Current Insights on Neurodegeneration by the Italian Proteomics Community

The growing number of patients affected by neurodegenerative disorders represents a huge problem for healthcare systems, human society, and economics. In this context, omics strategies are crucial for the identification of molecular factors involved in disease pathobiology, and for the discovery of biomarkers that allow early diagnosis, patients’ stratification, and treatment response prediction. The integration of different omics data is a required step towards the goal of personalized medicine. The Italian proteomics community is actively developing and applying proteomics approaches to the study of neurodegenerative disorders; moreover, it is leading the mitochondria-focused initiative of the Human Proteome Project, which is particularly important given the central role of mitochondrial impairment in neurodegeneration. Here, we describe how Italian research groups in proteomics have contributed to the knowledge of many neurodegenerative diseases, through the elucidation of the pathobiology of these disorders, and through the discovery of disease biomarkers. In particular, we focus on the central role of post-translational modifications analysis, the implementation of network-based approaches in functional proteomics, the integration of different omics in a systems biology view, and the development of novel platforms for biomarker discovery for the high-throughput quantification of thousands of proteins at a time

꿀벌과 제브라피쉬에서 대사체학과 단백질체학을 이용한 살충제 abamectin의 독성학적 비교

학위논문 (박사) -- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2021. 2. 김정한.Abamectin is an abamectin insecticide known to be isolated from fermentation of Streptomyces avermitilis, a naturally occurring soil Actinomycete. Due to its high toxicity in bees and fish, this study investigated the toxic mechanisms of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio) using targeted metabolomics approach by gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-high resolution mass spectrometry (LC-orbitrap-HRMS). All homogenized samples were extracted with 80% methanol (honeybee) or 50% methanol (zebrafish) and derivatized with TMS for GC-MS/MS analysis. In the targeted metabolomics approach, multiple reaction monitoring (MRM) mode of a GC-MS/MS for 396 metabolites was used to detect 239 metabolites in honeybee and 243 metabolites in zebrafish. With the results of metabolites detected in each sample, statistical analysis such as partial least squares‐discriminant analysis (PLS-DA), variable importance in the projection (VIP) and analysis of variance (ANOVA) were performed to identify important biomarkers. Metabolic pathways associated with those biomarkers were constructed using MetaboAnalyst 5.0. In the exposure experiment of honeybee to abamectin as a targeted metabolomics using GC-MS/MS and non-targeted metaboloimcs using LC-orbitrap-HRMS, metabolic pathways such as tyrosine metabolism, phenylalanine/tyrosine/tryptophan biosynthesis, citrate cycle, ascorbate/aldarate metabolism, and alanine/aspartate/glutamate metabolism were identified as the significantly perturbed pathways. While, zebrafish showed several metabolic pathways such as aminoacyl tRNA biosynthesis, glyoxylate/dicarboxylate metabolism, citrate cycle, and tryptophan metabolism were identified by exposure of abamectin. Such significant disturbance of important metabolites within key biochemical pathways by abamectin could result in biologically hazardous effects in honeybee and zebrafish. In toxicoproteomics study, the toxicological effects of abamectin in honeybee and adult zebrafisha were investigated using a label-free quantitative proteomic approach on LC-HRMS. The proteins of honeybee and zebrafish samples were extracted with 0.1M phosphate buffer (pH 7.4) and 200 μg of proteins were digested with trypsin using the in-solution filter-aided sample preparation (FASP) method. After LC-HRMS analysis, a total of 670 proteins were identified and 32 proteins were selected as biomarkers through volcano analysis in honeybee. In zebrafish, 2189 proteins were identified and 1050 proteins were selected as biomarkers by statistically analysis.Abamectin은 자연적으로 발생하는 토양 방선균인 Streptomyces avermitilis의 발효에서 분리 된 것으로 알려진 살충제다. 꿀벌과 어류의 독성이 높기 때문에 본 연구는 가스크로마토그래피-탠덤 질량 분석법 (GC-MS/MS) 및 액체크로마토그래피-고분해능 질량 분석법(LC-orbitrap-HRMS)에 의한 비표적 및 표적 대사 체학 접근법을 사용하여 꿀벌(Apis mellifera) 및 제브라피시 (Danio rerio)에서 abamectin의 독성 메커니즘을 조사했다. 모든 균질화 된 샘플을 80 % 메탄올(꿀벌) 또는 50 % 메탄올(제브라피쉬)로 추출하고 GC-MS/MS 분석을 위해 추출액을 TMS로 유도체화반응을 적용하였다. 표적 대사체학 접근법에서는 396 개의 대사산물에 대한 GC-MS/MS의 다중 반응 모니터링(MRM) 모드를 사용하여 꿀벌에서 239개의 대사산물과 제브라피시에서 243개의 대사산물을 검출했다. 각 샘플에서 검출 된 대사 산물의 결과를 바탕으로 PLS-DA 패턴분석, VIP score 및 ANOVA 분산 분석과 같은 통계 분석을 수행하여 중요한 바이오마커를 식별했다. 이러한 바이오 마커와 관련된 대사경로는 MetaboAnalyst 5.0을 사용하여 확인 할 수 있었다. GC-MS/MS를 사용하는 표적 대사체와 LC-orbitrap-HRMS를 사용하는 비표적 대사체학을 통하여 꿀벌의 노출 실험에서 tyrosine metabolism, phenylalanine/tyrosine/tryptophan biosynthesis, citrate cycle, ascorbate/aldarate metabolism, and alanine/aspartate/glutamate metabolism가 상당히 교란 된 것으로 확인되었다. 반면, 제브라피쉬는 aminoacyl tRNA biosynthesis, glyoxylate/dicarboxylate metabolism, citrate cycle, and tryptophan metabolism이 상당히 변화함을 확인하였다. Abamectin에 의한 중요한 대사산물의 심각한 교란은 꿀벌과 제브라피쉬에 생물학적으로 위험한 영향을 미칠 수 있음을 확인 할 수 있었다. 