enhanced the growth inhibitory effects of alectinib, increased cleaved PARP levels, and decreased pERK, pAKT, and BRCA1 levels with a combination of alectinib. In addition, the combination of LCS-1, an SOD1 inhibitor, and alectinib synergistically suppressed the growth in ABC-11/CHR2, but not in ABC-11. ConclusionsSOD1 overexpression is thought to be a mechanism for alectinib resistance, suggesting the possibility of overcoming resistance using SOD1 inhibitors.

Cryoprotectant-specific alterations in the proteome of Siberian sturgeon spermatozoa induced by cryopreservation

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability.

Plasma-based proteomic profiling identifies the distinct regulation of proteins in hyperplasia and endometrial cancer

BackgroundAmong gynaecological malignancies, endometrial cancer (EC) is the most prevalent type of uterine cancer affecting women. This study explored the proteomic profiles of plasma samples obtained from EC patients, those with hyperplasia (Hy), and a control group (CO). A combination of techniques, such as 2D-DIGE, mass spectrometry, and bioinformatics, including pathway analysis, was used to identify proteins with modified expression levels, biomarkers and their associated metabolic pathways in these groups.MethodsThirty-four patients, categorized into three groups—10 with EC, 12 with Hy, and 12 CO—between the ages of 46 and 75 years old were included in the study. Untargeted proteomic analysis was carried out using two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).ResultsIn all three groups, 114 proteins that were significantly (p ≤ 0.05 and fold change ≥ 1.5) altered were successfully identified using peptide mass fingerprints (PMFs). Compared with those in the control group (CO), the EC samples had 85 differentially expressed proteins (39 upregulated and 46 downregulated), and in the Hy group, 81 proteins were dysregulated (40 upregulated and 41 downregulated) compared to those in the CO group, while 33 proteins exhibited differential regulation (12 upregulated and 21 downregulated) in the EC plasma samples compared to those in the Hy group. Vitamin D binding protein and complement C3 distinguished Hy and EC from CO with the greatest changes in expression. Among the differentially expressed proteins identified, enzymes with catalytic activity represented the largest group (42.9%). In terms of biological processes, most of the proteins were involved in cellular processes (28.8%), followed by metabolic processes (16.7%). STRING analysis for protein interactions revealed that the significantly differentially abundant proteins in the three groups are involved in three main biological processes: signalling of complement and coagulation cascades, regulation of insulin-like growth factor (IGF) transport and uptake by insulin-like growth factor binding proteins (IGFBPs), and plasma lipoprotein assembly, remodelling, and clearance.ConclusionThe identified plasma protein markers have the potential to serve as biomarkers for differentiating between EC and Hy, as well as for early diagnosis and monitoring of cancer progression.

Proteomics of Penicillium chrysogenum for a Deeper Understanding of Lead (Pb) Metal Bioremediation.

Penicillium chrysogenum (P. chrysogenum), a ubiquitous filamentous fungus, has demonstrated remarkable potential in the bioremediation of lead-contaminated environments. Its inherent tolerance and bioaccumulation capacity for lead (Pb), coupled with its relatively rapid growth rate, make it an attractive candidate for bioremediation applications. This study aims to identify the proteomic changes in P. chrysogenuminduced by Pb metal stress and unravel the roles of identified proteins in molecular mechanisms and cellular responses. Untargeted proteomic analysis was carried out using a two-dimensional difference in gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). This study reported the identification of 43 statistically significant proteins (24 upregulated and 19 downregulated, ANOVA, p ≤ 0.05; fold change ≥1.5) in P. chrysogenum as a consequence of Pb treatment. Proteins were grouped according to their function into 18 groups from which 13 proteins were related to metabolism, 11 were related to cellular process and signaling, and 19 proteins were related to information storage and processing. The current study is considered the first report about the proteomics study of P. chrysogenum under Pb stress conditions, where upregulated proteins could better explain the mechanism of tolerance and Pb toxicity removal. Our research has provided a thorough understanding of the molecular and cellular processes involved in fungal-metal interactions, paving the way for the development of innovative molecular markers for heavy metal myco-remediation. To the best of our knowledge, this study of P. chrysogenum provides valuable insights toward growing research in comprehending the metal-microbe interactions. This will facilitate development of novel molecular markers for metal bioremediation.

