Protein phosphorylation is a central mechanism of cellular signal transduction in living organisms. Phosphoproteomic studies systematically catalogue and characterize alterations in phosphorylation states across multiple cellular conditions and are often incorporated into global proteomics experiments. Previously, we found that spin column-based Fe3+-NTA enrichment integrated well with our workflow but remained a bottleneck for methods that require higher throughput or a scale that is beyond the capacity of these columns. Here, we compare our well-established spin column-based enrichment strategy with one encompassing magnetic beads. Our data show little difference when using either method in terms of the number of identified phosphopeptides as well as their physicochemical properties. In all, we illustrate how the potentially scalable and automation-friendly magnetic Fe3+-NTA beads can seamlessly substitute spin column-based Fe3+-NTA agarose beads for global phosphoproteome profiling. SIGNIFICANCE: Protein phosphorylation plays a key role in regulating a multitude of biological processes and can lead to insights into disease pathogenesis. Methodologies which can efficiently enrich phosphopeptides in a scalable and high-throughput manner are essential for profiling dynamic phosphoproteomes. Here we compare two phosphopeptide enrichment workflows, a well-established spin column-based strategy with agarose Fe3+-NTA beads and a strategy using magnetic Fe3+-NTA beads. Our data suggest that the scalable and automation-friendly magnetic bead-based workflow is an equivalent, but more flexible, enrichment strategy for phosphoproteome profiling experiments.

Protein phosphorylation plays an important role in animal reproduction. However, its role in the onset of puberty in goats remains largely unexplored. Accordingly, in the present study, the molecular changes controlling the onset of puberty in goats were investigated by identifying the differentially phosphorylated proteins (DPPs) and phosphorylation sites (DPSs) in the hypothalami of prepubertal and pubertal female goats using LC-MS/MS and tandem mass tag labelling. A total of 3265 phosphopeptides corresponding to 1628 phosphoproteins were identified, including 234 upregulated and 342 downregulated phosphopeptides. The DPSs HTT, MAP1B, CAMKK1, MAP2, DNAJC5, and GAP43 were identified. These DPPs are enriched in the endocytosis, cAMP signaling, Rap1 signaling, melanogenesis, and insulin secretion pathways. These pathways are related to gonadotropin-releasing hormone and puberty. In particular, glucose-6-phosphate isomerase, fructose-bisphosphate aldolase C, and fructose-bisphosphate aldolase A occupy important locations in the protein-protein interaction network. These data provide evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, they may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals. SIGNIFICANCE: This study provides evidence for a complex interaction network in goat hypothalamus proteins that affects puberty. Furthermore, our data may help identify new puberty-regulating candidates and/or serve as an important resource for exploring the physiological mechanism of puberty onset in mammals.

Erratum in J Proteomics. 2022 Jun 15;261:104590.

BACKGROUND: Verrucous epidermal nevus (VEN) are keratinocytic epidermal nevus that appear at birth or in early childhood. They exhibit a range of manifestations, depending on the patient's age. VEN are rarely encountered in clinical practice, and the systemic and comprehensive clinical characteristics of VEN have not been well investigated. Furthermore, the association between tandem mass tag (TMT)-based quantitative proteomics and the VEN phenotype is still unclear. OBJECTIVES: This study investigated the differences in the clinical characteristics and lesion proteomics between inflammatory linear VEN (ILVEN) and local VEN. METHODS: This retrospective study enrolled 125 patients with histopathologically diagnosed VEN who presented to our hospital between 2019 and 2021. We collected the clinical data of all patients with VEN using a self-designed questionnaire. The expression of proteins in VEN lesions was analyzed using TMT proteomics technology. RESULTS: In total, there were 125 patients with VEN that were evaluated, including 67 (53.60%) patients with local VEN and 58 (46.40%) with ILVEN. No significant differences were found in sex, onset age, and lesion location between patients with local VEN and those with ILVEN (all P > 0.05). Significant differences were found in the onset site and pruritus scores between patients with ILVEN and those with local VEN (all P < 0.05). According to the TMT proteomics results, 89 proteins were up or downregulated with at least 1.3-fold (upregulated: 38, downregulated: 51; P < 0.05) in ILVEN lesions relative to VEN lesions. The top 10 differentially expressed proteins between ILVEN and local VEN lesions were OGN, NT5C3A, ADD1, OLFML1, DHRS1, CALML5, SAMHD1, SFRP2, SPRR1B, and SERPINB13. The upregulated proteins are mainly involved in neutrophil activation, neutrophil-mediated immunity, and p53 signaling pathway (hsa04115). The downregulated proteins are mainly involved in cellular response to cytokine stimulus, cell adhesion, Th1 and Th2 cell differentiation. In total, based on the differentially expressed proteins between ILVEN and local VEN, five pathways that may be associated with the pathogenesis of inflammation, including CAMs (P = 0.006), Th1 and Th2 cell differentiation (P = 0.017), PPAR signaling pathway (P = 0.023), Th17 cell differentiation (P = 0.024), and p53 signaling pathway (P = 0.041). CONCLUSIONS: Clinical data of the patients revealed that ILVEN lesions presented with intense pruritus and inflammatory change. Differentially expressed proteins between ILVEN and local VEN are mainly involved in multiple inflammation related pathways associated with the pathogenesis mechanisms of pruritus. LIMITATIONS: The small sample size in clinical characteristic and proteomics study is one of the most significant limitations in our study. The inflammation associated proteins and signal pathways in the pathogenesis of pruritus in ILVEN is not explored. SIGNIFICANCE: In this study, we found the lesions of ILVEN patients presented with intense pruritus and inflammational change. A total of 89 proteins were up or downregulated with at least 1.3-fold (upregulated: 38, downregulated: 51; P < 0.05) in ILVEN lesions relative to VEN lesions. On the other hand, the etiology of itch in ILVEN mainly associated with inflammation, but the exact mechanisms was still unclear. We found the differentially expressed proteins between ILVEN and local VEN enriched five pathways that may be associated with the pathogenesis of inflammation and pruritus.