한편, 독성 단백질체학 연구에서는 꿀벌과 제브라피쉬에서 LC-HRMS를 통하여 비표지 정량을 활용한 단백질체학적 접근방식을 사용하였다. 꿀벌과 제브라피시 샘플의 단백질은 0.1M phosphate buffer (pH 7.4)로 추출하고 200μg의 단백질은 in-solution filter-aided sample preparation (FASP) 방법을 사용하여 트립신으로 분해하였다. LC-HRMS 분석 후 총 670개의 단백질이 확인되었고, 화산 분석을 통해 32개의 단백질이 바이오 마커로 선정되었다. 제브라 피쉬에서는 2189개의 단백질이 확인되었고 통계 분석을 통해 1050개의 단백질이 바이오 마커로 선택되었다.Abstract.............................................................................................................i Table of Contents...................................................................iii List of Tables...........................................................................vi List of Figures.................................................................................................viii Preface.......................................................................................................................1 Chapter I. Toxicometabolomics of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio).............................................................................................3 Introduction.........................................................................................................5 Methodology of metabolomics............................................................................8 Application of metabolomics.............................................................................21 Purose of study………………………………………………………………….23 Materials and Methods......................................................................................24 Chemicals and reagents........................................................................................24 Experimental animals…………………………………………………………24 Chemical exposure...............................................................................................24 Sample preparation..............................................................................................25 Targeted profiling and identification of metabolites by GC-MS/MS...................26 Non-targeted profiling and identification of metabolites by LC-Orbitrap-HRMS…………………………………………………………..………………26 Statistical analysis and biomarker selection.........................................................30 Metabolic pathway analysis.................................................................................34 Measurement of MDA contents………………………………………………...34 Results and Discussion.......................................................................................36 Chemical exposure and sample preparation…….................................................36 Targeted profiling and identification of metabolites by GC-MS/MS...................36 Non-targeted profiling and identification of metabolites by LC-Orbitrap-HRMS…………………………………………………………………………..52 Statistical analysis and biomarker selection…………………………………….58 Metabolic pathway analysis…………….............................................................74 Abamectin effects on MDA in zebrafish………………………………………..84 Conclusions........................................................................................................88 Chapter II. Toxicoproteomics of abamectin in honeybee (Apis mellifera) and zebrafish (Danio rerio)...........................................................................................91 Introduction.......................................................................................................93 Proteomics...........................................................................................................93 Methodology of proteomics.................................................................................94 Application of proteomics..................................................................................105 Purpose of study.................................................................................................180 Materials and Methods....................................................................................109 Chemicals and reagents......................................................................................109 Experimental animals and chemical exposure……………………………….109 Sample preparation............................................................................................109 Profiling and identification of proteomes by LC-Orbitrap-HRMS.....................111 Statistical analysis……………………….......................................................114 Enrichment analysis...........................................................................................114 Results and Discussion.....................................................................................115 Protein profiling using LC-Orbitrap-HRMS......................................................115 Proteomic alteration induced by abamectin exposure........................................120 Enrichment analysis...........................................................................................186 Conclusions......................................................................................................199 References.............................................................................................................200 초록...............................................................................................................212Docto