Iron Supplementation Increases Tumor Burden and Alters Protein Expression in a Mouse Model of Human Intestinal Cancer.

Iron supplements are widely consumed. However, excess iron may accelerate intestinal tumorigenesis. To determine the effect of excess iron on intestinal tumor burden and protein expression changes between tumor and normal tissues, ApcMin/+ mice were fed control (adequate) and excess iron (45 and 450 mg iron/kg diet, respectively; n = 9/group) for 10 wk. Tumor burden was measured, and two-dimensional fluorescence difference gel electrophoresis was used to identify differentially expressed proteins in tumor and normal intestinal tissues. There was a significant increase (78.3%; p ≤ 0.05) in intestinal tumor burden (mm2/cm) with excess iron at wk 10. Of 980 analyzed protein spots, 69 differentially expressed (p ≤ 0.05) protein isoforms were identified, representing 55 genes. Of the isoforms, 56 differed (p ≤ 0.05) between tumor vs. normal tissues from the adequate iron group and 23 differed (p ≤ 0.05) between tumors from the adequate vs. excess iron. Differentially expressed proteins include those involved in cell integrity and adaptive response to reactive oxygen species (including, by gene ID: ANPEP, DPP7, ITGB1, PSMA1 HSPA5). Biochemical pathway analysis found that iron supplementation modulated four highly significant (p ≤ 0.05) functional networks. These findings enhance our understanding of interplay between dietary iron and intestinal tumorigenesis and may help develop more specific dietary guidelines regarding trace element intake.

Methods comparison of two-dimensional gel electrophoresis for host cell protein characterization.

Two-dimensional electrophoresis (2DE) is a gel-based protein separation method based on size and charge which is commonly used for the characterization of host cell proteins (HCPs) during drug development in biotech and pharmaceutical companies. HCPs are a heterogenous mixture of proteins produced by host cells during a biologics drug manufacturing process. Different gel electrophoresis methods including traditional 2D SDS-PAGE with silver and SYPRO Ruby fluorescent dye staining as well as two-dimensional difference gel electrophoresis (2D-DIGE) were compared for their relative abilities to characterize HCPs. SYPRO Ruby was shown to be more sensitive than silver stain in the traditional 2D gels both with and without product protein present. Silver stain also displayed a significant preference for staining acidic proteins over basic ones while SYPRO Ruby was more consistent in imaging proteins across different isoelectric points. The non-traditional method of 2D-DIGE provides high resolution and reproducibility when comparing samples with similar protein profiles but was limited in imaging HCP spots due to its narrow dynamic range. Overall, 2DE is a powerful tool to separate and characterize HCPs and is optimized by choosing the best stain or method for each specific application. Using a combination of two or more different 2DE staining methods, when possible, provides the most comprehensive coverage to support the characterization of a complex mixture like HCPs. However, in instances where only one staining method can be used, SYPRO Ruby is shown to be the more reliable, more sensitive, and easier to use traditional staining method for most HCP-based applications.

Differential Protein Expression between the Motor and Sensory Fascicles in Rat Femoral Nerve Injury.

It is important to distinguish between motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery. However, there are no biomarkers currently available for effective identification of motor or sensory fascicles. The objective of this study was to identify differentially expressed proteins between motor and sensory fascicles of rats in response to injury. The study was carried out using a rat femoral nerve injury model. A proteomic analysis was performed to detect differential protein expression using samples of bilateral motor and sensory branches of intact and injured rat femoral nerves through fluorescent two-dimensional difference gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Chi-square tests and t-tests were performed for comparison between motor or sensory nerve groups. The data identified six proteins that were differentially expressed between motor and sensory fascicles (>1.5-fold, P < 0.05), including apolipoprotein E, neurofilament light polypepticle, TEC kinase, serine protease inhibitor A3N, peroxiredoxin-2, and TPM1. The proteomic results were consistent with the mRNA expression levels of these genes as determined by quantitative reverse transcription polymerase chain reaction. Our study suggests that these proteins may play roles in nerve regeneration and repair. Importantly, apolipoprotein E and Serpina3n may serve as specific biomarkers for distinguishing motor and sensory fascicles of the peripheral nerves for nerve alignment in surgery.

