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Peptidomics is the detection and identification of the peptides present in a sample, and quantitative peptidomics provides additional information about the amounts of these peptides. It is possible to perform absolute quantitation of peptide levels in which the biological sample is compared to synthetic standards of each peptide. More commonly, relative quantitation is performed to compare peptide levels between two or more samples. Relative quantitation can measure differences between all peptides that are detectable, which can exceed 1000 peptides in a complex sample. In this chapter, various techniques used for quantitative peptidomics are described along with discussion of the advantages and disadvantages of each approach. A guide to selecting the optimal quantitative approach is provided, based on the goals of the experiment and the resources that are available.

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Hepatocellular carcinoma (HCC) is associated with high rates of metastasis and recurrence, and is one of the most common causes of cancer-associated death worldwide. This study examined the protein changes within circulating exosomes in patients with HCC against those in healthy people using isobaric tags for a relative or absolute quantitation (iTRAQ)-based quantitative proteomics analysis. The protein levels of von Willebrand factor (VWF), cathelicidin antimicrobial peptide (CAMP), and proteasome subunit beta type-2 (PSMB2) were altered in HCC. The increased levels of VWF and PSMB2 but decreased CAMP levels in the serum of patients with HCC were validated by enzyme-linked immunosorbent assays. The level of CAMP (the only cathelicidin found in humans) also decreased in the circulating exosomes and buffy coat of the HCC patients. The serum with reduced levels of CAMP protein in the HCC patients increased the cell proliferation of Huh-7 cells; this effect was reduced following the addition of CAMP protein. The depletion of CAMP proteins in the serum of healthy people enhances the cell proliferation of Huh-7 cells. In addition, supplementation with synthetic CAMP reduces cell proliferation in a dose-dependent manner and significantly delays G1-S transition in Huh-7 cells. This implies that CAMP may act as a tumor suppressor in HCC.

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OBJECTIVE: This study aims to investigate the effect of methylprednisolone (MP) intrathecal injection on a rat model of acute spinal cord injury (ASCI). METHODS: Allen's frame was used to establish a rat model of ASCI. MP and normal saline were intrathecally injected to Sprague-Dawley rats at 0, 3, 6, 8, 12, and 24 hours after ASCI, and injured spinal cord tissues were sterilely extracted after 24 hours of treatment. Isobaric tags for relative and absolute quantitation (iTRAQ) were coupled with 2-dimensional liquid chromatography tandem mass spectrometry to separate and identify differentially expressed proteins. RESULTS: The expression of growth factor receptor-bound protein 2 (Grb2) was downregulated in the MP groups at 0 hours (iTRAQ ratio = 0.996), 3 hours (iTRAQ ratio = 0.737), 8 hours (iTRAQ ratio = 0.763), and 24 hours (iTRAQ ratio = 0.908) after injury compared with that in the control groups. No significant difference in Grb2 expression was observed between the control groups at 6 and 12 hours after ASCI. CONCLUSIONS: Standardized MP intrathecal injection after ASCI treatment reduces Grb2 activation in a rat ASCI model. Further studies should determine whether or not the same effect can be observed in human ASCIs.

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Spiroplasma eriocheiris is one of the major pathogenic bacteria in crustaceans, featuring high infectivity, rapid transmission, and an absence of effective control strategies, resulting in significant economic losses to the aquaculture industry. Research into virulence-related factors provides an important perspective to clarify how Spiroplasma eriocheiris is pathogenic to shrimps and crabs. Therefore, in this study, isobaric tags for relative and absolute quantitation (iTRAQ) technology was utilized to undertake a differential proteomic analysis of high- and low-virulence Spiroplasma eriocheiris strains at different growth phases. A total of 868 differentially expressed proteins (DEPs) were obtained, of which 31 novel proteins were identified by proteogenomic analysis. There were 62, 61, 175, and 235 DEPs between the log phase (YD) and non-log phase (YFD) of the high-virulence strain, between the log phase (CD) and non-log phase (CFD) of the low-virulence strain, between YD and CD, and between CFD and YFD, respectively. All the DEPs were compared with virulence protein databases (MvirDB and VFDB), and 68 virulence proteins of Spiroplasma eriocheiris were identified, of which 12 were involved in a total of 21 metabolic pathways, including motility, chemotaxis, growth, metabolism and virulence of the bacteria. The results of this study form the basis for further research into the molecular mechanism of virulence and physiological differences between high- and low-virulence strains of Spiroplasma eriocheiris, and provide a scientific basis for a detailed understanding of its pathogenesis.