Application of Different Bioanalytical Workflows for Proteomics of Prostate Cancer

In the current dissertation, we focused on the development and application of multiple mass spectrometry-based bioanalytical platforms for phosphoproteomic characterization in cell culture and clinical specimens of prostate cancer; and on the application of optimized methods to analysis of differential protein expression to reveal molecular mechanism of drug action in animal model of prostate cancer. Characterization of the phosphoproteome in prostate cancer Our study in phosphoprotein signatures on a large scale in prostate cancer focused on the LNCaP human prostate cancer cell line, and on human prostate cancer tissue. For the LNCaP prostate cancer cell line, we applied a combination of analytical platforms: (1) a novel in-gel isoelectric focusing (IEF) LC-MS/MS analytical platform; (2) a 2-DE based platform combined with phospho-specific staining. The in-gel IEF LC-MS/MS analytical methodology used in the study included separation of the LNCaP proteins by in-gel isoelectric focusing; digestion of the proteins with trypsin; enrichment of the digests for phosphopeptides with immobilized metal ion affinity chromatography (IMAC); analysis of the enriched digests by LC-MS/MS; and identification of the phosphorylated peptides/proteins through searches of the Swiss-Prot protein sequence database. With in-gel IEF based analytical platform, we have characterized over 600 different phosphorylation sites in 296 phosphoproteins in the LNCaP prostate cancer cell line. This panel of the LNCaP phosphoproteins was 3-fold larger than the panel obtained in our previous work, and is the largest phosphoprotein panel in prostate cancer reported to date. The phosphoproteins identified in this study belonged to various locations within the cell and were involved in various processes including cell differentiation, transcription regulation, and intercellular signal transduction. We also developed a 2-DE based platform, in combination with multiplexed staining and LC-MS/MS, for the identification of LNCaP phosphoproteins. In this study, we applied 2-DE as separation technique, Pro-QTM Diamond stain as phosphoprotein detection method, LC-MS/MS and database searches for protein identification to investigate the phosphoproteins in the LNCaP prostate cancer cell line. Proteins identified from spots of interest were shown to be highly relevant to prostate cancer. We demonstrated the feasibility of using 2-DE with phospho-specific stain and mass spectrometry to investigate the phosphoproteins in the LNCaP cell line. This methodology complements the in-gel IEF LC-MS/MS platform that we used for phosphoproteomics study; it will be of particular value for future comparative studies of phosphoproteins in various physiological states. For prostate cancer tissue, a gel-free approach was performed to analyze five prostate cancer tissue specimens to obtain phosphoproteomic signature of prostate cancer for biomarker discovery. Proteins were extracted with Trizol reagent, and then in-solution digested with trypsin. Phosphopeptides were enriched with IMAC, and analyzed the phosphorylated peptides/proteins by LC-MS/MS with identification through searches of the Swiss-Prot protein sequence database. The panels obtained for prostate cancer tissue contain 15-24 phosphoproteins. Some of the characterized phosphoproteins were present in all five specimens; in addition, each specimen also produced a unique set of phosphoproteins. The findings provided a direct glimpse into the phosphoprotein machinery operating within the human prostate cancer tissue. This pilot study focused on a small set of specimens. The phosphoprotein panels that were obtained contained a number of proteins that were unique to a particular specimen. Comparative proteomics study of drug effects in prostate cancer We carried out the first comparative proteomics study for the examination of the effects of bicalutamide/embelin combination treatment on prostate tumors by characterizing the alterations in protein expression that was induced upon treatment of mice bearing prostate tumors with anticancer combination therapy. A comparative proteomic strategy based on 2-DE coupled with LC-MS/MS was performed on mouse prostate tumor tissue. Proteins from the mouse prostate tumors were extracted with Trizol, and the protein mixtures were separated by 2-DE. Differences in the protein profiles for the different treatment groups were evaluated by computer-assisted analysis of SYPRO Ruby stained 2-DE gels. LC-MS/MS and database searches were used to identify differentially expressed proteins. Pathway analysis was carried out on the dataset of identified proteins with the Ingenuity bioinformatics tool. Out of the 33 differentially expressed protein spots, 30 protein spots were identified and grouped into various functional classes. The major protein categories were metabolism (52%), stress response (12%), protein biosynthesis (13%) and apoptosis (11%), suggesting that alterations in these processes may be involved in the mechanism of drug action. Proteins associated with oxidative stress were up-regulated, which indicated that treatment with bicalutamide/embelin may affect the redox balance within the prostate tumor, and this effect may contribute to tumor suppression