Nonalcoholic fatty liver disease (NAFLD) is the leading risk factor for common chronic liver disease and is often regarded as a prevalent metabolic disorder tightly associated with obesity. However, the existence of metabolically healthy obesity (MHO) indicates that some important factors may participate in protecting individuals with MHO free of NAFLD, even with excessive adiposity. To explore factors independent of obesity that may be involved in the occurrence of NAFLD, we performed an iTRAQ-based proteomic study to identify proteins differentially expressed in serum between NAFLD and MHO subjects. Compared with the MHO group, ten proteins were upregulated and five were downregulated significantly in the NAFLD group. Gene Ontology analysis indicated significant changes in the immune response and triglyceride metabolism-related pathways between MHO and NAFLD. We further validated three candidates markedly dysregulated in NAFLD by Western blotting and ELISA, including two upregulated proteins (afamin and apolipoprotein H) and one downregulated protein (apolipoprotein C-1). Detection of serum apolipoprotein H levels in a large-scale cohort with MHO and different stages of NAFLD indicated that apolipoprotein H may be a potential blood biomarker for distinguishing NAFLD from MHO and an independent risk factor for predicting NAFLD.

This study aimed to investigate the characteristic proteomic pattern of plasma from sows supplemented with low dietary ω6:ω3 fatty acids (FAs) ratio during gestation and lactation. Two dietary treatments (n = 8 each) comprised either a control ratio of ω6:ω3 FAs (CR, 13:1 during gestation and 10:1 during lactation) or a low ratio (LR, 4:1 during gestation and lactation) by adding soybean oil or linseed oil, respectively. High-resolution mass spectrometry-based quantitative proteomics was applied on plasma (n = 5 each) at day 108 of gestation (G108) and at the end of lactation (L-End), and a total of 379 proteins and 202 master proteins were identified. Out of these, four differentially abundant proteins between LR and CR samples at G108 may relate to serine-type endopeptidase inhibitor activity. Differentially abundant proteins in L-End versus G108 (12 up-regulated and 10 down-regulated) were positively correlated with the events that regulate plasma lipoproteins, stimulus- and defence-responses. These findings demonstrate the benefit of increased dietary ω3 FAs in modifying proteins involved in protective mechanisms against increased stresses in key life cycle phases in pigs. In addition, proteome changes from late gestation to late lactation disclosed the underlying mechanism of pigs in response to reproduction-related stimuli. SIGNIFICANCE: This study aimed to provide a proteomics insight into the beneficial effects of maternal diet supplementation with a low ω6:ω3 fatty acids ratio, based on previously reported performance and zootechnical data. The results suggest that a low dietary ω6:ω3 fatty acids ratio could enhance the cellular defence mechanisms against increased stresses and in particular to oxidative stress in sows during gestation and lactation, as reflected in proteomic changes of haptoglobin (HP), alpha-1-antitrypsin (SERPINA1) and serum amyloid P-component (APCS). Furthermore, significantly changed proteome profiles in sow plasma between late gestation and lactation phases have been revealed for the first time. This finding identified the adaptation mechanisms of sows to changing physiological events during reproduction.

