The use of proteomics in biomarker discovery in neurodegenerative diseases

Biomarkers for neurodegenerative diseases should reflect the central pathogenic processes of the diseases. The field of clinical proteomics is especially well suited for discovery of biomarkers in cerebrospinal fluid (CSF), which reflects the proteins in the brain under healthy conditions as well as in several neurodegenerative diseases. Known proteins involved in the pathology of neurodegenerative diseases are, respectively, normal tau protein, beta-amyloid (1-42), synaptic proteins, amyloid precursor protein (APP), apolipoprotein E (apoE), which previously have been studied by protein immunoassays. The objective of this paper was to summarize results from proteomic studies of differential protein patterns in neurodegenerative diseases with focus on Alzheimer's disease (AD). Today, discrimination of AD from controls and from other neurological diseases has been improved by simultaneous analysis of both beta-amyloid (1-42), total-tau, and phosphorylated tau, where a combination of low levels of CSF-beta-amyloid 1-42 and high levels of CSF-tau and CSF-phospho-tau is associated with an AD diagnosis. Detection of new biomarkers will further strengthen diagnosis and provide useful information in drug trials. The combination of immunoassays and proteomic methods show that the CSF proteins express differential protein patterns in AD, FTD, and PD patients, which reflect divergent underlying pathophysiological mechanisms and neuropathological changes in these diseases.     

Protein profiling in unlocking the basis of varicocele-associated infertility

Varicocele is one of the major causes of male infertility and has a negative impact on spermatogenesis. The conventional semen analysis does not reveal the underlying subcellular mechanisms associated with defects in spermatozoa. Proteomics and bioinformatics analysis can be used to identify the molecular aetiologies associated with poor semen quality in varicocele patients. Mitochondrial dysfunction has been identified as the main factor affecting normal physiological functions of spermatozoa. This article discusses the proteomic studies of spermatozoa and seminal plasma in varicocele patients. Proteomics can identify potential spermatozoa and seminal plasma biomarkers in varicocele-mediated male infertility. In future, these protein biomarkers can be useful in the development of noninvasive diagnostic and therapeutic strategies for varicocele patients.     

Utility of peptide–protein affinity complexes in proteomics: identification of interaction partners of a tumor suppressor peptide*

Abstract: We used a N‐biotinylated peptide analog of the C‐terminal domain of the tumor suppressor protein, p21^cip1/waf1 to elucidate peptide/protein interacting partners. The C‐terminal domain of p21^cip1/waf1 protein spanning 141–160 amino acid residues is known to bind PCNA and this interaction is important in many biological processes including cell‐cycle control. This C‐terminal 20‐mer efficiently extracts PCNA in the presence of a variety of N‐ or C‐terminally attached affinity tags. Using difference silver stained 2D gels combined with in‐gel tryptic digests, we identified the difference spots using MALDI‐TOF mass spectrometry‐based peptide mass fingerprinting followed by a database search using profound against NCBIs human nonredundant protein sequence data bank. Identified spots include the p48 subunit of chromatin assembly factor‐1, the heat shock 70 protein analog BiP, calmodulin, nucleolin and a spot similar in size to dimeric PCNA. In contrast, microcapillary ion‐trap LC‐MS/MS analysis of a tryptic digest of entire affinity extracts derived from both control and experimental runs followed by database searches using sequest confirmed the presence of most of the above proteins. This strategy also identified hnRNPA1, HPSP90α, HSP40 and T‐complex protein 1, a protein similar to prothymosin, and a possible allelic variant of the p21^cip1/waf1 protein. The use of N‐biotinylated peptide derived from the C‐terminal domain of p21^cip1/waf1 protein in proteomic analysis exemplified here suggests that peptides obtained from intracellular functional screens could also potentially serve as efficient baits to discover new drug targets.     

应用基质辅助激光解析电离飞行时间质谱源后衰变技术鉴定蛋白质

目的应用源后衰变(PSD)技术结合数据库检索鉴定二维蛋白质电泳(2DE)胶上的蛋白质斑点。方法 用已知多肽(ACTH)18~39肽段和TPA被胰酶降解后的1个肽段建立了PSD-MALDI-TOF-MS方法。结果应用已建立的PSD-MALDI-TOF-MS法分别将2DE分离后胶上的2个未知蛋白质斑点鉴定为40S核糖体蛋白S12和dnaK抑制蛋白。结论PSD技术在蛋白质组学研究中有较大的应用前景。     

蛋白质组学在中医药现代化研究中的应用探讨

蛋白质组学是后基因组时代的最重要研究课题，能够深化对生命现象本质的了解。对于中医药现代化研究，蛋白质组学作为高通量平行筛选技术在理论体系上与中医药的整体观相契合，能够深化证候的内在本质研究及中药复方的组成与作用机理研究，并能有力促进中药新药开发。随着蛋白质组学相关技术的发展完善，其对中医药现代化研究的帮助将进一步扩大。     

蛋白质组学在肝癌研究中的应用

肝癌是世界上流行最广的恶性肿瘤之一,但发病机制至今仍不明了,且缺乏有效的早期诊断方法和治疗靶点。随着人类基因组计划的实施和推进,生命科学研究已进入了后基因组时代。研究的重点从基因组学转向蛋白质组学(proteomics)。蛋白质组学将成为研究肿瘤及相关疾病的最有效方法之一。本文着重就蛋白质组学在肝癌研究的应用作一综述。     

