Mass spectrometry methods to study protein-metabolite interactions

Introduction: To ????understand and manipulate biochemical processes and signaling pathways, the knowledge of endogenous protein-metabolite interactions would be extremely helpful. Recent developments in precision mass spectrometry, high-throughput proteomics and sensitive metabolomic profiling are beginning to converge on a possible solution, heralding a new era of global metabolome-proteome ‘interactome’ studies that promise to change biomedical research and drug discovery.

Areas covered: Here, we review innovative mass spectrometry-based methods and recent pioneering studies aimed at elucidating the physical associations of small molecule ligands with cellular proteins. The technologies covered belong to two main categories: tag-based and tag-free methods. We emphasize the latter in this review, and outline promising experimental workflows and key data analysis considerations involved.

Expert opinion: Recent ground-breaking advances in chemical-proteomics technology and allied computational methods now make the global detection of protein-ligand engagement an increasingly attractive research problem. Despite ongoing challenges, rapid progress in the field is expected these coming next few years, leading to a refreshed systems biology research paradigm and much needed new opportunities for improving sparse drug discovery pipelines.     

Proteomic analysis of synovial fluid as an analytical tool to detect candidate biomarkers for knee osteoarthritis

We conducted research to detect the proteomic profiles in synovial fluid (SF) from knee osteoarthritis (OA) patients to better understand the pathogenesis and aetiology of OA. Our long-term goal is to identify reliable candidate biomarkers for OA in SF. The SF proteins obtained from 10 knee OA patients and 10 non-OA patients (9 of whom were patients with a meniscus injury in the knee; 1 had a discoid meniscus in the knee, and all exhibited intact articular cartilage) were separated by two-dimensional electrophoresis (2-DE). The repeatability of the obtained protein spots regarding their intensity was tested via triplicate 2-DE of selected samples. The observed protein expression patterns were subjected to statistical analysis, and differentially expressed protein spots were identified via matrix-assisted laser desorption/ionisation-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Our analyses showed low intrasample variability and clear intersample variation. Among the protein spots observed on the gels, there were 29 significant differences, of which 22 corresponded to upregulation and 7 to downregulation in the OA group. One of the upregulated protein spots was confirmed to be haptoglobin by mass spectrometry, and the levels of haptoglobin in SF are positively correlated with the severity of OA (r = 0.89, P < 0.001). This study showed that 2-DE could be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA. Spots of interest identified by mass spectrometry, such as haptoglobin, may be associated with OA severity.     

肝癌肿瘤标志物的研究

目的 肝癌是恶性程度最高的肿瘤之一,当前急切需要肝癌的标志物.但是当前的所用的标志物缺乏敏感性和特异性,有一些新的候选标志物近年来被发现.方法 我们回顾了传统和新的肝癌血清标志物相关文献.结果 有几种有希望的新的肝癌血清标志物被认识.它们包括单个蛋白、蛋白组学合成物、肿瘤自身抗体.但是它们还没有一个能通过由EDRN所设定的最严厉的标准.结论 新的肝癌血清标志物被批准前还需等待合适的对照临床研究.     

Building a pipeline to discover and validate novel therapeutic targets and lead compounds for Alzheimer's disease

Cognitive decline, Alzheimer's disease (AD) and other causes are major public health problems worldwide. With changing demographics, the number of persons with dementia will increase rapidly. The treatment and prevention of AD and other dementias, therefore, is an urgent unmet need. There have been considerable advances in understanding the biology of many age-related disorders that cause dementia. Gains in understanding AD have led to the development of ante-mortem biomarkers of traditional neuropathology and the conduct of several phase III interventions in the amyloid-β cascade early in the disease process. Many other intervention strategies are in various stages of development. However, efforts to date have met with limited success. A recent National Institute on Aging Research Summit led to a number of requests for applications. One was to establish multi-disciplinary teams of investigators who use systems biology approaches and stem cell technology to identify a new generation of AD targets. We were recently awarded one of three such grants to build a pipeline that integrates epidemiology, systems biology, and stem cell technology to discover and validate novel therapeutic targets and lead compounds for AD treatment and prevention. Here we describe the two cohorts that provide the data and biospecimens being exploited for our pipeline and describe the available unique datasets. Second, we present evidence in support of a chronic disease model of AD that informs our choice of phenotypes as the target outcome. Third, we provide an overview of our approach. Finally, we present the details of our planned drug discovery pipeline.     