Towards discovering novel aspects of nuclear biology in the malaria parasite "Plasmodium falciparum"

The apicomplexan parasite P. falciparum continues to cause morbidity and mortality imposing a significant health and economic burden on human society. In light of antimalarial drug resistance and the lack of an effective vaccine there is an urgent need to understand the basic biology of Plasmodium parasites in much greater detail. In particular, basic nuclear processes such as those remain surprisingly unsought despite their importance in parasite survival and life cycle progression. Thus, identification and localisation of novel parasite proteins to areas of the nucleus is an important first step towards giving new insights into nuclear architecture and function. The main aim of this thesis was to compile an inventory of the nuclear proteome across the intra-erythrocytic cell cycle using high accuracy mass spectrometry coupled with bioinformatics and in vivo localisation experiments. The dataset was analysed for accuracy and retention of true nuclear proteins revealing a final list of 802 potential nuclear proteins with an estimated precision of 76%. Interestingly, the informational pool of this study was able to identify a large number of novel nuclear components including novel protein domains possibly involved in gene regulation, members of the nuclear pores, the nucleolus and the proteasome (chapter 2). Several transgenic parasite lines used for the experimental validation part of the nuclear core proteome were further investigated in more detail. One of these transgenic cell lines expresses the C-terminally tagged bromo-domain protein PF10_0328 and was investigated by co-immuoprecipitation experiments followed by LC-MS/MS to identify interacting proteins. Bromodomain proteins bind specifically to acetylated lysine residues in histone tails and are important regulators of transcription. Our results suggest that PF10_0328 acts in concert with two additional bromo-domain proteins in regulating transcription in P. falciparum (chapter 3). Further characterisation on the functional level of these three important regulators is currently ongoing in a collaborative effort. Characterisation of bromo-domain proteins could establish new intervention strategies against malaria as the recognition of acetylated histone tails by bromo-domains can be selectively prevented by small molecules. Furthermore, several proteins residing in the nuclear pores and the nucleolus of P. falciparum were used to visualise these structures in relation to chromosome end clusters based on fluorescence microscopy. We show that both structures, involved in nuclear-cytoplasmic trafficking and ribosomal biogenesis, respectively, do not appear to ‘cross-talk’ with silenced chromosome ends at the nuclear periphery of P. falciparum (chapter 4). In conclusion, I believe that my work about several aspects of gene regulation and nuclear architecture increases the understanding of the biology of this medically important pathogen and could have potential to identify new avenues for interventions against malaria