Analysis of the in vitro synergistic effect of 5-fluorouracil and cisplatin on cervical carcinoma cells

5-Fluorouracil (5-FU) is currently being used as an anticancer drug to reduce tumor bulk in order to increase the operability rate and postoperative survival in patients with cervical cancer, which has been combined with cisplatin (CP) because of its superior activities observed in human carcinoma cells. However, the combined anticancer effect of 5-FU and CP in cervical carcinoma cells is poorly understood. Therefore, we conducted a study to investigate whether anticancer drugs 5-FU and CP may exhibit the combined antiproliferative effect in cervical carcinoma cells. Using proteomics analysis, including two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS), we investigated the antiproliferative effect-related proteins after treatment with 5-FU and/or CP. Our experiments showed that the combination of 5-FU and CP engaged both the apoptotic pathways: the membrane death receptor-mediated apoptosis pathway and the mitochondrial apoptotic pathway. Moreover, the combination of 5-FU and CP resulted in remarkable increasing susceptibility to apoptosis. We suggest that the combination of 5-FU and CP suppresses the growth of cervical carcinoma cells by synergistic effect with the induction of apoptosis. In vitro synergistic effect of 5-FU and CP supports the basis of the clinical application of the combination chemotherapy to the patients with cervical cancer.     

The attenuation effect of potassium 2‐(1‐hydroxypentyl)‐benzoate in a mouse model of diabetes‐associated cognitive decline: The protein expression in the brain

Aims dl‐PHPB (potassium 2‐(1‐hydroxypentyl)‐benzoate) has been shown to have neuroprotective effects against acute cerebral ischemia, vascular dementia, and Alzheimer''s disease. The aim of this study was to investigate the effects of dl‐PHPB on memory deficits and preliminarily explore the underlying molecular mechanism.MethodsBlood glucose and behavioral performance were evaluated in the KK‐A^y diabetic mouse model before and after dl‐PHPB administration. Two‐dimensional difference gel electrophoresis (2D‐DIGE)‐based proteomics was used to identify differentially expressed proteins in brain tissue. Western blotting was used to study the molecular mechanism of the related signaling pathways.ResultsThree‐month‐old KK‐A^y mice were given 150 mg/kg dl‐PHPB by oral gavage for 2 months, which produced no effect on the level of serum glucose. In the Morris water maze test, KK‐A^y mice treated with dl‐PHPB showed significant improvements in spatial learning and memory deficits compared with vehicle‐treated KK‐A^y mice. Additionally, we performed 2D‐DIGE to compare brain proteomes of 5‐month KK‐A^y mice treated with and without dl‐PHPB. We found 14 altered proteins in the cortex and 11 in the hippocampus; two of the 25 altered proteins and another four proteins that were identified in a previous study on KK‐A^y mice were then validated by western blot to further confirm whether dl‐PHPB can reverse the expression levels of these proteins. The phosphoinositide 3‐kinase/protein kinase B/glycogen synthase kinase‐3β (PI3K/Akt/GSK‐3β) signaling pathway was also changed in KK‐A^y mice and dl‐PHPB treatment could reverse it.ConclusionsThese results indicate that dl‐PHPB may play a potential role in diabetes‐associated cognitive impairment through PI3K/Akt/GSK‐3β signaling pathway and the differentially expressed proteins may become putative therapeutic targets.     

Proteomic analysis of the effects of caffeine in a neonatal rat model of hypoxic‐ischemic white matter damage

AimWhite matter damage (WMD) is the main cause of cerebral palsy and cognitive impairment in premature infants. Although caffeine has been shown to possess neuroprotective effects in neonatal rats with hypoxic‐ischemic WMD, the mechanisms underlying these protective effects are unclear. Herein, proteins modulated by caffeine in neonatal rats with hypoxic‐ischemic WMD were evaluated.MethodsWe identified differential proteins and performed functional enrichment analyses between the Sham, hypoxic‐ischemic WMD (HI), and HI+caffeine‐treated WMD (Caffeine) groups. Confirmed the changes and effect of proteins in animal models and determined cognitive impairment via water maze experiments.ResultsIn paraventricular tissue, 47 differential proteins were identified between the Sham, HI, and Caffeine groups. Functional enrichment analyses showed that these proteins were related to myelination and axon formation. In particular, the myelin basic protein (MBP), proteolipid protein, myelin‐associated glycoprotein precursor, and sirtiun 2 (SIRT2) levels were reduced in the hypoxic‐ischemic WMD group, and this effect could be prevented by caffeine. Caffeine alleviated the hypoxic‐ischemic WMD‐induced cognitive impairment and improved MBP, synaptophysin, and postsynaptic density protein 95 protein levels after hypoxic‐ischemic WMD by preventing the HI‐induced downregulation of SIRT2; these effects were subsequently attenuated by the SIRT2 inhibitor AK‐7.ConclusionCaffeine may have clinical applications in the management of prophylactic hypoxic‐ischemic WMD; its effects may be mediated by proteins related to myelin development and synapse formation through SIRT2.