Proteomics Investigation of the Impact of the Enterococcus faecalis Secretome on MCF-7 Tumor Cells.

Breast cancer is the most prevalent form of cancer among women. The microenvironment of a cancer tumor is surrounded by various cells, including the microbiota. An imbalance between microbes and their host may contribute to the development and spread of breast cancer. Therefore, the objective of this study is to investigate the influence of Enterococcus faecalis on a breast cancer cell line (MCF-7) to mimic the luminal A subtype of breast cancer, using an untargeted proteomics approach to analyze the proteomic profiles of breast cancer cells after their treatment with E. faecalis in order to understand the microbiome and its role in the development of cancer. The breast cancer cell line MCF-7 was cultured and then treated with a 10% bacterial supernatant at two time points (24 h and 48 h) at 37 °C in a humidified incubator with 5% CO2. Proteins were then extracted and separated using two-dimensional difference (2D-DIGE) gel electrophoresis, and the statistically significant proteins (p-value < 0.05, fold change > 1.5) were identified via matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The protein fingerprints showed a differential protein expression pattern in the cells treated with E. faecalis for 24 and 48 h compared with the control. We found 58 statistically significant proteins changes in the MCF-7 breast cancer cells affected by E. faecalis. Kilin and transgelin were upregulated after 24 h of treatment and could be used as diagnostic and prognostic markers for breast cancer. In addition, another protein involved in the inhibition of cell proliferation was coiled-coil domain-containing protein 154. The protein markers identified in this study may serve as possible biomarkers for breast cancer progression. This promotes their future uses as important therapeutic goals in the treatment and diagnosis of cancer and increases our understanding of the breast microbiome and its role in the development of cancer.

Identification of proteins that contribute to flour strength among elite breeding lines in a breeding program

Gluten, which is a protein aggregate specific to wheat, is crucial in determining the physical properties of wheat flour dough. The gluten is primarily composed of glutenin and gliadin, which are multigenic and comprise diverse proteoforms. Consequently, it is difficult to analyze the quality of gluten by genetic analysis alone. Here, comprehensive protein analysis of wheat cultivars/strains was performed using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) that allows proteoforms to be resolved by spot position. AlveoConsistograph deformation energy (Fb value) was used as a metric for the strength of the dough. Major proteins resolved by 2D-DIGE were identified by LC/MS/MS. We verified that the Glu-B1al allele significantly influences the physical properties of the dough. Next, excluding the influence of the Glu-B1al allele, we identified proteins that contribute to the physical properties of the dough.Two γ-gliadins and five low molecular weight glutenins were identified. A positive correlation was observed between the spot volume ratio of γ-gliadin and its corresponding Fb value. Thus, γ-gliadin exists as multiple proteoforms, one of which may contribute to the physical properties of dough. Combining the proteins identified in this study will help breeders develop high quality flour.

Phospho-DIGE Identified Phosphoproteins Involved in Pathways Related to Tumour Growth in Endometrial Cancer.