Protein-protein interaction networks (PPIs) govern the majority of biological processes, but how oncogenic mutations impact these interactions and their functions at a network scale is poorly understood. Mutations of epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) is a pre-requisition for EGFR tyrosine kinase inhibitor (TKI) treatment. Identification of interaction partners that bind to mutated EGFR can help understand the mechanism of action and pathways that mediate drug resistance. In this study, we characterized the dynamic interaction network of a pair of EGFR wildtype and mutant NSCLC cell lines. We performed immunoprecipitation of endogenous EGFR at various time points following EGF treatment and analyzed the associated proteins by quantitative mass spectrometry. Our results showed that the core signaling modules and key downstream pathways are maintained in the mutant cell line, but receptor internalization and intracellular trafficking in the mutant is delayed. Furthermore, we identified mutant EGFR-associated proteins that could affect EGFR functions in lung adenocarcinoma. SIGNIFICANCE: We analyzed the dynamic EGFR interaction network in NSCLC cell lines expressing wild-type and mutant EGFR. By comparing the similarities and differences in the EGFR proteome, we gained a better understanding of EGFR signal transduction network, and identified new factors for further functional characterizations and clinical significance assessment.

Anoxygenic phototrophic bacteria display phenomenal metabolic plasticity leading to distinct phenotypes. Extracellular elevated glucose levels limit photosynthesis in photosynthetic organisms; diversely, cause oxidative stress with ROS generation and "diabetic" like situation in non-photosynthetic organisms. In this study, longer incubations of externally provided glucose (22 mM) inhibited photosynthetic machinery in a phototrophic bacterium, Rubrivivax benzoatilyticus. Data analysis at three time points- exponential, early and late stationary phase, uncovered dynamic protein and metabolite abundance implying metabolic rewiring led non-cultivable state in response to glucose. Protein dynamics datum suggested that proteins related to primary metabolism down-regulated prior to those of secondary metabolism. Numerous proteins for metabolism and energy generation were highly expressed during exponential phase whereas those for membrane transport/translocation and DNA repair accumulated at early and late stationary phase respectively, suggesting a programmed knock-off of phototrophic growth mode and a switch to non-cultivable state. Overall, the omics analyses explicated the metabolic adjustment associated with glucose grown cells of R. benzoatilyticus. Further, our investigation unravelled creation of oxidative stress suggesting physiological stress (oxygen limitation) might be a key player leading to a non-cultivable state in this phototrophic organism. The study, emphasizing microbial glucose intolerance, unlocks the doorway to perceive microorganisms with new perspective. SIGNIFICANCE: Anoxygenic photosynthetic bacteria (APB), thriving under diverse habitat, exhibits magnificent metabolic flexibility. Generally, phototrophy is the preferred growth mode and energy generating route for APB. But, our analyses implicated that the glucose, under phototrophic growth conditions, triggered photobleaching in an APB member, Rubrivivax benzoatilyticus. However, retention of growth along with pigmentation under chemotrophic growth mode supports that glucose gradually knocked off the phototrophic growth mode of R. benzoatilyticus and switched to an alternate energy driving route or less energy demanding non-cultivabile state. Thus, the change in lifestyle i.e. photoheterotrophic growth instead of chemotrophic perhaps, might be the prime culprit and key player in inducing the said state of non-cultivability, akin to diabetes. The study, shedding light on the plausible regulation of cultivability, unveils the programmed regulated switching between different growth modes of the organism and illuminates the importance of glucose intolerance by microorganisms. Through this investigation, we appeal that the studies on 'glucose intolerance in microorganisms' also need due attention that will perhaps change our outlook to perceive micro-organisms in relation to their physiological life style.

Phosphorylation is a major regulatory mechanism controlling protein and cell function. Phosphoproteomics is continuing to reveal the extent and complexity of protein phosphorylation. In particular, most proteins are emerging to contain multiple phosphorylation sites. However, phosphoproteomics has outpaced current understanding of the functional roles of individual phospho-sites. In this paper the Phos-tag gel method is presented and discussed in the context of other available tools for phosphorylation research. Strengths and weaknesses of Phos-tag gels are outlined and an integrated approach to phosphorylation research is proposed. SIGNIFICANCE: The Phos-tag gel method has unique strengths especially regarding the analysis of multi-site phosphorylation. A combined approach including Phos-tag gels together with other methods like isotope labelling, phospho-specific antibodies, and mass spectrometry is required to advance current understanding of protein phosphorylation.

