Comprehensive Analysis Of Protein Expression Research Articles

Mass spectrometry‐based proteomics for biomarker discovery in the Drosophila model of Parkinson's disease

AbstractParkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide. Despite extensive research, the etiology of both familial and sporadic PD remains unclear. While most PD cases are sporadic, a significant minority are linked to genetic mutations, notably in the synuclein‐alpha (SNCA) and leucine‐rich repeat kinase 2 (LRRK2) genes. Animal models, such as Drosophila melanogaster (D. melanogaster), enable detailed study of these genetic mutations and their neurotoxic effects. Recent advancements in mass spectrometry‐based proteomics have enhanced our understanding of PD by facilitating comprehensive analysis of protein expression and interactions in mutant and wild‐type organisms, potentially revealing novel therapeutic targets. This review highlights the pivotal role of mass spectrometry‐based proteomics in advancing PD research, emphasizing the contributions of D. melanogaster models in identifying potential biomarkers.

Innovative thiosemicarbazones that induce multi-modal mechanisms to down-regulate estrogen-, progesterone-, androgen- and prolactin-receptors in breast cancer

The estrogen receptor-α (ER-α) is a key driver of breast cancer (BC) and the ER-antagonist, tamoxifen, is a central pillar of BC treatment. However, cross-talk between ER-α, other hormone and growth factor receptors enables development of de novo resistance to tamoxifen. Herein, we mechanistically dissect the activity of a new class of anti-cancer agents that inhibit multiple growth factor receptors and down-stream signaling for the treatment of ER-positive BC. Using RNA sequencing and comprehensive protein expression analysis, we examined the activity of di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), on the expression and activation of hormone and growth factor receptors, co-factors, and key resistance pathways in ER-α-positive BC. DpC differentially regulated 106 estrogen-response genes, and this was linked to decreased mRNA levels of 4 central hormone receptors involved in BC pathogenesis, namely ER, progesterone receptor (PR), androgen receptor (AR), and prolactin receptor (PRL-R). Mechanistic investigation demonstrated that due to DpC and Dp44mT binding metal ions, these agents caused a pronounced decrease in ER-α, AR, PR, and PRL-R protein expression. DpC and Dp44mT also inhibited activation and down-stream signaling of the epidermal growth factor (EGF) family receptors, and expression of co-factors that promote ER-α transcriptional activity, including SRC3, NF-κB p65, and SP1. In vivo, DpC was highly tolerable and effectively inhibited ER-α-positive BC growth. Through bespoke, non-hormonal, multi-modal mechanisms, Dp44mT and DpC decrease the expression of PR, AR, PRL-R, and tyrosine kinases that act with ER-α to promote BC, constituting an innovative therapeutic approach.

Long-term Changes in the Central Amygdala Proteome in Rats with a History of Chronic Cocaine Self-administration

The central nucleus of the amygdala (CeA) is a striatum-like structure that contains mainly inhibitory circuits controlling a repertoire of (mal)adaptive behaviors related to pain, anxiety, motivation, and addiction. Neural activity in the CeA is also necessary for the expression of persistent and robust drug seeking, also termed ‘incubation of drug craving.’ However, neuroadaptations within this brain region supporting incubated drug craving have not been characterized. Here, we conducted a comprehensive analysis of protein expression in the CeA of male rats after prolonged (45-day) abstinence from extended-access cocaine self-administration using a quantitative proteomic approach. The proteomic analysis identified 228 unique proteins altered in cocaine rats relative to animals that received saline. Out of the identified proteins, 160 were downregulated, while 68 upregulated. Upregulation of tyrosine hydroxylase and downregulation of neural cell-adhesion protein contactin-1 were validated by immunoblotting. Follow-up analysis by the Ingenuity Pathway Analysis tool revealed alterations in protein networks associated with several neurobehavioral disorders, cellular function and morphology, as well as axogenesis, long-term potentiation, and receptor signaling pathways. This study suggests that chronic cocaine self-administration, followed by a prolonged abstinence results in reorganization of specific protein signaling networks within the CeA that may underlie incubated cocaine craving and identifies potential novel ‘druggable’ targets for the treatment of cocaine use disorder (CUD).

Proteomics analysis of the amygdala in rats with CFA-induced pain aversion with electro-acupuncture stimulation.