Endometrial cancer (EC) is the most common gynecologic malignancy of the endometrium. This study focuses on EC and normal endometrium phosphoproteome to identify differentially phosphorylated proteins involved in tumorigenic signalling pathways which induce cancer growth. We obtained tissue samples from 8 types I EC at tumour stage 1 and 8 normal endometria. We analyzed the phosphoproteome by two-dimensional differential gel electrophoresis (2D-DIGE), combined with immobilized metal affinity chromatography (IMAC) and mass spectrometry for protein and phosphopeptide identification. Quantities of 34 phosphoproteins enriched by the IMAC approach were significantly different in the EC compared to the endometrium. Validation using Western blotting analysis on 13 patients with type I EC at tumour stage 1 and 13 endometria samples confirmed the altered abundance of HBB, CKB, LDHB, and HSPB1. Three EC samples were used for in-depth identification of phosphoproteins by LC-MS/MS analysis. Bioinformatic analysis revealed several tumorigenic signalling pathways. Our study highlights the involvement of the phosphoproteome in EC tumour growth. Further studies are needed to understand the role of phosphorylation in EC. Our data shed light on mechanisms that still need to be ascertained but could open the path to a new class of drugs that could hinder EC growth.

Screening Serum Biomarkers for Rats Preconditioned with Hyperbaric Oxygen: Potential of Predicting Prognosis for Stroke.

Stroke is a major health concern and a leading cause of mortality and morbidity. We and other groups have documented that hyperbaric oxygen preconditioning could significantly alleviate neuronal damage in ischemia‒reperfusion models through various mechanisms. However, we found that some of the subjects did not benefit from preconditioning with hyperbaric oxygen. The preconditioning phenomenon is similar to vaccination, in which the endogenous survival system is activated to fight against further injuries. However, with vaccine inoculations, we could test for specific antibodies against the pathogens to determine if the vaccination was successful. Likewise, this experiment was carried out to explore a biomarker that can reveal the effectiveness of the preconditioning before neuronal injury occurs. Middle cerebral artery occlusion (MCAO) was used to induce focal cerebral ischemia-reperfusion injury. 2D-DIGE-MALDI-TOF-MS/MS proteomic technique was employed to screen the differentially expressed proteins in the serum of rats among the control (Con) group (MCAO model without hyperbaric oxygen (HBO) preconditioning), hyperbaric oxygen protective (HBOP) group (in which the infarct volume decreased after HBO preconditioning vs. Con), and hyperbaric oxygen nonprotective (HBOU) group (in which the infarct volume remained the same or even larger after HBO preconditioning vs. Con). Candidate biomarkers were confirmed by western blot and enzyme linked immunosorbent assay (ELISA), and the relationship between the biomarkers and the prognosis of cerebral injury was further validated. Among the 15 differentially expressed protein spots detected in the HBOP group by Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), 3 spots corresponding to 3 different proteins (haptoglobin, serum albumin, and haemopexin) products were identified by MALDI-TOF-MS/MS. Serum albumin and haemopexin were upregulated, and haptoglobin was downregulated in the HBOP group (p < 0.05 vs. Con and HBOU groups). After the western blot study, only the changes in haemopexin were validated and exhibited similar changes in subjects from the HBOP group in accordance with MALDI-TOF-MS/MS proteomic analysis and enzyme linked immunosorbent assay (ELISA) analysis. The serum level of the hemopexin (HPX) at 2 h after HBO preconditioning was correlated with the infarct volume ratio after MCAO. Haemopexin may be developed as a predictive biomarker that indicated the effectiveness of a preconditioning strategy against cerebral ischaemic injury.

Carbonylated Proteins as Key Regulators in the Progression of Metabolic Syndrome.

Based on the known role of oxidative stress in the pathogenesis and progression of metabolic syndrome, we used two-dimensional gel electrophoresis with immunochemical detection of protein carbonyls (2D-Oxyblot) to characterize the carbonylated proteins induced by oxidative stress in spontaneously hypertensive rats/NDmcr-cp (CP), an animal model of metabolic syndrome. We also profiled the proteins that showed change of expression levels in their epididymal adipose tissue at the pre-symptomatic (6-week-old) and the symptomatic (25-week-old) stages of the metabolic syndrome. Two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) was used to analyze proteins extracted from the epididymal adipose tissue. The up-regulated proteins identified at the pre-symptomatic stage were mainly associated with ATP production and redox reaction, while the down-regulated proteins found at the symptomatic stage were involved in antioxidant activity and the tricarboxylic acid (TCA) cycle. Further analysis using the 2D-Oxyblot showed significantly high carbonylation levels of gelsolin and glycerol-3-phosphate dehydrogenase [NAD+] at the symptomatic stage. These results suggest that reduced antioxidant capacity underlies the increased oxidative stress state in the metabolic syndrome. The identified carbonylated proteins, including gelsolin, are potential targets that may act as key regulators in the progression of the metabolic syndrome.

