11th Asia Oceania Human Proteome Organization Congress Report.

As the first in-person Asia Oceania Human Proteomics Organization (AOHUPO) congress since 2018, the 11th AOHUPO congress was an opportune time for the research community to reconnect and to renew friendships after the long period of restricted travel due to the global pandemic. Moreover, this congress was a great opportunity for the many AO regional proteomics and mass spectrometry scientists to meet in Singapore to exchange ideas and to present their latest findings. Cohosted by the Singapore Society for Mass Spectrometry and the Malaysian Proteomics Society and held in conjunction with the seventh Asia Oceania Agricultural Proteomics Organization Congress and Singapore Society for Mass Spectrometry 2023, the meeting featured both human and agricultural proteomics. Over five hundred scientists from the AO region converged on the MAX Atria @ Singapore EXPO, Changi, Singapore from May 8 to 10 for the main congress. The diverse program was made up of 64 invited speakers and panellists for seven plenary lectures, 27 concurrent symposia, precongress and postcongress workshops, and 174 poster presentations. The AOHUPO society were able to celebrate not only their 20th anniversary but also the outstanding academic research from biological and agricultural proteomics and related 'omics fields being conducted across the Asia-Oceania region.

A simple biomarker scoring matrix for early gastric cancer detection.

Gastric cancer (GC) is a major cause of death in many parts of the world. While 90% of early GC is curable by resection, only about 5% of patients diagnosed in the late stages survive beyond five years. This provides strong impetus to push for early GC detection through the use of non-invasive biomarkers, before metastatic complications arise. It is also of strong medical interest to identify patients of the diffuse subtype at the earliest time possible, since the disease variant progresses very rapidly and is associated with much higher mortality rate. In this study, we compared quantitatively the gastric fluid proteome of 70 GC patients to 17 individuals with benign gastritis in search of potential biomarkers that aid in GC diagnosis, prognosis and subtype stratification. We report that as much as half of the gastric fluid proteome is subject to regulation in diseased states, and propose a simple biomarker panel scoring matrix for early GC detection with diagnostic sensitivity of 95.7%. We also demonstrate as proof-of-concept that a digitised record generated with SWATH-MS based on 380 protein abundance signatures from the gastric fluid could segregate patients with diffuse-type GC.

Unravelling the proteome of degenerative human mitral valves.

Degenerative mitral valve disease (DMVD), which includes the syndromes of mitral valve prolapse (MVP) and flail leaflet, is a common valvular condition which can be complicated by mitral regurgitation and adverse cardiovascular outcomes. Although several genetic and other studies of MVP in dog models have provided some information regarding the underlying disease mechanisms, the proteins and molecular events mediating human MVP pathogenesis have not been unraveled. In this study, we report the first large-scale proteome profiling of mitral valve tissue resected from patients with MVP. A total of 1134 proteins were identified, some of which were validated using SWATH-MS and western blotting. GO annotation of these proteins confirmed the validity of this proteome database in various cardiovascular processes. Among the list of proteins, we found several structural and extracellular matrix proteins, such as asporin, biglycan, decorin, lumican, mimecan, prolargin, versican, and vinculin, that have putative roles in the pathophysiology of MVP. These proteins could also be involved in the cardiac remodeling associated with mitral regurgitation. All MS data have been deposited in the ProteomeXchange with identifier PXD000774 (http://proteomecentral.proteomexchange.org/dataset/PXD000774).

Analysis of colorectal cancer glyco-secretome identifies laminin ß-1 (LAMB1) as a potential serological biomarker for colorectal cancer.

The high mortality rate in colorectal cancer is mostly ascribed to metastasis, but the only clinical biomarker available for disease monitoring and prognosis is the carcinoembryonic antigen (CEA). However, the prognostic utility of CEA remains controversial. In an effort to identify novel biomarkers that could be potentially translated for clinical use, we collected the secretomes from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, using the hollow fiber culture system, and utilized the multilectin affinity chromatography approach to enrich for the secreted glycoproteins (glyco-secretome). The HCT-116 and E1 glyco-secretomes were compared using the label-free quantitative SWATH-MS technology, and a total of 149 glycoproteins were differentially secreted in E1 cells. Among these glycoproteins, laminin ß-1 (LAMB1), a glycoprotein not previously known to be secreted in colorectal cancer cells, was observed to be oversecreted in E1 cells. In addition, we showed that LAMB1 levels were significantly higher in colorectal cancer patient serum samples as compared to healthy controls when measured using ELISA. ROC analyses indicated that LAMB1 performed better than CEA at discriminating between colorectal cancer patients from controls. Moreover, the diagnostic performance was further improved when LAMB1 was used in combination with CEA.

iTRAQ analysis of colorectal cancer cell lines suggests Drebrin (DBN1) is overexpressed during liver metastasis.