Molecular mechanisms and targets of new anticancer treatments

The work presented in this thesis is an effort to decipher and understand the mechanism of action (MOA) of anticancer agents by building on and complementing chemical proteomics methods. The backbone of the thesis relies on a recent method called Functional Identification of Target by Expression Proteomics (FITExP) developed in Zubarev lab, where drug induced proteomic signatures are analyzed in various cell lines and top differentially regulated proteins with consistent behavior are determined, among which the drug target and mechanistic proteins are usually present. FITExP relies on the assumption that proteins most affected with a perturbation have a higher probability of being involved in that process. In this regard, Paper I aimed to enhance the performance of FITExP analysis by merging proteomic data from drug-treated matrix attached and detached cells. This is while the majority if not all proteomics and molecular biology experiments are performed in matrix attached cells, as the general belief is that detached cells lose their structural integrity and do not harbor valuable information. However, detached cells are those that are more sensitive to chemotherapeutics and might reflect the proteome changes better. The comparative proteomics of living and dying cells improved FITExP performance with regards to identification of targets and provided insight about proteins involved in cellular life and death decisions. Furthermore, the orthogonal partial least squares-discriminant analysis (OPLS-DA) paradigm presented in this study, was used throughout the thesis for contrasting and visualizing the proteomic signature of a molecule against others, to reveal targets and specific proteins changing in response to the molecule of interest. In Paper II, as a further development of FITExP and to demonstrate its applicability in a broader context, we built a proteome signature library of 56 clinical and experimental anticancer agents in A549 lung adenocarcinoma cell line. This resource called ProTargetMiner can be used for different purposes. The proximity of compounds in hierarchical clustering or t-SNE could be used for prediction of the mechanism of new compounds. Contrasting each molecule against other treatments using the OPLS-DA scheme presented in Paper I, revealed drug targets, mechanistic proteins, resistance factors, drug metabolizing enzymes and effects on protein complexes. Representative examples were used to demonstrate that the specificity factors extracted from the OPLS-DA models can help identify subtle but biologically significant processes, even when such an effect is as low as 15% fold change. Furthermore, we showed that the inclusion of 8-10 contrasting molecules in the OPLS-DA models can produce enough specificity for drug target deconvolution, which offered a miniaturization opportunity. Therefore, we built three deeper datasets using 9 compounds that showed the most diverse proteome changes in the orthogonal space in three cell lines from major cancer types: A549 lung, MCF-7 breast and RKO colon cancers. These datasets provide a unique depth of 7398, 8735 and 8551 respectively, with no missing values. Subsequently, a Shiny package was created in R, which can employ these datasets as a resource and merge it with user data and provide OPLS-DA output and target deconvolution opportunity for new compounds. Finally, using the original ProTargetMiner data, we also built a first of its kind proteomic correlation database which can find applications in deciphering the function of uncharacterized proteins. Moreover, the resource helped to identify a set of core or untouchable proteins with stable expression across all the treatments, revealing essential functions within the cells. Such proteins could be used as house-keeping controls in molecular biology experiments. In paper III, we combined FITExP with other chemical proteomics tools Thermal Proteome Profiling (TPP) and multiplexed redox proteomics, to study the target and mechanism space of auranofin. This would also allow to assess the power, orthogonality and complementarity of these techniques in the realm of chemical proteomics. TPP is a recently developed technique that can monitor changes in the stability of proteins upon binding to small molecules. Redox proteomics is a method by which the oxidation level of protein cysteinome can be quantitatively analyzed. Auranofin is an FDA-approved anti-inflammatory drug for treatment of rheumatoid arthritis, but due to its potent antitumor activity, it is currently in clinical trials against cancer. Although several MOAs have been suggested for auranofin, uncertainties exist regarding its cellular targets; therefore, this molecule was chosen as a challenging candidate to test the chemical proteomics tools. A combination of the above mentioned tools confirmed thioredoxin reductase 1 (TXNRD1) (ranking 3rd) as the cognate target of auranofin and demonstrated that perturbation of oxidoreductase pathway is the main route of auranofin cytotoxicity. We next showed that changes in the redox state of specific cysteines can be linked to protein stability in TPP. Some of these cysteines were mapped to the active sites of redox-active enzymes. In Paper IV, using quantitative multiplexed proteomics, we helped to show that b-AP15, a bis-benzylidine piperidone compound inhibiting deubiquitinases USP14 and UCHL5, produces a similar perturbation signature as bortezomib in colon cancer cells. However, in comparison with bortezomib, b-AP15 induces chaperone expression to a significantly higher level and leads to a more extensive accumulation of polyubiqutinated proteins. The polyubiqutinated proteins co-localize with mitochondrial membrane and subsequently reduce oxidative phosphorylation. These results help define the atypical cell death induced by b-AP15 and describe why this molecule is effective against apoptosis resistant cells in variety of tumor models. Finally, in Paper V, we extended the applications of TPP and combined it with specificity concept for proteome-wide discovery of specific protein substrates for enzymes. We developed a universal method called System-wide Identification of Enzyme Substrates by Thermal Analysis (SIESTA) that relies on the hypothesis that enzymatic post-translational modification of substrate proteins can potentially change their stability against thermal denaturation. Furthermore, we applied the concept of specificity similar to the above papers, to reveal potential substrates using OPLS-DA. SIESTA was applied to two enzyme systems, namely TXNRD1 and poly-(ADP-ribose) polymerase-10 (PARP10), identifying known and putative candidate substrates. A number of these candidate proteins were validated as PARP10 substrates by targeted mass spectrometry, chemiluminescence and other assays. SIESTA is an unbiased and system wide approach and its broad application can improve our understanding of enzyme function in homeostasis and disease. In turn, specific protein substrates can serve as readouts in high throughput screening and facilitate drug discovery. Taken together, in this thesis, FITExP methodology was improved in two directions. In paper I, we improved the performance of FITExP by combining the proteomics data from detached and attached cells. In Paper II, we demonstrated how the proteomics data on a multitude of drugs in a single cell line enables the discovery of compound targets and MOA. Furthermore, we built an R Shiny package which can serve as a resource for the cancer community in target and MOA deconvolution. In Papers III and IV, we applied an arsenal of chemical proteomics tools for characterization of two anticancer compounds. In Paper V, we expanded the applications of TPP to identification of specific protein substrates for enzymes in a system-wide manner