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High-throughput quantitative analysis of the cells' proteomes across multiple conditions such as various perturbations and different time points is essential for gaining insights into treatment-induced biological responses or disease pathological states. The advancements in mass spectrometry instrumentation and isobaric labeling methods provided useful tools to help address such demands. However, the current widely adopted isobaric labeling methods such as tandem mass tag (TMT) and isobaric tags for relative and absolute quantitation (iTRAQ) are based on low-mass reporter ions, which are indistinguishable among different peptide analytes, to achieve relative quantification. Therefore, these methods intrinsically suffer from severe ratio distortion when analyzing complex samples due to peptide coelution and cofragmentation. Here, we developed a novel set of isobaric tags named dimethylated leucine complementary ion (DiLeuC) and relied on complementary ions for relative quantification, in which the complementary ions are the remanent peptide segments after fragmentation in the high-mass range. Since those residual peptide fragments are precursor-specific, they retain the relative abundance information in an interference-free manner even in a complex matrix environment. The quantification accuracy of our method was validated in a two-proteome model where the yeast proteome was spiked with a strong background human proteome as interference. In addition, we also applied this strategy to single-cell proteome analysis, demonstrating its potential utility for sensitive high-throughput quantitative proteomics.

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A growing body of evidence has linked the gut microbiota to liver metabolism. The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health. However, the effects of Lactobacillus gasseri LA39, a potential probiotic, on liver metabolism remain unclear. Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes, and used the germ-free (GF) mouse model to evaluate host-microbe interaction. Here, we explored the effects of L. gasseri LA39 gavage on the protein expression profiles of the liver of GF mice. Our results showed that a total of 128 proteins were upregulated, whereas a total of 123 proteins were downregulated by treatment with L. gasseri LA39. Further bioinformatics analyses suggested that the primary bile acid (BA) biosynthesis pathway in the liver was activated by L. gasseri LA39. Three differentially expressed proteins (cytochrome P450 family 27 subfamily A member 1 (CYP27A1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1)) involved in the primary BA biosynthesis pathway were further validated by western blot assay. In addition, targeted metabolomic analyses demonstrated that serum and fecal ß|-muricholic acid (a primary BA), dehydrolithocholic acid (a secondary BA), and glycolithocholic acid-3-sulfate (a secondary BA) were significantly increased by L. gasseri LA39. Thus, our data revealed that L. gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation. Based on these findings, we suggest that L. gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

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Cellular health, functionality, response to environment, and other variables affecting cell, tissue, or organ viability are reflected in the cellular proteomes and metabolomes. These "omic" profiles are in constant flux even during normal cellular functioning, to maintain cellular homeostasis, in response to small environmental changes and maintenance of optimal cell viability. However proteomic "fingerprints" can also provide insight into cellular ageing, response to disease, adjustment to environmental changes, and other variables that impact cellular viability. A variety of proteomic methods can be used to determine qualitative and quantitative proteomic change. In this chapter, we will focus on a labeling method called isobaric tags for relative and absolute quantification (iTRAQ), which is frequently used to identify and quantify proteomic expression changes in cells and tissues.

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Many studies have been conducted to improve technology for semen cryopreservation in pigs. However, computer-assisted analysis of sperm motility and morphology is insufficient to predict the molecular function of frozen-thawed semen. More accurate expression patterns of boar sperm proteins may be derived using the isobaric tags for relative and absolute quantification (iTRAQ) technique. In this study, the iTRAQ-labeling system was coupled with liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis to identify differentially expressed CM10-fractionated proteins between fresh and frozen-thawed boar semen. A total of 76 protein types were identified to be differentially expressed, among which 9 and 67 proteins showed higher and lower expression in frozen-thawed than in fresh sperm samples, respectively. The classified functions of these proteins included oxidative phosphorylation, mitochondrial inner membrane and matrix, and pyruvate metabolic processes, which are involved in adenosine triphosphate (ATP) synthesis; and sperm flagellum and motile cilium, which are involved in sperm tail structure. These results suggest a possible network of biomarkers associated with survival after the cryopreservation of Duroc boar semen.