A Practical and Analytical Comparative Study of Gel-Based Top-Down and Gel-Free Bottom-Up Proteomics Including Unbiased Proteoform Detection.

Proteomics is an indispensable analytical technique to study the dynamic functioning of biological systems via different proteins and their proteoforms. In recent years, bottom-up shotgun has become more popular than gel-based top-down proteomics. The current study examined the qualitative and quantitative performance of these two fundamentally different methodologies by the parallel measurement of six technical and three biological replicates of the human prostate carcinoma cell line DU145 using its two most common standard techniques, label-free shotgun and two-dimensional differential gel electrophoresis (2D-DIGE). The analytical strengths and limitations were explored, finally focusing on the unbiased detection of proteoforms, exemplified by discovering a prostate cancer-related cleavage product of pyruvate kinase M2. Label-free shotgun proteomics quickly yields an annotated proteome but with reduced robustness, as determined by three times higher technical variation compared to 2D-DIGE. At a glance, only 2D-DIGE top-down analysis provided valuable, direct stoichiometric qualitative and quantitative information from proteins to their proteoforms, even with unexpected post-translational modifications, such as proteolytic cleavage and phosphorylation. However, the 2D-DIGE technology required almost 20 times as much time per protein/proteoform characterization with more manual work. Ultimately, this work should expose both techniques' orthogonality with their different contents of data output to elucidate biological questions.

Downregulation of Heat Shock Protein 72 Contributes to Fibrostenosis in Crohn's Disease.

Crohn's disease (CD) with recurrent inflammation can cause intestinal fibrostenosis due to dysregulated deposition of extracellular matrix. However, little is known about the pathogenesis of fibrostenosis. Here, we performed a differential proteomic analysis between normal, inflamed, and fibrostenotic specimens of patients with CD and investigated the roles of the candidate proteins in myofibroblast activation and fibrosis. We performed two-dimensional difference gel electrophoresis and identified candidate proteins using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and orbitrap liquid chromatography-mass spectrometry. We also verified the levels of candidate proteins in clinical specimens and examined their effects on 18Co myofibroblasts and Caco-2 intestinal epithelial cells. We identified five of 30 proteins (HSP72, HSPA5, KRT8, PEPCK-M, and FABP6) differentially expressed in fibrostenotic CD. Among these proteins, the knockdown of heat shock protein 72 (HSP72) promoted the activation and wound healing of myofibroblasts. Moreover, knockdown of HSP72 induced the epithelial-mesenchymal transition of intestinal epithelial cells by reducing E-cadherin and inducing fibronectin and α-smooth muscle actin, which contribute to fibrosis. HSP72 is an important mediator that regulates myofibroblasts and epithelial-mesenchymal transition in fibrosis of CD, suggesting that HSP72 can serve as a target for antifibrotic therapy.

In Vitro Exposure to Glucose Alters the Expression of Phosphorylated Proteins in Platelets

Diabetes mellitus (DM) is a pro-thrombotic state that can potentially cause serious cardiovascular complications. Platelet hyperactivation plays an important role in these pathological processes, however there is little or no information on the effect of hyperglycemia on platelet proteins. The aim of this study was to identify the molecular targets associated with platelet reactivity under hyperglycemia. Towards this goal, we examined the effects of the exposure of platelets to 1 and 2 h glucose (300 mg/dL) and control (vehicle and osmolality control using mannitol) on platelet proteins (n = 4 samples per group) using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) combined with MALDI-TOF/TOF tandem mass spectrometry. Two-hour exposure to glucose significantly up-regulated the expression of ATP synthase subunit beta, filamin-A, and L-lactate dehydrogenase A chain in platelets. Pro-Q Diamond staining confirmed the effect of 2 h glucose on vinculin, heat shock protein HSP 90-alpha, filamin-A, and fructose-bisphosphate aldolase A (platelet phosphorylated proteins). The identified proteins are involved in various cellular processes and functions and possibly in platelet reactivity under hyperglycemic conditions.