Investigating the role and regulation of human mitochondrial poly(A) polymerase

PhD ThesisPolyadenylation by the mitochondrial poly(A) polymerase (mtPAP) is a crucial step of post-transcriptional modification in mammalian gene expression. In human mitochondria, polyadenylation is required for completion of seven UAA stop codons following complete processing of the major polycistronic RNA unit. Patients homozygous for a 1432A>G mutation in the PAPD1 gene, which encodes mtPAP, suffer from symptoms consistent with mitochondrial disease including autosomal-recessive spastic ataxia and optic atrophy. The principal defect of the 1432A>G mutation is short adenylate tails on mt-mRNAs. Fibroblast lines from patients harboring the 1432A>G PAPD1 mutation were established, and analysis of mitochondrial gene expression showed non-uniform dysregulation. For mt-mRNAs and translation products, there is a mix of depletion, stabilization and no effect, leading to major deficits at steady-state protein levels and of respiratory complexes. To confirm the pathological nature of the mutation, a complementation experiment was performed, which showed that expression of the WT PAPD1 gene rescued the mutant phenotype. To assess whether catalytic activity was altered in the mutant enzyme, in vitro polyadenylation assays with WT and N478D recombinant mtPAP were undertaken. The N478D mtPAP was found to generate the short oligo(A) tails as observed in vivo. In addition, the presence of the LRPPRC/SLIRP complex increased the maximal poly(A) extensions generated by both WT and mutant mtPAP. Finally, experiments were undertaken to identify factors potential interacting with mtPAP. The major interacting factor was found to be ATAD3, a protein reported to be involved with multiple mitochondrial processes involving DNA and translation machinery in the form of nucleoids or mitoribosomes respectively. In summary, these investigations provide insights into the impact and regulation of mitochondrial polyadenylation, and contribute towards unraveling the complexities of post-transcriptional maturation in human mitochondrial gene expression.The Pathological Society of Great Britain and Ireland, and Newcastle University

