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.

Next Generation Proteomics for Clinical Biomarker Detection Using SWATH-MS.

The technology of "sequential windowed acquisition of all theoretical fragment ion spectra," known as SWATH-MS, is rapidly gaining popularity as a next generation proteomics technology for comprehensive proteome quantitation. In this chapter, we describe the use of SWATH-MS as a label-free quantitative technique in a proteomics study to identify novel serological biomarker for colorectal cancer. We compared the secreted glycoprotein profiles (glyco-secretomes) enriched from the colon adenocarcinoma cell line HCT-116 and its metastatic derivative, E1, and observed that laminin ß-1 (LAMB1) was oversecreted in E1 cells. This novel oversecretion of LAMB1 was validated in colorectal cancer patient serum samples, and ROC analyses showed that LAMB1 performed better than carcinoembryonic antigen (CEA) as a clinical diagnostic biomarker for colorectal cancer. We focus here on the sample preparation methodology and data processing workflow for SWATH-MS studies.

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.

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.

The prognostic value of the stem-like group in colorectal cancer using a panel of immunohistochemistry markers.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the Western world. It is becoming increasingly clear that CRC is a diverse disease, as exemplified by the identification of subgroups of CRC tumours that are driven by distinct biology. Recently, a number of studies have begun to define panels of diagnostically relevant markers to align patients into individual subgroups in an attempt to give information on prognosis and treatment response. We examined the immunohistochemical expression profile of 18 markers, each representing a putative role in cancer development, in 493 primary colorectal carcinomas using tissue microarrays. Through unsupervised clustering in stage II cancers, we identified two cluster groups that are broadly defined by inflammatory or immune-related factors (CD3, CD8, COX-2 and FOXP3) and stem-like factors (CD44, LGR5, SOX2, OCT4). The expression of the stem-like group markers was associated with a significantly worse prognosis compared to cases with lower expression. In addition, patients classified in the stem-like subgroup displayed a trend towards a benefit from adjuvant treatment. The biologically relevant and poor prognostic stem-like group could also be identified in early stage I cancers, suggesting a potential opportunity for the identification of aggressive tumors at a very early stage of the disease.

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).

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).

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.

Proteomics discovery of biomarkers for mitral regurgitation caused by mitral valve prolapse.

Mitral regurgitation (MR) is a common valvular lesion frequently caused by mitral valve prolapse (MVP). Surgical intervention in MVP patients with significant MR is predicated on symptoms and measures of left ventricular dysfunction. Because these indicators may be subjective or imprecise, serological biomarkers of disease could be a valuable adjunct to standard evaluation. This study aimed to identify such biomarkers by a proteomics approach. Two pooled plasma samples from 24 MVP subjects with MR (MVP/MR) and 24 non-MVP individuals were treated with the combinatorial peptide ligand library (CPLL) beads prior to iTRAQ labeling and ESI-MS/MS. Lower levels of haptoglobin, platelet basic protein (PBP), and complement component C4b were observed in the MVP/MR as compared to the control sample. These findings were verified by ELISA testing of each of the 24 paired samples, and another 42 matched cases and controls. The AUC values, sensitivities and specificities for (i) haptoglobin, (ii) PBP, (iii) C4b, and (iv) all 3 proteins in combination were (i) 0.813, 76%, 74%; (ii) 0.721, 56%, 77%; (iii) 0.689, 83%, 49%; and (iv) 0.840, 89%, 67%, respectively. In conclusion, haptoglobin, PBP, and C4b are down-regulated in MVP/MR. Their value as serological biomarkers of valvular pathology should be further explored. BIOLOGICAL SIGNIFICANCE: We report the first study that performed comparative proteomics of clinical human plasma samples to identify novel diagnostic biomarkers for mitral valve prolapse (MVP) patients with moderate to severe mitral regurgitation (MR). MR is a common valvular lesion that can be complicated by heart failure, sudden death and atrial fibrillation, yet many patients with severe MR are asymptomatic. Our results revealed reduced levels of haptoglobin, platelet basic protein (PBP), and complement component C4b in the MVP/MR patients as compared to the matched control cases. The plasma proteomics findings were subsequently confirmed by ELISA. Each of these candidate biomarkers has a putative role in the pathophysiology of MVP/MR, further supporting their roles in detection and possibly surveillance and prognostication of this disease.

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.

The gastric fluid proteome as a potential source of gastric cancer biomarkers.

Gastric cancer is a significant cause of death in many parts of the world. Although timely intervention is associated with better clinical outcome, early gastric cancer detection is frequently not possible given its asymptomatic nature. As such, sensitive and specific gastric cancer biomarkers are highly sought after as diagnostic surrogates that may replace invasive endoscopic and histological examinations. Unlike gastric cancer tissue and serum which are heterogeneous and overloaded with abundant proteins, the gastric fluid contains a concentrated molecular biopsy of the stomach that accurately reflects gastric oncology. This review attempts to (i) summarise the state of proteomics-based gastric cancer biomarker discovery from patient gastric fluids, (ii) outline key considerations in working with the body fluid, and (ii) discuss how the challenges in gastric cancer diagnosis may be overcome with new perspectives in gastric cancer screening.

