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.

Global analysis of RNA-binding proteins identifies a positive feedback loop between LARP1 and MYC that promotes tumorigenesis.

In addition to genomic alterations, aberrant changes in post-transcriptional regulation can modify gene function and drive cancer development. RNA-binding proteins (RBPs) are a large class of post-transcriptional regulators that have been increasingly implicated in carcinogenesis. By integrating multi-omics data, we identify LARP1 as one of the most upregulated RBPs in colorectal cancer (CRC) and demonstrate its oncogenic properties. We perform LARP1:RNA interactome profiling and unveil a previously unexplored role for LARP1 in targeting the 3'UTR of oncogenes in CRC. Notably, we identify the proto-oncogenic transcription factor MYC as a key LARP1-regulated target. Our data show that LARP1 positively modulates MYC expression by associating with its 3'UTR. In addition, antisense oligonucleotide-mediated blocking of the interaction between LARP1 and the MYC 3'UTR reduces MYC expression and in vitro CRC growth. Furthermore, a systematic analysis of LARP1:protein interactions reveals IGF2BP3 and YBX1 as LARP1-interacting proteins that also regulate MYC expression and CRC development. Finally, we demonstrate that MYC reciprocally modulates LARP1 expression by targeting its enhancer. In summary, our data reveal a critical, previously uncharacterized role of LARP1 in promoting CRC tumorigenesis, validate its direct regulation of the proto-oncogene MYC and delineate a model of the positive feedback loop between MYC and LARP1 that promotes CRC growth and development.

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.

Subcellular Proteome Landscape of Human Embryonic Stem Cells Revealed Missing Membrane Proteins.

Human embryonic stem cells (hESCs) have the capacity for self-renewal and multilineage differentiation, which are of clinical importance for regeneration medicine. Despite the significant progress of hESC study, the complete hESC proteome atlas, especially the surface protein composition, awaits delineation. According to the latest release of neXtProt database (January 17, 2018; 19 658 PE1, 2, 3, and 4 human proteins), membrane proteins present the major category (1047; 48%) among all 2186 missing proteins (MPs). We conducted a deep subcellular proteomics analysis of hESCs to identify the nuclear, cytoplasmic, and membrane proteins in hESCs and to mine missing membrane proteins in the very early cell status. To our knowledge, our study achieved the largest data set with confident identification of 11 970 unique proteins (1% false discovery rate at peptide, protein, and PSM levels), including the most-comprehensive description of 6 138 annotated membrane proteins in hESCs. Following the HPP guideline, we identified 26 gold (neXtProt PE2, 3, and 4 MPs) and 87 silver (potential MP candidates with a single unique peptide detected) MPs, of which 69 were membrane proteins, and the expression of 21 gold MPs was further verified either by multiple reaction monitoring mass spectrometry or by matching synthetic peptides in the Peptide Atlas database. Functional analysis of the MPs revealed their potential roles in the pluripotency-related pathways and the lineage- and tissue-specific differentiation processes. Our proteome map of hESCs may provide a rich resource not only for the identification of MPs in the human proteome but also for the investigation on self-renewal and differentiation of hESC. All mass spectrometry data were deposited in ProteomeXchange via jPOST with identifier PXD009840.

S-Nitrosylation of Divalent Metal Transporter 1 Enhances Iron Uptake to Mediate Loss of Dopaminergic Neurons and Motoric Deficit.