BackgroundClinical patients suffering from pain usually exhibit aversion to pain-associated environments (pain aversion). Electro-acupuncture (EA) has been proven to be effective for the treatment of pain aversion in our previous studies. The amygdala could have substantial consequences on emotion and pain consolidation as well as general pain aversion behavior, however, the underlying mechanism remains unclear.PurposeThe current study was performed to investigate Isobaric tags for relative and absolute quantitation (iTRAQ) based quantitative proteomic analysis of the amygdala in rats with complete Freund’s adjuvant (CFA)-induced pain aversion, and comprehensive analysis of protein expression were performed to explore the underlying mechanism by which EA affects pain aversion.Materials and methodsInflammatory pain was induced with an intraplantar injection of 100 μL of CFA in the plantar surface of the left hind paw of the male Spragure-Dawley (SD) rats. Then the CFA-induced conditioned place aversion (C-CPA) test was performed. EA stimulation on the bilateral Zusanli and Sanyinjiao acu-points was used for 14 days and the EA stimulation frequency is 2 Hz. Based on iTRAQ-based proteomics analysis, we investigated the protein expression in the amygdala.ResultsEA can increase the paw withdrawal threshold in inflammatory pain induced by noxious stimulation. A total of 6319 proteins were quantified in amygdala. Of these identified proteins, 123 were identified in the pain aversion group relative to those in the saline group, and 125 significantly altered proteins were identified in the pain aversion + EA group relative to the pain aversion group. A total of 11 proteins were found to be differentially expressed in the amygdala of pain aversion and EA-treated rats. The expression of three proteins, glyceraldehyde-3-phosphate dehydrogenase, glutamate transporter-1, and p21-activated kinase 6, were confirmed to be consistent with the results of the proteome.ConclusionOur investigation demonstrated the possible mechanism of central nerve system by which EA intervetion on pain aversion.

411P - Comparative analysis of protein profiles of prognosis-associated proteins and KIT-related proteins in gastrointestinal stromal tumour

BackgroundGastrointestinal stromal tumor (GIST) is a type of mesenchymal tumor and has KIT mutations in 80–90% of the cases. The development of tyrosine kinase inhibitors have greatly improved patient outcomes. We have previously described association between lower pfetin (KCTD12) expression and poor prognosis in the GIST patients (Ref 1. Suenara Y et al. Clin Cancer Res2008). In this study, to investigate the protein profiles of kit expression, we performed comprehensive protein expression analysis using the gastric tumor cell lines and surgical specimens in GIST. MethodsWe performed KITgene knockdown using siRNA inthe GIST cell line (T1 cell line) and generated the protein expression profiling data by i-TRAQ. Based on this i-TRAQ protein profiling data from the T1 cell line, we referenced our previously published protein expression analysis databases associated with GIST prognosis in the patient samples (Ref 1.) and compared the protein profile of KIT-associated proteins (T1 cell line) and prognosis-associated protein (patient samples). ResultsIn our study, the i-TRAQ profiling method detected expression of approximately 1500–2000 proteins. In KITknockdown cells, 125 proteins were identified to have statistically different expression (p < 0.05). Based on our previously published GIST prognosis protein profile database (using 17 patient tissue samples), 25 proteins were identified to be statistically significant (p < 0.05). Upon comparing the prognosis database with theKIT knockdown database, 6 proteins were identified as common proteins.In particular, the heat shock protein (HSP) 90-beta, which has been previously shown to contribute to the growth of cancer cells, was identified as a critical protein in the biological behavior of GIST. ConclusionsHSP 90-beta is currently considered as a therapeutic target for HSP 90 inhibitors against imatinib-resistant GIST. Our data suggests that the differentiation and cell growth of GIST may be enhanced by the identified proteins, which are in turn induced by KIT. We believe that our established protein profile data will help in improving the understanding of malignant behaviors in GIST. Clinical trial identificationHSP 90-betaplay an important role in the tumor progression of GISTs. Legal entity responsible for the studyJuntendo University. FundingThis study was supported by Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number #19K22694, #19H03789 and #15KK0353 to Y.S. ; #17K08730 to T.S.; #17K10987to K.K.; #19K16753 to K.A.; #18K15329 to T.O., # 18K16634 to Y.K.) and Leading Advanced Projects for Medical Innovation (LEAP) (Grant Number # JP18am0001009) and the Practical Research for Innovative Cancer Control (Grant Number # JP18ck0106252) from the Japan Agency for Medical Research and Development. DisclosureAll authors have declared no conflicts of interest.