Mass spectrometry-based label-free proteomics is becoming the analytical tool of interest to identify and quantitate the biomarkers for cancer. The oral squamous cell carcinoma (OSCC) which is one of the leading cancers worldwide, lacks biomarkers for the early prognosis and diagnosis. The present study profiled plasma proteome of the Indian OSCC human patients using a label-free mass spectrometry proteomics approach. The study first time utilized the three most widely used data analysis software MaxQuant (MQ), Proteome discoverer (PD), and Trans proteomic pipeline (TPP) together for label-free quantitation of the proteins. The study identified 16 proteins which can be used as a signature protein panel for OSCC. The pathway analysis showed predominant involvement of the immune system, hemostasis as the major pathways that are indicative of the disease progression. The network analysis showed maximum interaction for the Fibronectin and C-reactive protein. The study demonstrates that plasma proteins contain signatures that can be used as putative biomarkers for OSCC. Based on the label-free quantitation and the mechanistic analysis C-reactive protein, Carbonic anhydrase-1, and Fibronectin are identified as putative biomarkers of OSCC. Once these findings are validated in the large cohorts these protein signatures can be used as a biomarker for OSCC. SIGNIFICANCE: The oral squamous cell carcinoma (OSCC) is the eighteenth most prevalent malignancy in the world and ranks second in India. There are no biomarkers that could be indicative of the diseased state. Various studies have been carried out on saliva and tumors of OSCC patients in India, but none of the studies have profiled the plasma. We utilized the label-free approach for the first time on the Indian population in generating the panel of plasma proteins which could be indicative of the diseased state. The study identified Carbonic anhydrase 1, C-reactive protein, and Fibronectin proteins as putative biomarkers for the OSCC. The study obtained the signature panel by utilizing the 3 most widely used software for the label-free quanatitation (LFQ) namely MaxQuant, Proteome Discoverer, and Trans proteomic pipeline. The utilization of 3 software for the LFQ reduced the bias and provided a comprehensive list of proteins. All the differential proteins were mechanistically studied for their biological relevance and the pathway and network analysis were carried out. The study helps us in increasing the understanding of the proteins which are involved in the progression of the diseases. Studying the panel of proteins from all biofluids along with plasma in large cohorts of the population will help in understanding the disease in greater depth and help in identifying the relevant biomarkers for the OSCC.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with no efficient treatment. Researchers have indicated the importance of quantitative approaches on proteome and different post-translation modifications studies both in diagnosis and treatment purposes. Sialic acid-containing glycopeptides (the sialiome) is one of these modifications which can be used as a tool in cancer diagnosis or therapeutic strategies since the sialylation is strongly associated with cancer migration and metastasis. Based on our study, safranal, which is a non-toxic compound in orally intakes, exhibits a significant cytotoxic effect on MDA-MB-231 in comparison to normal cells. We conducted a comprehensive proteomics and sialiomics analysis of safranal treated MDA-MB-231 cells by using a combination of TMT labeling and titanium dioxide enrichment of sialylated N-linked glycopeptides to investigate the underlying molecular mechanism behind safranal-induced apoptosis. Safranal has main effect on the inhibition of metabolism and mitochondrial dysfunction. It regulates proteins considered as activator of DNA fragmentation and apoptosis mediators. Moreover, safranal regulates sialylation of glycoproteins involving in cellular adhesion, migration and survival. It suppresses cell survival and metastasis through the alteration of the sialylation level on important signaling receptors. These results highlight the impact of safranal as a potent anticancer compound on TNBCs which also can be strongly used in daily diets. SIGNIFICANCE: In first step, we evaluated the cell viability of MDA-MB-231 cell lines against the purified saffron components (total crocin, picrocrocin, crocin I and safranal). Safranal was the only compound demonstrated the anti-proliferation effect. In order to obtain an understanding of safranal cytotoxic effect on MDA-MB-231, we designed the three set of treated cell lines in 30 min, 12 h and 24 h time-points in three replicates and a combination of TMT-based labeling quantitative proteomics and titanium dioxide (TiO2)-based enrichment of sialylated N-linked glycopeptides for sialiomics analysis as a strategy to follow the more detailed mechanisms of safranal effect. The results of bioinformatics analysis revealed the multifunction role of safranal on MDA-MB-231 cell lines. Safranal mainly dysregulates mitochondrial function, inhibits metabolism and starts initial signaling of apoptosis which lead to DNA fragmentation. Moreover, safranal caused the majority of down-regulation in sialylation profile in all time-points. Safranal also declines the cell survival, adhesion and migration by dysregulation of the sialylation level in important proteins including integrins, tumor necrosis factor receptor and cell adhesion molecules (CAMs). The results provide a set of therapeutic targets for triple negative breast cancer which can help designing of effective anticancer drugs specially in targeted therapies.