Novel urinary and serological markers of prostate cancer using proteomics techniques: an important tool for early cancer diagnosis and treatment monitoring

In Africa, Prostate cancer (PCa) is the most frequently diagnosed solid organ tumour in males and use of prostate specific antigen (PSA) is presently fraught with diagnostic inaccuracies. Not least, in a multi-ethnic society like South Africa, proteome differences between African, Caucasian and Mixed-Ancestry PCa patients are largely unknown. Hence, discovery and validation of affordable, non-invasive and reliable diagnostic biomarkers of PCa would expand the frontiers of PCa management. We have employed two high-throughput proteomics technologies to identify novel urine- and blood-based biomarkers for early diagnosis and treatment monitoring of prostate cancer in a South African cohort as well as elucidate proteome differences in patients from our heterogeneous cohort. We compared the urinary proteomes of PCa, Benign Prostatic Hyperplasia (BPH), disease controls comprising patients with other uropathies (DC) and normal healthy controls (NC) both by pooling and individual discovery shotgun proteomic assessment on a nano-Liquid chromatography (nLC) coupled Hybrid Quadrupole-Orbitrap Mass Spectrometer platform. In-silico verification of identified biomarkers was performed using the Human Protein Atlas (HPA) as well as SRMAtlas; and verified potential biomarkers were experimentally prevalidated using a targeted parallel reaction monitoring (PRM) proteomics approach. Further, we employed the CT100+ antigen microarray platform to assess the differential humoral antibody response of PCa, DC and BPH patients in our cohort to a panel of 123 tumour-associated cancer antigens. Candidate antigen biomarkers were analyzed for ethnic group variation in our cohort and potential cancer diagnostic and immunotherapeutic inferences were drawn. Using these approaches, we identified 5595 and 9991 non-redundant peptides from the pooled and individual experiments respectively. While nine proteins demonstrated ethnic trend, 37 and 73 proteins were differentially expressed by pooled and individual analysis respectively. All 32 verified biomarkers were prevalidated with parallel reaction monitoring. Good PRM signals for 12 top ranking biomarker was observed, including PSA and prostatic acid phosphatase. We also identified 41 potential diagnostic and immunotherapeutic antigen biomarkers. Proteogenomic functional pathway analyses of differentially expressed antigens showed similar enrichments of biologic processes. We identified herein novel urinary and blood-based potential diagnostic biomarkers and immunotherapeutic targets of PCa in a South African PCa Cohort using multiple proteomics approaches

A brave new world of RNA-binding proteins

RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple globular RNA-binding domains (RBDs) and change the fate or function of the bound RNAs. Several hundred such RBPs have been discovered and investigated over the years. Recent proteome-wide studies have more than doubled the number of proteins implicated in RNA binding and uncovered hundreds of additional RBPs lacking conventional RBDs. In this Review, we discuss these new RBPs and the emerging understanding of their unexpected modes of RNA binding, which can be mediated by intrinsically disordered regions, protein–protein interaction interfaces and enzymatic cores, among others. We also discuss the RNA targets and molecular and cellular functions of the new RBPs, as well as the possibility that some RBPs may be regulated by RNA rather than regulate RNA