Cytokines in adipose-derived mesenchymal stem cells promote the healing of liver disease.

Adipose-derived mesenchymal stem cells (ADSCs) are a treatment cell source for patients with chronic liver injury. ADSCs are characterized by being harvested from the patient’s own subcutaneous adipose tissue, a high cell yield (i.e., reduced immune rejection response), accumulation at a disease nidus, suppression of excessive immune response, production of various growth factors and cytokines, angiogenic effects, anti-apoptotic effects, and control of immune cells via cell-cell interaction. We previously showed that conditioned medium of ADSCs promoted hepatocyte proliferation and improved the liver function in a mouse model of acute liver failure. Furthermore, as found by many other groups, the administration of ADSCs improved liver tissue fibrosis in a mouse model of liver cirrhosis. A comprehensive protein expression analysis by liquid chromatography with tandem mass spectrometry showed that the various cytokines and chemokines produced by ADSCs promote the healing of liver disease. In this review, we examine the ability of expressed protein components of ADSCs to promote healing in cell therapy for liver disease. Previous studies demonstrated that ADSCs are a treatment cell source for patients with chronic liver injury. This review describes the various cytokines and chemokines produced by ADSCs that promote the healing of liver disease.

Detection of titin fragments in urine in response to exercise-induced muscle damage.

Many studies have attempted to determine the associations between blood biomarkers and exercise-induced muscle damage. However, poor correlations between the changes in biomarker levels and the magnitude of muscle symptoms have been reported. Recent advances in proteomic tools offer a strategy for the comprehensive analysis of protein expression, which can be used to identify biomarkers. Here, we used a proteomic analysis to identify urinary proteins that appear in response to a calf-raise exercise, including repetitive eccentric muscle contractions, and found that a titin (also known as connectin) N-terminal fragment molecule appears in the urine after eccentric exercise. We measured the titin fragment in urine samples from nine individuals before and after eccentric exercise using a newly-established enzyme-linked immunosorbent assay and found that the titin fragment excretion rate increased 96 h after the exercise (5.1 to 77.6 pg/min, p <0.01). The changes in the titin fragment excretion rate were correlated strongly with blood markers of muscle damage and with muscle symptoms. These findings suggest that the urinary titin fragment is potentially a noninvasive biomarker of muscle damage.

Microengineered embryonic stem cells niche to induce neural differentiation.

A major challenge in therapeutic use of embryonic stem cells (ESCs) for treating neurodegenerative diseases is creating a niche in vitro for controlled neural-specific differentiation of ESCs. We employ a niche microengineering approach to derive neural cells from ESCs by mimicking embryonic development in terms of direct intercellular interactions. Using a polymeric aqueous two-phase system (ATPS) microprinting technology, murine ESCs (mESCs) are precisely localized over a monolayer of supporting stromal cells to allow formation of individual mESC colonies. Polyethylene glycol (PEG) and dextran (DEX) are dissolved in culture media to form two immiscible aqueous solutions. A robotic liquid handler is used to print a nanoliter-volume drop of the denser DEX phase solution containing mESCs onto a confluent layer of supporting PA6 stromal cells submerged in the aqueous PEG phase. mESCs proliferate into isolated colonies of uniform size. For the first time, a comprehensive protein expression analysis of individual mESC colonies is performed over a two-week culture period to track temporal progression of cells from a pluripotent stage to specific neural cells. Starting from day 4, the expression of nestin, neural cell adhesion molecule (NCAM), and beta-III tubulin shows a significant increase but then levels off after the first week of culture. The expression of specific neural cell markers glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and tyrosine hydroxylase (TH) is elevated during the second week of culture. This microengineering approach to control ESCs differentiation niche combined with the time-course protein expression analysis of individual differentiating colonies facilitates understanding of evolution of specific neural cells from ESCs and identifying underlying molecular markers.

Pro-Insulin-Like Growth Factor-II Ameliorates Age-Related Inefficient Regenerative Response by Orchestrating Self-Reinforcement Mechanism of Muscle Regeneration.