Urinary Proteome Differences in Patients with Type 2 Diabetes Pre and Post Liraglutide Treatment.

Diabetes mellitus is a chronic multisystem disease with a high global prevalence. The glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide is known to lower glucose levels and reduce weight. However, the mechanisms underlying the benefits of liraglutide treatment in patients with type 2 diabetes mellitus (T2DM) remain unclear. Twelve male patients with T2DM (pre and post liraglutide treatment) and HbA1c between 8% and 11% were recruited. In the present study, a two-dimensional difference gel electrophoresis (2D-DIGE) matrix-assisted laser desorption/ionization-time of flight (MALDI TOF) mass spectrometric approach combined with bioinformatics and network pathway analysis was used to explore the urine proteomic profile. The mean age of the patients was 52.4 ± 7.5 years. After treatment with liraglutide, a statistically significant change (p < 0.006) was observed in HbA1c with no significant changes in body weight or markers of dyslipidemia. Two-dimensional difference gel electrophoresis identified significant changes (≥1.5-fold change, ANOVA, p ≤ 0.05) in 32 proteins (4 down- and 28 upregulated) in liraglutide post treatment compared to the pre-treatment state. Albumin, serotransferrin, metallothionein-2 (MT-2), and keratins K1 and K10 were found to be upregulated after liraglutide treatment. The patients showed significant improvement in glycemic control after the 12-week treatment with liraglutide. The renoprotective effect of liraglutide may be linked to the increased urinary abundance of MT-2 and the decreased abundance of zinc alpha 2-glycoprotein (ZAG) and Alpha-1 antitrypsin (α1-AT). More studies are needed to elucidate the molecular mechanisms behind the renoprotective effects of liraglutide.

A characteristic N-glycopeptide signature associated with diabetic cognitive impairment identified in a longitudinal cohort study

BackgroundIdentifying a biomarker for the decline in cognitive function in patients with diabetes is important. Therefore, we aimed to identify the N-glycopeptides on plasma proteins associated with diabetic cognitive impairment in participants in a longitudinal study using N-glycoproteomics. MethodsWe used samples from the 3-year SONIC (Septuagenarians, Octogenarians, Nonagenarians Investigation with Centenarians) longitudinal cohort study of older Japanese people in the general population. First, we placed the participants with diabetes into two groups: those that did or did not have cognitive decline over a 6-year period. Next, their plasma protein profiles were compared between baseline and the 6-year time point using two-dimensional fluorescence difference gel electrophoresis. Finally, an N-glycoproteomic study of the focused proteins was performed using an enrichment technique and liquid chromatography-tandem mass spectrometry. ResultsApproximately 500 N-glycopeptides, derived from 18 proteins, were identified in each sample, from among which we identified the N-glycopeptides that were associated with diabetic cognitive impairment using multivariate analysis. We found that N-glycopeptides with sialylated tri- or tetra-antennary glycans on alpha-2-macroglobulin, clusterin, serum paraoxonase/arylesterase 1, and haptoglobin were less abundant, whereas 3-sialylated tri-antennary N-glycopeptides on serotransferrin were more abundant. ConclusionN-glycopeptides with sialylated multi-antennary glycans comprise a characteristic signature associated with diabetic cognitive impairment. General significanceThe characterized N-glycopeptides represent potential biomarker candidates for diabetic cognitive impairment.