Colorectal cancer is currently the third in cancer incidence worldwide and the fourth most common cause of cancer deaths. Mortality in colorectal cancer is often ascribed to liver metastasis. In an effort to elucidate the proteins involved in colorectal cancer liver metastasis, we compared the proteome profiles of the human colon adenocarcinoma cell line HCT-116 with its metastatic derivative E1, using the iTRAQ labelling technology, coupled to 2D-LC and MALDI-TOF/TOF MS. A total of 547 proteins were identified, of which 31 of them were differentially expressed in the E1 cell line. Among these proteins, the differential expressions of translationally controlled tumour protein 1, A-kinase anchor protein 12 and Drebrin (DBN1) were validated using Western blot. In particular, DBN1, a protein not previously known to be involved in colorectal cancer metastasis, was found to be overexpressed in E1 as compared to HCT-116 cells. The overexpression of DBN1 was further validated using immunohistochemistry on colorectal cancer tissue sections with matched lymph node and liver metastasis tissues. DBN1 is currently believed to be involved in actin cytoskeleton reorganisation and suppresses actin filament cross-linking and bundling. Since actin reorganisation is an important process for tumour cell migration and invasion, DBN1 may have an important role during colorectal cancer metastasis.

Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12.

Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP).

Sieving through the cancer secretome.

Cancer is among the most prevalent and serious health problems worldwide. Therefore, there is an urgent need for novel cancer biomarkers with high sensitivity and specificity for early detection and management of the disease. The cancer secretome, encompassing all the proteins that are secreted by cancer cells, is a promising source of biomarkers as the secreted proteins are most likely to enter the blood circulation. Moreover, since secreted proteins are responsible for signaling and communication with the tumor microenvironment, studying the cancer secretome would further the understanding of cancer biology. Latest developments in proteomics technologies have significantly advanced the study of the cancer secretome. In this review, we will present an overview of the secretome sample preparation process and summarize the data from recent secretome studies of six common cancers with high mortality (breast, colorectal, gastric, liver, lung and prostate cancers). In particular, we will focus on the various platforms that were employed and discuss the clinical applicability of the key findings in these studies. This article is part of a Special Issue entitled: An Updated Secretome.

Proteomic analysis of the oil palm fruit mesocarp reveals elevated oxidative phosphorylation activity is critical for increased storage oil production.

Palm oil is a highly versatile commodity with wide applications in the food, cosmetics, and biofuel industries. Storage oil in the oil palm mesocarp can make up a remarkable 80% of its dry mass, making it the oil crop with the richest oil content in the world. As such, there has been an ongoing interest in understanding the mechanism of oil production in oil palm fruits. To identify the proteome changes during oil palm fruit maturation and factors affecting oil yield in oil palm fruits, we examined the proteomic profiles of oil palm mesocarps at four developing stages--12, 16, 18, and 22 weeks after pollination--by 8-plex iTRAQ labeling coupled to 2D-LC and MALDI-TOF/TOF MS. It was found that proteins from several important metabolic processes, including starch and sucrose metabolism, glycolysis, pentose phosphate shunt, fatty acid biosynthesis, and oxidative phosphorylation, were differentially expressed in a concerted manner. These increases led to an increase in carbon flux and a diversion of resources such as ATP and NADH that are required for lipid biosynthesis. The temporal proteome profiles between the high-oil-yielding (HY) and low-oil-yielding (LY) fruits also showed significant differences in the levels of proteins involved in the regulation of the TCA cycle and oxidative phosphorylation. In particular, the expression level of the ß subunit of the ATP synthase complex (complex IV of the electron transport chain) was found to be increased during fruit maturation in HY but decreased in the LY during the fruit maturation. These results suggested that increased energy supply is necessary for augmented oil yield in the HY oil palm trees.

Identification and functional validation of caldesmon as a potential gastric cancer metastasis-associated protein.