Sarcopenia, age-related muscle weakness, increases the frequency of falls and fractures in elderly people, which can trigger severe muscle injury. Rapid and successful recovery from muscle injury is essential not to cause further frailty and loss of independence. In fact, we showed insufficient muscle regeneration in aged mice. Although the number of satellite cells, muscle stem cells, decreases with age, the remaining satellite cells maintain the myogenic capacity equivalent to young mice. Transplantation of young green fluorescent protein (GFP)-Tg mice-derived satellite cells into young and aged mice revealed that age-related deterioration of the muscle environment contributes to the decline in regenerative capacity of satellite cells. Thus, extrinsic changes rather than intrinsic changes in satellite cells appear to be a major determinant of inefficient muscle regeneration with age. Comprehensive protein expression analysis identified a decrease in insulin-like growth factor-II (IGF-II) level in regenerating muscle of aged mice. We found that pro- and big-IGF-II but not mature IGF-II specifically express during muscle regeneration and the expressions are not only delayed but also decreased in absolute quantity with age. Supplementation of pro-IGF-II in aged mice ameliorated the inefficient regenerative response by promoting proliferation of satellite cells, angiogenesis, and suppressing adipogenic differentiation of platelet derived growth factor receptor (PDGFR)α(+) mesenchymal progenitors. We further revealed that pro-IGF-II but not mature IGF-II specifically inhibits the pathological adipogenesis of PDGFRα(+) cells. Together, these results uncovered a distinctive pro-IGF-II-mediated self-reinforcement mechanism of muscle regeneration and suggest that supplementation of pro-IGF-II could be one of the most effective therapeutic approaches for muscle injury in elderly people.

Comprehensive Protein Expression Analysis of Chronic Lymphocytic Leukemia Uncovers a Correlation of CDK4, P27 and P53 with Hierarchical Risk and Suggests a Dichotomy of Apoptosis and Proliferation.

Abstract 1255Poster Board I-277Chronic lymphocytic leukemia (CLL) has a variable clinical course. Genomic aberrations identify prognostic subgroups, pointing towards distinct underlying biological mechanisms. However, for a subset of aberrations, the affected cellular pathways are unknown. Even though the importance of posttranscriptional regulation, e.g. by micro RNAs, has become evident, the proteome of CLL remains little characterized. Therefore, the expression of 23 proteins involved in cell cycle, DNA damage, apoptosis and signaling was investigated in a total of 206 cytogenetically well characterized CLL cases by Western blotting. CLL cases were categorized according to the hierarchical model by Döhner et al. (NEJM 2000) into del(17p) (N=41), del(11q) (N=39), del(13q) (N=39) as sole abnormality, trisomy 12 (N=58) or normal karyotype (N=28). VH mutation status was available in 198 of 206 cases. For statistical analyses, protein expression was quantified by densitometry in 185 patients. Interestingly, in CLL subgroups with a good prognosis, differentially expressed proteins were rather involved in apoptosis. In contrast, in cytogenetic subgroups with a worse prognosis, deregulated proteins were associated with DNA damage and cell cycle control (see figure). In addition, higher levels of CDK4, P27 and P53 significantly correlated with increasing hierarchical risk as defined by genetic subgroups. Inter-protein correlations pointed to either STAT6-signaling or AKT1-signaling to be affected in CLL, but the low number of significant partial correlations between proteins underlines the major influence of genetic aberrations on the cellular CLL phenotype. In summary, differences in protein levels define distinct molecular phenotypes with a dichotomy of apoptosis and proliferation in CLL associated with prognostic subgroups. These findings open new therapeutic options for patient-tailored treatment of CLL.Figure:Overview of protein regulations in hierarchical subgroups of CLL suggesting a dichotomy of apoptosis and proliferation in CLL associated with benign and aggressive subgroups, respectively. The figure illustrates an increasing number of deregulated proteins associated with proliferation/DNA damage and a decreasing number of deregulated proteins that control apoptosis with higher genetic risk category (arrows indicate upregulation or downregulation when compared to other risk categories; two arrows: proteins with significant differential expression; single arrows: significant before adjustment for multiple testing (trend)). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Identification of protein biomarkers in nipple aspiration fluid