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.

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.

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.

Heart extracellular matrix supports cardiomyocyte differentiation of mouse embryonic stem cells.

We have evaluated the effect of heart extracellular matrix (ECM) on the cardiomyocyte differentiation of mouse embryonic stem cells (ES cells) using de-cellularized heart tissue. Several lines of evidence indicate that ECM plays significant roles in cell proliferation, cell death and differentiation, but role of ECM possessing a 3D structure in differentiation has not been studied in detail. We found that there are substantial differences in the quantitative protein profiles of ECM in SDS-treated heart tissue compared to that of liver tissue, as assessed by iTRAQ™ quantitative proteomics analysis. When mouse ES cells were cultured on thin (60 µm) sections of de-cellularized tissue, the expression of cardiac myosin heavy chain (cMHC) and cardiac troponin I (cTnI) was high in ES cells cultured on heart ECM compared with those cultured on liver ECM. In addition, the protein expression of cMHC and cTnI was detected in cells on heart ECM after 2 weeks, which was not detectable in cells on liver ECM. These results indicate that heart ECM plays a critical role in the cardiomyocyte differentiation of ES cells. We propose that tissue-specific ECM induced cell lineage specification through mechano-transduction mediated by the structure, elasticity and components of ECM.

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.

A network-based maximum link approach towards MS identifies potentially important roles for undetected ARRB1/2 and ACTB in liver cancer progression.

Hepatocellular Carcinoma (HCC) ranks among the deadliest of cancers and has a complex etiology. Proteomics analysis using iTRAQ provides a direct way to analyse perturbations in protein expression during HCC progression from early- to late-stage but suffers from consistency and coverage issues. Appropriate use of network-based analytical methods can help to overcome these issues. We built an integrated and comprehensive Protein-Protein Interaction Network (PPIN) by merging several major databases. Additionally, the network was filtered for GO coherent edges. Significantly differential genes (seeds) were selected from iTRAQ data and mapped onto this network. Undetected proteins linked to seeds (linked proteins) were identified and functionally characterised. The process of network cleaning provides a list of higher quality linked proteins, which are highly enriched for similar biological process gene ontology terms. Linked proteins are also enriched for known cancer genes and are linked to many well-established cancer processes such as apoptosis and immune response. We found that there is an increased propensity for known cancer genes to be found in highly linked proteins. Three highly-linked proteins were identified that may play an important role in driving HCC progression - the G-protein coupled receptor signalling proteins, ARRB1/2 and the structural protein beta-actin, ACTB. Interestingly, both ARRB proteins evaded detection in the iTRAQ screen. ACTB was not detected in the original dataset derived from Mascot but was found to be strongly supported when we re-ran analysis using another protein detection database (Paragon). Identification of linked proteins helps to partially overcome the coverage issue in shotgun proteomics analysis. The set of linked proteins are found to be enriched for cancer-specific processes, and more likely so if they are more highly linked. Additionally, a higher quality linked set is derived if network-cleaning is performed prior. This form of network-based analysis complements the cluster-based approach, and can provide a larger list of proteins on which to perform functional analysis, as well as for biomarker identification.

S100A9, GIF and AAT as potential combinatorial biomarkers in gastric cancer diagnosis and prognosis.

PURPOSE: We have mined the gastric fluid proteome for potential gastric cancer (GC) biomarkers that may enhance disease detection and facilitate prognostic monitoring. EXPERIMENTAL DESIGN: In biomarker discovery, a total of 12 patient gastric fluid samples (stages I, III, IV and gastritis) were analysed by 2DE for expression changes that correlated with GC status or disease progression. Gastric fluid proteins showing differential expression with GC were identified by MALDI-TOF/TOF MS as putative biomarkers. Levels of these potential biomarker candidates were independently validated by Western blotting in further 60 gastritis and GC patients. A targeted approach that recruits biomarker candidates for panel consideration was adopted to test if two or more biomarkers in combination improved diagnostic power. RESULTS: From the 15 differentially regulated proteins identified, expression levels of S100A9, GIF and AAT in the gastric fluid clearly correlated with GC status. S100A9/AAT (AUC = 0.81) and S100A9/GIF (AUC = 0.92) were revealed as promising biomarker pairs for early GC diagnosis and disease monitoring, respectively. CONCLUSION AND CLINICAL RELEVANCE: Early diagnosis, accurate staging and constant disease monitoring remain the prerequisites for effective treatment against GC. As current biomarkers like CA19-9 and carcinoembryonic antigen (CEA) lack sensitivity and specificity, there is a pressing need for novel GC detection and monitoring methods. To this end, S100A9, GIF and AAT from the gastric fluid may significantly augment existing methods of GC detection and monitoring, and eliminate the need for invasive tissue biopsies.

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.