In this study, we aim to identify biomarkers for gastric cancer metastasis using a quantitative proteomics approach. The proteins extracted from a panel of 4 gastric cancer cell lines, two derived from primary cancer (AGS, FU97) and two from lymph node metastasis (AZ521, MKN7), were labeled with iTRAQ (8-plex) reagents and analyzed by 2D-LC-MALDI-TOF/TOF MS. In total, 641 proteins were identified with at least a 95% confidence. Using cutoff values of >1.5 and <0.67, 19 proteins were found to be up-regulated and 34 were down-regulated in the metastatic versus primary gastric cancer cell lines respectively. Several of these dysregulated proteins, including caldesmon, were verified using Western blotting. It was found that caldesmon expression was decreased in the two metastasis-derived cell lines, and this was confirmed by further analysis of 7 gastric cancer cell lines. Furthermore, immunohistochemical staining of 9 pairs of primary gastric cancer and the matched lymph node metastasis tissue also corroborated this observation. Finally, knockdown of caldesmon using siRNA in AGS and FU97 gastric cancer cells resulted in an increase in cell migration and invasion, while the overexpression of caldesmon in AZ521 cells led to a decrease in cell migration and invasion. This study has thus established the potential role of caldesmon in gastric cancer metastasis, and further functional studies are underway to delineate the underlying mechanism of action of this protein.

Integrative toxicoproteomics implicates impaired mitochondrial glutathione import as an off-target effect of troglitazone.

Troglitazone, a first-generation thiazolidinedione of antihyperglycaemic properties, was withdrawn from the market due to unacceptable idiosyncratic hepatotoxicity. Despite intensive research, the underlying mechanism of troglitazone-induced liver toxicity remains unknown. Here we report the use of the Sod2(+/-) mouse model of silent mitochondrial oxidative-stress-based and quantitative mass spectrometry-based proteomics to track the mitochondrial proteome changes induced by physiologically relevant troglitazone doses. By quantitative untargeted proteomics, we first globally profiled the Sod2(+/-) hepatic mitochondria proteome and found perturbations including GSH metabolism that enhanced the toxicity of the normally nontoxic troglitazone. Short- and long-term troglitazone administration in Sod2(+/-) mouse led to a mitochondrial proteome shift from an early compensatory response to an eventual phase of intolerable oxidative stress, due to decreased mitochondrial glutathione (mGSH) import protein, decreased dicarboxylate ion carrier (DIC), and the specific activation of ASK1-JNK and FOXO3a with prolonged troglitazone exposure. Furthermore, mapping of the detected proteins onto mouse specific protein-centered networks revealed lipid-associated proteins as contributors to overt mitochondrial and liver injury when under prolonged exposure to the lipid-normalizing troglitazone. By integrative toxicoproteomics, we demonstrated a powerful systems approach in identifying the collapse of specific fragile nodes and activation of crucial proteome reconfiguration regulators when targeted by an exogenous toxicant.

Cancer proteomics.

Cancer presents high mortality and morbidity globally, largely due to its complex and heterogenous nature, and lack of biomarkers for early diagnosis. A proteomics study of cancer aims to identify and characterize functional proteins that drive the transformation of malignancy, and to discover biomarkers to detect early-stage cancer, predict prognosis, determine therapy efficacy, identify novel drug targets, and ultimately develop personalized medicine. The various sources of human samples such as cell lines, tissues, and plasma/serum are probed by a plethora of proteomics tools to discover novel biomarkers and elucidate mechanisms of tumorigenesis. Innovative proteomics technologies and strategies have been designed for protein identification, quantitation, fractionation, and enrichment to delve deeper into the oncoproteome. In addition, there is the need for high-throughput methods for biomarker validation, and integration of the various platforms of oncoproteome data to fully comprehend cancer biology.

Mining the gastric cancer secretome: identification of GRN as a potential diagnostic marker for early gastric cancer.

Gastric cancer is the second leading cause of cancer deaths worldwide, and currently, there are no clinically relevant biomarkers for gastric cancer diagnosis or prognosis. In this study, we applied a 2D-LC-MS/MS based approach, in combination with iTRAQ labeling, to study the secretomes of the gastric cancer cell lines AGS and MKN7. By performing a comparative analysis between the conditioned media and the whole cell lysates, our workflow allowed us to differentiate the bona fide secreted proteins from the intracellular contaminants within the conditioned media. Ninety proteins were found to have higher abundance in the conditioned media as compared to the whole cell lysates of AGS and MKN7 cells. Using a signal peptide and nonclassical secretion prediction tool and an online exosome database, we demonstrated that up to 92.2% of these 90 proteins can be exported out of the cells by classical or nonclassical secretory pathways. We then performed quantitative comparisons of the secretomes between AGS and MKN7, identifying 43 differentially expressed secreted proteins. Among them, GRN was found to be frequently expressed in gastric tumor tissues, but not in normal gastric epithelia by immunohistochemistry. Sandwich ELISA assay also showed elevation of serum GRN levels in gastric cancer patients, particularly those with early gastric cancer. Receiver operating characteristic (ROC) curves analysis confirmed that serum GRN can provide diagnostic discriminations for gastric cancer patients.