Panomics reveals patient individuality as the major driver of colorectal cancer progression

BackgroundColorectal cancer (CRC) is one of the most prevalent cancers, with over one million new cases per year. Overall, prognosis of CRC largely depends on the disease stage and metastatic status. As precision oncology for patients with CRC continues to improve, this study aimed to integrate genomic, transcriptomic, and proteomic analyses to identify significant differences in expression during CRC progression using a unique set of paired patient samples while considering tumour heterogeneity.MethodsWe analysed fresh-frozen tissue samples prepared under strict cryogenic conditions of matched healthy colon mucosa, colorectal carcinoma, and liver metastasis from the same patients. Somatic mutations of known cancer-related genes were analysed using Illumina's TruSeq Amplicon Cancer Panel; the transcriptome was assessed comprehensively using Clariom D microarrays. The global proteome was evaluated by liquid chromatography-coupled mass spectrometry (LC‒MS/MS) and validated by two-dimensional difference in-gel electrophoresis. Subsequent unsupervised principal component clustering, statistical comparisons, and gene set enrichment analyses were calculated based on differential expression results.ResultsAlthough panomics revealed low RNA and protein expression of CA1, CLCA1, MATN2, AHCYL2, and FCGBP in malignant tissues compared to healthy colon mucosa, no differentially expressed RNA or protein targets were detected between tumour and metastatic tissues. Subsequent intra-patient comparisons revealed highly specific expression differences (e.g., SRSF3, OLFM4, and CEACAM5) associated with patient-specific transcriptomes and proteomes.ConclusionOur research results highlight the importance of inter- and intra-tumour heterogeneity as well as individual, patient-paired evaluations for clinical studies. In addition to changes among groups reflecting CRC progression, we identified significant expression differences between normal colon mucosa, primary tumour, and liver metastasis samples from individuals, which might accelerate implementation of precision oncology in the future.

Proteome derangement in malignant epithelial cells and its stroma following exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Discovering novel changes in the proteome of malignant lung epithelial cells and/or the tumor-microenvironment is paramount for diagnostic, prognostic, and/or therapy development. A time-dependent 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced mouse lung tumor model was used to screen the proteome of lung tumors. NNK-transformed human lung epithelial BEAS-2B cells were then established to evaluate the epithelial cell-specific protein changes. A duration-dependent increase of tumor burden was observed in NNK-treated mice, 2/12 (17%), 8/12 (67%), 9/12 (75%), and 10/10 (100%) at weeks 8, 12, 16, and 20 after the NNK exposure, respectively. A total of 25 differentially expressed proteins (≥ twofold change), predominantly structural, signaling, and metabolic proteins, were detected by two-dimensional difference gel electrophoresis and identified by mass spectrometry. Calregulin, ezrin, histamine releasing factor (HRF), and inorganic pyrophosphatase 1 (PPA1) exhibited changes and were further confirmed via immunoblotting. In addition, immunohistochemistry (IHC) analysis indicated upregulated E-cadherin and decreased vimentin expression in epithelial cells of tumor tissues. Acquisition of a neoplastic phenotype in NNK-transformed BEAS-2B cells was demonstrated by enhanced wound closure and increased anchorage independent colony formation. In transformed BEAS-2B cells, protein expression of E-cadherin, ezrin, and PPA1 (but not calregulin and HRF) was upregulated, as was observed in tumor tissues IHC staining using mouse lung tumor tissues further revealed that HRF upregulation was not lung epithelial cell specific. Altogether, tumorigenesis after NNK exposure may be initiated by protein dysregulation in lung epithelial cells together with proteome derangement derived from other cell types existing in the tumor-microenvironment.

040 Vildagliptin administration alters mouse skin proteome

Bullous pemphigoid (BP) is a subcutaneous blistering skin disease in which IgG autoantibodies are formed against the hemidesmosomal protein BP180. Epidemiological studies have revealed a strong association between an increased risk of BP and the use of dipeptidyl peptidase 4 inhibitors (DPP4i/gliptins), especially vildagliptin. We performed a proteomic study on vildagliptin-exposed mice to reveal factors for the development of DPP4i-associated BP. We administered vildagliptin via drinking water to mice (control group, n = 5, treatment group n = 6) for three months and used minimum two-dimensional difference gel electrophoresis (2D-DIGE) to study the effects of vildagliptin on the skin proteome.