Elevated iron deposition has been reported in Parkinson's disease (PD). However, the route of iron uptake leading to high deposition in the substantia nigra is unresolved. Here, we show a mechanism in enhanced Fe2+ uptake via S-nitrosylation of divalent metal transporter 1 (DMT1). While DMT1 could be S-nitrosylated by exogenous nitric oxide donors, in human PD brains, endogenously S-nitrosylated DMT1 was detected in postmortem substantia nigra. Patch-clamp electrophysiological recordings and iron uptake assays confirmed increased Mn2+ or Fe2+ uptake through S-nitrosylated DMT1. We identified two major S-nitrosylation sites, C23 and C540, by mass spectrometry, and DMT1 C23A or C540A substitutions abolished nitric oxide (NO)-mediated DMT1 current increase. To evaluate in vivo significance, lipopolysaccharide (LPS) was stereotaxically injected into the substantia nigra of female and male mice to induce inflammation and production of NO. The intranigral LPS injection resulted in corresponding increase in Fe2+ deposition, JNK activation, dopaminergic neuronal loss and deficit in motoric activity, and these were rescued by the NO synthase inhibitor l-NAME or by the DMT1-selective blocker ebselen. Lentiviral knockdown of DMT1 abolished LPS-induced dopaminergic neuron loss.SIGNIFICANCE STATEMENT Neuroinflammation and high cytoplasmic Fe2+ levels have been implicated in the initiation and progression of neurodegenerative diseases. Here, we report the unexpected enhancement of the functional activity of transmembrane divalent metal transporter 1 (DMT1) by S-nitrosylation. We demonstrated that S-nitrosylation increased DMT1-mediated Fe2+ uptake, and two cysteines were identified by mass spectrometry to be the sites for S-nitrosylation and for enhanced iron uptake. One conceptual advance is that while DMT1 activity could be increased by external acidification because the gating of the DMT1 transporter is proton motive, we discovered that DMT1 activity could also be enhanced by S-nitrosylation. Significantly, lipopolysaccharide-induced nitric oxide (NO)-mediated neuronal death in the substantia nigra could be ameliorated by using l-NAME, a NO synthase inhibitor, or by ebselen, a DMT1-selective blocker.

Label-Free Quantitative Phosphoproteomics Reveals Regulation of Vasodilator-Stimulated Phosphoprotein upon Stathmin-1 Silencing in a Pair of Isogenic Colorectal Cancer Cell Lines.

In this communication, we present the phosphoproteome changes in an isogenic pair of colorectal cancer cell lines, viz., the poorly metastatic HCT-116 and the highly metastatic derivative E1, upon stathmin-1 (STMN1) knockdown. The aim was to better understand how the alterations of the phosphoproteins in these cells are involved in cancer metastasis. After the phosphopeptides were enriched using the TiO2 HAMMOC approach, comparative proteomics analysis was carried out using sequential window acquisition of all theoretical mass spectra-MS. Following bioinformatics analysis using MarkerView and OneOmics platforms, we identified a list of regulated phosphoproteins that may play a potential role in signaling, maintenance of cytoskeletal structure, and focal adhesion. Among these phosphoproteins, was the actin cytoskeleton regulator protein, vasodilator-stimulated phosphoprotein (VASP), where its change in phosphorylation status was found to be concomitant with STMN1-associated roles in metastasis. We further showed that silencing of stathmin-1 altered the expression, subcellular localization and phosphorylation status of VASP, which suggested that it might be associated with remodeling of the cell cytoskeleton in colorectal cancer metastasis.

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).

Unbiased proteomic and transcript analyses reveal that stathmin-1 silencing inhibits colorectal cancer metastasis and sensitizes to 5-fluorouracil treatment.

UNLABELLED: Colorectal cancer metastasis is a major cause of mortality worldwide, which may only be controlled with novel methods limiting tumor dissemination and chemoresistance. High stathmin-1 (STMN1) expression was previously established as a hallmark of colorectal cancer progression and predictor of poor survival; however, the mechanism of action is less clear. This work demonstrates that STMN1 silencing arrests tumor-disseminative cascades by inhibiting multiple metastatic drivers, and repressing oncogenic and mesenchymal transcription. Using a sensitive iTRAQ labeling proteomic approach that quantified differential abundance of 4562 proteins, targeting STMN1 expression was shown to reinstate the default cellular program of metastatic inhibition, and promote cellular adhesion via amplification of hemidesmosomal junctions and intermediate filament tethering. Silencing STMN1 also significantly improved chemoresponse to the classical colorectal cancer therapeutic agent, 5FU, via a novel caspase-6 (CASP6)-dependent mechanism. Interestingly, the prometastatic function of STMN1 was independent of p53 but required phosphorylations at S25 or S38; abrogating phosphorylative events may constitute an alternative route to achieving metastatic inhibition. These findings establish STMN1 as a potential target in antimetastatic therapy, and demonstrate the power of an approach coupling proteomics and transcript analyses in the global assessment of treatment benefits and potential side-effects. IMPLICATIONS: Stathmin-1 is a potential candidate in colorectal cancer therapy that targets simultaneously the twin problems of metastatic spread and chemoresistance.