Proteomic analysis of colorectal cancer metastasis: stathmin-1 revealed as a player in cancer cell migration and prognostic marker.

Metastasis accounts largely for the high mortality rate of colorectal cancer (CRC) patients. In this study, we performed comparative proteome analysis of primary CRC cell lines HCT-116 and its metastatic derivative E1 using 2-D DIGE. We identified 74 differentially expressed proteins, many of which function in transcription, translation, angiogenesis signal transduction, or cytoskeletal remodeling pathways, which are indispensable cellular processes involved in the metastatic cascade. Among these proteins, stathmin-1 (STMN1) was found to be highly up-regulated in E1 as compared to HCT-116 and was thus selected for further functional studies. Our results showed that perturbations in STMN1 levels resulted in significant changes in cell migration, invasion, adhesion, and colony formation. We further showed that the differential expression of STMN1 correlated with the cells' metastatic potential in other paradigms of CRC models. Using immunohistochemistry, we also showed that STMN1 was highly expressed in colorectal primary tumors and metastatic tissues as compared to the adjacent normal colorectal tissues. Furthermore, we also showed via tissue microarray analyses of 324 CRC tissues and Kaplan-Meier survival plot that CRC patients with higher expression of STMN1 have poorer prognosis.

Identification of potential pathways involved in induction of apoptosis by butyrate and 4-benzoylbutyrate in HT29 colorectal cancer cells.

Butyrate and its analogues have long been investigated as potential chemotherapeutic agents. Our previous structure-activity relationship studies of butyrate analogues revealed that 4-benzoylbutyrate had comparable in vitro effects to butyrate when used to treat HT29 and HCT116 colorectal cancer cell lines. The aim of this study was to identify potential mechanisms associated with the antitumorigenic effects of 4-benzoylbutyrate. In this study, butyrate, 3-hydroxybutyrate and 4-benzoylbutyrate were also investigated for their effects on histone deacetylase (HDAC) activity and histone H4 acetylation in HT29 and HCT116 cells. The biological effects of these analogues on HT29 cells were further investigated using quantitative proteomics to determine the proteins potentially involved in their apoptotic and antiproliferative effects. Because 3-hydroxybutyrate had minimal to no effect on apoptosis, proliferation or HDAC activity, this analogue was used to identify differentially expressed proteins that were potentially specific to the apoptotic effects of butyrate and/or 4-benzoylbutyrate. Butyrate treatment inhibited HDAC activity and induced H4 acetylation. 4-Benzoylbutyrate inhibited HDAC activity but failed to enhance H4 acetylation. Proteomic analysis revealed 20 proteins whose levels were similarly altered by both butyrate and 4-benzoylbutyrate. Proteins that showed common patterns of differential regulation in the presence of either butyrate or 4-benzoylbutyrate included c-Myc transcriptional targets, proteins involved in ER homeostasis, signal transduction pathways and cell energy metabolism. Although an additional 23 proteins were altered by 4-benzoylbutyrate uniquely, further work is required to understand the mechanisms involved in its apoptotic effects.

Network-based pipeline for analyzing MS data: an application toward liver cancer.

Current limitations in proteome analysis by high-throughput mass spectrometry (MS) approaches have sometimes led to incomplete (or inconclusive) data sets being published or unpublished. In this work, we used an iTRAQ reference data on hepatocellular carcinoma (HCC) to design a two-stage functional analysis pipeline to widen and improve the proteome coverage and, subsequently, to unveil the molecular changes that occur during HCC progression in human tumorous tissue. The first involved functional cluster analysis by incorporating an expansion step on a cleaned integrated network. The second used an in-house developed pathway database where recovery of shared neighbors was followed by pathway enrichment analysis. In the original MS data set, over 500 proteins were detected from the tumors of 12 male patients, but in this paper we reported an additional 1000 proteins after application of our bioinformatics pipeline. Through an integrative effort of network cleaning, community finding methods, and network analysis, we also uncovered several biologically interesting clusters implicated in HCC. We established that HCC transition from a moderate to poor stage involved densely connected clusters that comprised of PCNA, XRCC5, XRCC6, PARP1, PRKDC, and WRN. From our pathway enrichment analyses, it appeared that the HCC moderate stage, unlike the poor stage, is enriched in proteins involved in immune responses, thus suggesting the acquisition of immuno-evasion. Our strategy illustrates how an original oncoproteome could be expanded to one of a larger dynamic range where current technology limitations prevent/limit comprehensive proteome characterization.