OpenSWATH is an analysis toolkit commonly used for data independent acquisition (DIA). Although the output of OpenSWATH is controlled at 1% false discovery rate (FDR), the output report still contains many peptide precursors with low similarity fragments. At the last step of OpenSWATH for peptide quantification, researchers usually need to manually check the similarity of the extracted ion chromatograms (XICs) of fragments to distinguish the high confidence and the low confidence peptide precursors. Here we developed an algorithm with a Graphic User Interface named MSSort-DIAXMBD, which combines the deep convolutional neural network (CNN) and the double-threshold segmentation process, to automatically recognize the high confidence precursors and low confidence precursors. To train the model of MSSort-DIAXMBD, we built a database contained about 50,000 manually classified peptide precursors acquired from different instrument platforms and different species. With the double-threshold segmentation strategy, MSSort-DIAXMBD can reduce the number of the low confidence peptides required for manual inspections to less than 10% and be used as the last step of OpenSWATH to visualize and classify the MS/MS data of peptide precursors. SIGNIFICANCE: Although the output of OpenSWATH is controlled at 1% false discovery rate (FDR), the output report still contains many peptide precursors with low similarity fragments. At the last step of OpenSWATH for peptide quantification, researchers usually need to manually check the similarity of fragment XICs to distinguish the high confidence and the low confidence peptide precursors. However, manual inspection is inefficient. For instance, it takes about 50 h to sort even a small dataset of 1000 MS/MS spectra manually. In this paper we developed a software named MSSort-DIAXMBD to automatically recognize the high confidence precursors. We manually classify 50,000 peptide precursors as training set to train a convolutional neural network. After training finished, MSSort-DIAXMBD takes only a few minutes to automatically classify 20,000 peptide precursors, leaving a small portion of fuzzy ones for manual inspection. On the benchmarked dataset, MSSort-DIAXMBD can significantly improve the efficiency and accuracy of recognition of high confidence peptide precursors.

Madin-Darby canine kidney (MDCK) cells are widely used in basic research and for the propagation of influenza A viruses (IAV) for vaccine production. To identify targets for antiviral therapies and to optimize vaccine manufacturing, a detailed understanding of the viral life cycle is important. This includes the characterization of virus entry, the synthesis of the various viral RNAs and proteins, the transfer of viral compounds in the cell and virus budding. In case quantitative information is available, the analysis can be complemented by mathematical modelling approaches. While comprehensive studies focusing on IAV entry as well as viral mRNA, vRNA and cRNA accumulation in the nucleus of cells have been performed, quantitative data regarding IAV protein synthesis and accumulation was mostly lacking. In this study, we present a mass spectrometry (MS)-based method to evaluate whether an absolute quantification of viral proteins is possible for single-round replication in suspension MDCK cells. Using influenza A/PR/8/34 (H1N1, RKI) as a model strain at a multiplicity of infection of ten, defined amounts of isotopically labelled peptides of synthetic origin of four IAV proteins (hemagglutinin, neuraminidase, nucleoprotein, matrix protein 1) were added as an internal standard before tryptic digestion of samples for absolute quantification (AQUA). The first intracellular protein detected was NP at 1 h post infection (hpi). A maximum extracellular concentration of 7.7E+12 copies/mL was achieved. This was followed by hemagglutinin (3 hpi, maximum 4.1E+12 copies/mL at 13 hpi), matrix protein 1 (5 hpi, maximum 2.2E+12 copies/mL at 13 hpi) and neuraminidase (5 hpi, 6.0E+11 copies/mL at 13 hpi). In sum, for the first time absolute IAV protein copy numbers were quantified by a MS-based method for infected MDCK cells providing important insights into viral protein dynamics during single-round virus replication. SIGNIFICANCE: Influenza A virus is a significant human pathogen worldwide. To improve therapies against influenza and overcome bottlenecks in vaccine production in cell culture, it is critical to gain a detailed understanding of the viral life cycle. In addition to qPCR-based models, this study will examine the dynamics of influenza virus proteins during infection of producer cells to gain initial insights into changes in absolute copy numbers.