Nipple aspiration fluid (NAF) can be useful for studying relevant changes in breast parenchyma and identification of potential biomarkers for detection, monitoring therapy and risk assessment of breast cancer. NAF has been investigated extensively for identification of protein biomarkers using approaches such as ELISA, immunoassay, western blot for chosen individual protein markers such as prostate specific antigen, basic fibroblast growth factor, prostaglandin E2 etc., or by using comprehensive analysis of protein expression using platforms such as surface enhanced laser desorption ionization-time of flight mass spectrometry. The results of the former studies indicate the potential value of selected markers for detection of cancer which have not been validated in larger studies. The results of majority of studies that performed comprehensive protein analysis indicate that protein expression patterns are highly conserved between cancerous and non cancerous breasts and that protein profiling of NAF may have more value for risk assessment than for detection of breast cancer. Using a novel approach of protein microarray for seven markers with significant differences in protein levels were found between NAF obtained from cancerous and contralateral breast and healthy controls for markers such as cathepsin and urokinase. Overall, proteomic studies of NAF indicate the probable value of several protein biomarkers. There is a need for validation of the markers that have been identified so far, to be promising for risk assessment or early detection of breast cancer in large prospective clinical trials.

Is Proteomics the New Genomics?

Mass spectrometry (MS)-based proteomics has become a formidable tool for the investigation of posttranslational modifications to proteins, protein interactions, and organelles. Is it now ready to tackle comprehensive protein expression analysis?

Classification of Acute Myelogenous Leukemia (AML) Based on Patterns of Signal Transduction Pathway (STP) and Apoptosis Regulating Protein Activation Determined by Reverse Phase Proteins Arrays (RPPA).

We have previously shown that the protein expression profile and especially the activation state of apoptosis and STP proteins are highly prognostic in AML. The development of RPPA permits a more comprehensive analysis of protein expression and phosphorylation (phos) patterns in the STP and apoptotic cascades. We have generated a screening array using protein derived from the leukemia enriched fraction of 94 primary AML samples (with 21 concurrent blood and marrow specimens), comprised equally of patients that were primary refractory (PrimRef), or that achieved complete remission that was continuous (CCR) or which relapsed after 6 to 24 months (REL). These slides have been probed with 22 total and 15 phos-specific antibodies (ABs) against apoptosis and STP proteins. Spot intensities were quantified using MicroVigene and the data for each protein standardized data by subtracting the mean expression levels across samples and dividing by the standard deviation. Unsupervised hierarchical clustering analysis on the proteins was performed. We found one small cluster of 5 proteins: Cyclin D1, β-catenin, phos-NMP, p53, and AMPK. The remaining proteins formed another cluster, with a subset of 8 proteins somewhat further separated. In several cases (mTOR, JNK, and PTEN) the total protein amounts clustered as nearest neighbors to the phos version of the same protein. Using perturbation bootstrap resampling to assess the significance of clusters we found 4 reproducible clusters and the structure suggests additional clusters in this data set. A Fisher exact test showed that these were significantly associated with response (p = 0.03) and with the prognostic category defined by cytogenetics (p = 0.04) but was not associated with the source (blood vs. marrow) of the sample (p = 0.67). Ten proteins were differentially expressed between cytogenetically defined prognostic groups: total and phospho AKT, PTEN and JNK, p-mTOR, p-STAT3, pp38 and PS6.p24.44 (all with P<0.02). No protein was prognostic for response across the entire population but several were prognostic for response and survival within specific clusters. Clustering of ratios of phos-/total AB revealed 2 patterns: one with higher phos- of kinases (“kinase high”); another with low kinase expression (“kinase low”) but higher phos- of apoptosis regulating proteins. In the “kinase high” cluster single or concurrent increased activation of pAkt308, pAkt473, pPKCa, p-mTor (2448), pErk1,2 (42/44), p-p38 over their corresponding total protein was associated with worse outcome (~15% CCR rate). Those with increased Bcl2/Bax and pJNK/JNK levels fared worse with only 10% CCR. A favorable outcome was associated with concurrent high pAkt308/Akt and pGSK3/GSK3 levels (50% CCR) or being in the “kinase low” group while having high pStat3(705)/Stat3 levels (58% CCRs). The identified clusters and their correlation with cytogenetic group and outcome mainly driven by the activated, phospho protein forms, strongly supports the idea that the activation state of STP and apoptotic regulators determines biological behavior and clinical outcome. Distinct pathway activation patterns can classify AML, provide prognostic guidance and may serve to triage patients to emerging targeted therapies aimed at these pathways.

Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein

To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.