Novel proteomic biomarker panel for prediction of aggressive metastatic hepatocellular carcinoma relapse in surgically resectable patients.

The natural course of early HCC is unknown, and its progression to intermediate and advanced HCC can be diverse. Some early stage HCC patients enjoy prolonged disease-free survival, whereas others suffer aggressive relapse to stage IV metastatic cancer within a year. Comparative proteomics of HCC tumor tissues was carried out using 2D-DIGE and MALDI-TOF/TOF MS to identify proteins that can distinguish these two groups of stage I HCC patients. Twelve out of 148 differentially regulated protein spots were found to differ by approximately 2-fold for the relapse versus nonrelapse patient tissues. Four proteins, namely, heat shock 70 kDa protein 1, argininosuccinate synthase, isoform 2 of UTP-glucose-1-phosphate uridylyltransferase, and transketolase, were shown to have the potential to differentiate metastatic relapse (MR) from nonrelapse (NR) HCC patients after validation by western blotting and immunohistochemical assays. Subsequent TMA analysis revealed a three marker panel of HSP70, ASS1, and UGP2 to be statistically significant in stratifying the two groups of HCC patients. This combination panel achieved high levels of sensitivity and specificity, which has potential for clinical use in identifying HCC tumors prone to MR. This stratification will allow development of clinical management, including close follow-up and possibly treatment options, in the near future.

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).

Prognostic biomarkers for prediction of recurrence of hepatocellular carcinoma: current status and future prospects.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer death worldwide, with region specific etiologies. Despite improvements made in the diagnosis of HCC, the prognosis of HCC patients remains poor due to the high recurrence rate of HCC. There is an urgent need for development of prognostic biomarkers to predict the risk of recurrence in HCC patients after "curative" treatment. Such stratification may aid in patient management and development of personalized medicine for HCC treatment. Omics based studies facilitate the study of global changes in biomolecules in a disease in a high throughput manner, and hence are well poised to understand the complex changes which led to HCC recurrence. The quantitative nature of data obtained from omics based studies allow for development of prognostic biomarkers based on changes in gene, protein and metabolite expression. In this review, we surveyed the application of transcriptomics, proteomics and metabolomics in the study of HCC recurrence. We summarised the data in the literature from these three fields of studies that claimed to be prognostic for HCC recurrence. We critiqued on the limitations of each area of research and the challenges faced in translating the research results for clinical application in predicting HCC recurrence.

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.

Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum.

Conventional leaching (extraction) methods for gold recovery from electronic waste involve the use of strong acids and pose considerable threat to the environment. The alternative use of bioleaching microbes for gold recovery is non-pollutive and relies on the secretion of a lixiviant or (bio)chemical such as cyanide for extraction of gold from electronic waste. However, widespread industrial use of bioleaching microbes has been constrained by the limited cyanogenic capabilities of lixiviant-producing microorganisms such as Chromobacterium violaceum. Here we show the construction of a metabolically-engineered strain of Chromobacterium violaceum that produces more (70%) cyanide lixiviant and recovers more than twice as much gold from electronic waste compared to wild-type bacteria. Comparative proteome analyses suggested the possibility of further enhancement in cyanogenesis through subsequent metabolic engineering. Our results demonstrated the utility of lixiviant metabolic engineering in the construction of enhanced bioleaching microbes for the bioleaching of precious metals from electronic waste.

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.