Nano‑selenium (nano-Se) shows high biological activity and low toxicity, and has emerged as an ideal antioxidant. Our goal was to determine the underlying mechanism of nano-Se-mediated heat stress tolerance in rainbow trout (Oncorhynchus mykiss). Using liquid chromatography-mass spectrometry (LC-MS) metabolomics, histomorphology, and conventional biochemical assays, we investigated the physiological responses of heat-stressed rainbow trout to nano-Se. Fish were fed to two levels nano-Se at 18 °C for 9 days: CG18 (0 mg/kg) and Se18 (5 mg/kg). The water temperature of all groups was increased to 24 °C and maintained for 8 h (CG24, Se24). The results showed that most glycerophospholipids and CoA levels were decreased in CG18-CG24, and pathway enrichment analysis showed that it mainly interfered with glycerophospholipids and fatty acid metabolism. Meanwhile, hematoxylin and eosin and Oil Red O staining showed significant damage to CG18-CG24, which was ameliorated by Se18-Se24. The results combining analysis of antioxidant enzymes and heat shock proteins further support the notion that nano-Se supplementation inhibited galactose metabolism and activated the glutamate-glutamine metabolic pathway as the key metabolic strategy against heat stress. These results could establish heat stress defense strategies and increase our understanding of the mechanism of nutrient participation in fish's response to adverse environments. SIGNIFICANCE: Global warming is affecting the distribution and survival of cold-water fish worldwide, through seasonal water temperature increases and an increase in the frequency of extreme heat wave events. Surprisingly, Nano‑selenium (Nano-Se) with its outstanding advantages of high biological activity and low toxicity, making it a good Se nutrient supplement and free radical scavenger, and also an ideal and ecological way to supplement Se. How to utilize the metabolome to better address the complexity of the interactions that may occur with Nano-Se during the process of heat stress resistance is an important challenge. In the present study, this is the first publicly available metabonomics study of the anti-heat stress effect of Nano-Se as a nutrient on rainbow trout liver. These data indicated that Nano-Se effectively alleviated stress damage in rainbow trout, in which heat stress interfered with the metabolism of glycerophospholipids and fatty acids significantly, causing liver cell membrane damage and lipid metabolism disorders in rainbow trout. Meanwhile, supplementation of Nano-Se downregulated galactose metabolism and activated glutamate and glutamine metabolic pathways, which seems to be a key metabolic strategy to combat heat stress. The results provide a scientific basis for the development of an anti-heat-stress feed for rainbow trout that help maintain their health, productivity and welfare under unfavorable heat conditions.

This study focuses on comprehensive characterization of the venom proteome of the beaked sea snake (Hydrophis schistosus) from Songkhla Lake, Thailand. H. schistosus can be considered as the deadliest sea snake commonly found in the Pacific and Indian oceans. Their envenomation causes muscular paralysis and rhabdomyolysis. To develop effective treatment for this snakebite, it is necessary to understand the detailed venom composition. In this study, multiple mass spectrometry-based approaches were employed. Bottom-up proteomics revealed that tryptic digestion in-solution provided a higher number of toxin proteins identified and a larger sequence coverage, compared to in-gel digestion. In addition, a venom gland transcriptome-derived database was constructed and used as a reference, which 43 known and novel toxin proteins were identified using this database and the UniProtKB. Three-finger toxin and phospholipase A2 were shown to be top two most abundant protein families. Minor compositions included other toxin families and a number of non-toxin proteins. Moreover, a hybrid de novo sequencing was performed to enhance identification of the small proteins/peptides. Using non-digested samples, there were 46 predicted toxin peptides. The finding from this study could lead to a better understanding in pathological effects of the snakebite and the future development of effective antivenoms. SIGNIFICANCE: This study provides a better understanding of the venom proteome composition of the beaked sea snake (H. schistosus) found in the Gulf of Thailand, using a combination of different sample preparation techniques, Serpentes protein database searching, transcriptome-derived protein database searching, and a hybrid de novo peptide sequencing strategy. It revealed 13 toxin protein families and novel proteins in the beaked sea snake venom including new species of phospholipase A2s (PLA2s) and three-finger toxins (3FTxs). It could serve as a basis for the development of snakebite treatments and for the discovery of novel pharmaceutical drugs from the toxin peptides.