Identification of key players for colorectal cancer metastasis by iTRAQ quantitative proteomics profiling of isogenic SW480 and SW620 cell lines.

This study compared the whole cell proteome profiles of two isogenic colorectal cancer (CRC) cell lines (primary SW480 cell line and its lymph node metastatic variant SW620), as an in vitro metastatic model, to gain an insight into the molecular events of CRC metastasis. Using iTRAQ (isobaric tags for relative and absolute quantitation) based shotgun proteomics approach, we identified 1140 unique proteins, out of which 147 were found to be significantly altered in the metastatic cell. Ingenuity pathway analysis with those significantly altered proteins, revealed cellular organization and assembly as the top-ranked altered biological function. Differential expression pattern of 6 candidate proteins were validated by Western blot. Among these, the low expression level of ß-catenin combined with the up-regulation of CacyBP (Calcyclin binding Protein), a ß-catenin degrading protein, in the metastatic cell provided a rational guide for the downstream functional assays. The relative expression pattern of these two proteins was further validated in three other CRC cells by Western blot and quantitative immunofluorescence studies. Overexpression of CacyBP in three different primary CRC cell lines showed significant reduction in adhesion characteristics as well as cellular ß-catenin level as confirmed by our experiments, indicating the possible involvement of CacyBP in CRC metastasis. In short, this study demonstrates successful application of a quantitative proteomics approach to identify novel key players for CRC metastasis, which may serve as biomarkers and/or drug targets to improve CRC therapy.

The Sod2 mutant mouse as a model for oxidative stress: a functional proteomics perspective.

Oxidative stress has been implicated in the pathogenesis of numerous human diseases and disorders, but the mechanistic basis often remains enigmatic. The Sod2 mutant mouse, which is sensitized to mitochondrial stress, is an ideal mutant model for studying the role of oxidative stress in a diverse range of complications arising from mitochondrial dysfunction and diminished antioxidant defense. To fully appreciate the widespread molecular consequences under increased oxidative stress, a systems approach utilizing proteomics is able to provide a global overview of the complex biological changes, which a targeted single biomolecular approach cannot address fully. This review focuses on the applications of mass spectrometry and functional proteomics in the Sod2 mouse. The combinatorial approach provides novel insights into the interplay of chemistry and biology, free radicals and proteins, thereby augmenting our understanding of how redox perturbations influence protein dynamics. Ultimately, this knowledge can lead to the development of free radical-targeted therapies.

"Proteomineering" serum biomarkers. A study in scarlet.

The performance of sera pre-treatment for biomarker searching via combinatorial peptide ligand libraries (CPLL) has recently been challenged (Proteomics 2010, 10, 1416-1425) and stated to allow discovery of only medium to high-abundance proteins. We have thus investigated four elution protocols, as published in recent reports: (i) in 4 M urea+1% CHAPS; (ii) in 4 M urea+1% CHAPS+5% acetic acid; (iii) in 8 M urea+2% CHAPS+5% acetic acid; (iv) in boiling 4% SDS+25 mM DTT. One milliliter of serum, in all cases, was captured with 50 µL of CPLL beads, which were then eluted with the four eluants described above. In the first three cases, after the first elution, the beads were re-eluted with cocktail (iv), known to offer maximal release of proteins adsorbed by the CPLL ligands. Eluant (i) released only ca. 20% of the species adsorbed, eluant (ii) ca. 60%, eluant (iii) ca. 80%. Thus, the poor performance of the CPLL methodology, as reported in (i) is not due to any fault of the capture technique, but simply to the adoption of a very poor elution protocol. Even those using eluants (ii) and (iii) should know that a substantial fraction of the captured species still remains bound to the beads and is thus not available to biomarker discovery. Once more, eluant (iv) is recognized as the only one able to offer optimal recovery from the CPLL baits.

Subcellular fractionation methods and strategies for proteomics.

Developments in subcellular fractionation strategies have provided the means to profile and analyze the protein composition of organelles and cellular structures by proteomics. Here, we review the application of classical (e.g. density gradient centrifugation) and emerging sophisticated techniques (fluorescent-assisted organelle sorting) in the fractionation, and statistical/bioinformatics tools for the prediction of protein localization in subcellular proteomics. We also review the validation methods currently used (such as microscopy, RNA interference and multiple reaction monitoring) and discuss the importance of verification of the results obtained in subcellular proteomics. Finally, the numerous challenges facing subcellular proteomics including the dynamics of organelles are being examined. However, complementary approaches such as modern statistics, bioinformatics and large-scale integrative analysis are beginning to emerge as powerful tools to proteomics for analyzing subcellular organelles and structures.