Proteomic identification of proteins differentially expressed by melatonin in hepatic ischemia‐reperfusion injury

Abstract: Hepatic ischemia‐reperfusion (I‐R) injury induces hepatic dysfunction or failure. Melatonin is a potent free radical scavenger and a strong antioxidant. Although many studies have demonstrated the protective effect of melatonin in hepatic injury, the molecular mechanisms of this protection are unclear. We identified specific proteins that are differentially expressed by melatonin treatment in hepatic I‐R injury. Adult mice were subjected to 1 hr of ischemia and 3 hr of reperfusion. Animals were treated with vehicle or melatonin (10 mg/kg, i.p.) 15 min prior to ischemia and just before reperfusion. Serum aspartate aminotransferase and alanine aminotransferase levels were higher in I‐R group than in sham‐operated group, and these increases were reduced by melatonin treatment. Proteins that were differentially expressed following melatonin treatment during hepatic I‐R injury were detected using two‐dimensional gel electrophoresis. Hepatic I‐R injury induced down‐regulation of glyoxalase I, glutaredoxin‐3, spermidine synthase, proteasome subunit beta type‐4, and dynamin like protein‐1 (DLP‐1). However, melatonin prevented the reductions in these proteins induced by I‐R injury. Among the identified proteins, we focused on DLP‐1, which is essential for the maintenance of mitochondrial and endoplasmic reticulum morphology. Western blot analysis confirmed that melatonin prevents the hepatic I‐R injury‐induced decrease in DLP‐1. These results suggest that melatonin protects hepatic cells against hepatic I‐R injury and that its protective effects involve the regulation of specific proteins.     

基因组学和蛋白质组学及代谢组学在食管癌筛查与早期诊断应用研究现状

目的食管癌是常见的消化道恶性肿瘤,发病率较高而生存率较低。本文总结基因组学、蛋白质组学及代谢组学在食管癌早期筛查和诊断中的应用,为今后食管癌早期筛查和诊断提供新的平台和科学理论依据。方法以"基因组学、蛋白质组学、代谢组学、食管癌和早期诊断"为关键词检索CNKI期刊全文数据库,以"genomics、proteomics、metabolomics、esophagus cancer、early diagnosis"为关键词检索PubMed,检索建库至2018-12发表的相关文献。纳入标准:(1)基因组学、蛋白质组学和代谢组学相关研究;(2)食管癌早期筛查与诊断。排除标准:(1)综述类文献;(2)研究资料不全;(3)结果重复且相对陈旧的研究。根据纳入和排除标准选择29篇文献进行分析。结果既往研究证实,食管癌患者与健康者在基因、蛋白质表达及代谢表达方面存在差异,且发现TFF1、CCNA1和TFPI2等DNA甲基化,PA28β、AHSG和LRG等差异表达蛋白及蛋白特征组,多个氨基酸的代谢特征及其他物质的代谢失调均可作为食管癌早期筛查及诊断潜在的生物标志物。此外,利用各类组学技术及血液、尿液等生物样本在寻找生物标志物方面均表现出较好的可行性。结论基因组学、蛋白质组学及代谢组学技术有望为食管癌早期筛查及诊断提供新方法。     

Mushroom body‐preferential expression of proteins/genes involved in endoplasmic reticulum Ca2+‐transport in the worker honeybee (Apis mellifera L.) brain

To identify the molecular characteristics specific to the mushroom body (MB, a higher processing centre) neurones in the honeybee brain, we previously used proteomics to identify proteins that are preferentially expressed in these MBs. Here we continued our proteomic analysis to show that reticulocalbin, which is involved in endoplasmic reticulum (ER) Ca²⁺ transport, is also preferentially expressed in the MBs in the honeybee brain. Gene expression analysis revealed that reticulocalbin is preferentially expressed in the large‐type Kenyon cells, which are MB‐intrinsic neurones. In addition, the gene for the ryanodine receptor, which is also involved in ER Ca²⁺ transport, was also preferentially expressed in the large‐type Kenyon cells. In contrast, the expression of three other ER‐related genes, protein disulphide isomerase, sec61 and erp60, was not enriched in the MBs. These findings further support the notion that the function of ER Ca²⁺‐signalling, but not the mere intracellular density of ER, is specifically enhanced in the large‐type Kenyon cells in the honeybee brain.