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Glycans, which are widely distributed on most proteins and cell surfaces, are a class of important biomolecules playing crucial roles in various biological processes such as immune response and cellular communication. Modern mass spectrometry (MS) coupled with novel chemical probes greatly facilitates routine analysis of glycans. However, the requirement of high-throughput analysis still calls for advanced tools to be developed. Recently, we devised isobaric multiplex reagents for carbonyl-containing compound (SUGAR) tags for 4-plex N-glycan analysis. To further improve the throughput, we utilized the subtle mass differences among different isotopologues and expanded the multiplexing capacity to 12 channels, a 3-fold throughput improvement for the original SUGAR tag design and achieved high-throughput N-glycan analysis in a single LC-MS/MS injection. We then applied 12-plex SUGAR tags to profile the N-glycans in four subtypes of human Immunoglobulin G (IgG) and to investigate the N-glycan changes in the endometrial cancer cells (ECC1) treated with Atovaquone, a quinone antimicrobial medication, and a dihydroorotate dehydrogenase (DHODH) inhibitor. Data are available via ProteomeXchange with the identifier PXD038501.

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BACKGROUND: Neurofibrillary tangles comprising hyperphosphorylated tau are vital factors associated with the pathogenesis of Alzheimer's disease (AD). The elimination or reduction of hyperphosphorylated and abnormally aggregated tau is a valuable measure in AD therapy. Esculentoside A (EsA), isolated from Phytolacca esculenta, exhibits pharmacotherapeutic efficacy in mice with amyloid beta-induced AD. However, whether EsA affects tau pathology and its specific mechanism of action in AD mice remains unclear. PURPOSE: To investigate the roles and mechanisms of EsA in cognitive decline and tau pathology in a triple transgenic AD (3 × Tg-AD) mouse model. METHODS: EsA (5 and 10 mg/kg) was administered via intraperitoneal injection to 8-month-old AD mice for eight consecutive weeks. Y-maze and novel object recognition tasks were used to evaluate the cognitive abilities of mice. Potential signaling pathways and targets in EsA-treated AD mice were assessed using quantitative proteomic analysis. The NFT levels and hippocampal synapse numbers were investigated using Gallyas-Braak silver staining and transmission electron microscopy, respectively. Western blotting and immunofluorescence assays were used to measure the expression of tau-associated proteins. RESULTS: EsA administration attenuated memory and recognition deficits and synaptic damage in AD mice. Isobaric tags for relative and absolute quantitation proteomic analysis of the mouse hippocampus revealed that EsA modulated the expression of some critical proteins, including brain-specific angiogenesis inhibitor 3, galectin-1, and Ras-related protein 24, whose biological roles are relevant to synaptic function and autophagy. Further research revealed that EsA upregulated AKT/GSK3ß activity, in turn, inhibited tau hyperphosphorylation and promoted autophagy to clear abnormally phosphorylated tau. In hippocampus-derived primary neurons, inhibiting AMP-activated protein kinase (AMPK) activity through dorsomorphin could eliminate the effect of EsA, as revealed by increased tau hyperphosphorylation, downregulated activity AKT/GSK3ß, and blocked autophagy. CONCLUSIONS: To our knowledge, this study is the first to demonstrate that EsA attenuates cognitive decline by targeting the pathways of both tau hyperphosphorylation and autophagic clearance in an AMPK-dependent manner and it shows a high reference value in AD pharmacotherapy research.

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ObjectiveHenoch-Schönlein purpura（HSP） is one of the dominant diseases in Mongolian medicine. Qishun Baolier（QSBLE）， as the main prescription for the treatment of HSP， has significant clinical effect， but its mechanism is not yet clear. Baed on this， this study is intended to screen the differentially expressed proteins before and after treatment， and preliminarily explore the molecular mechanism of QSBLE in the treatment of HSP. MethodTaking oneself as the control， 30 HSP patients aged 6-45 years were collected， and QSBLE was taken orally at 12：00 and 24：00， respectively. The dose was adjusted according to age and the course of treatment was one week. The distribution of proteinuria， hematuria and skin purpura of all patients were determined before and after treatment. The serum samples of 10 patients with clinically significant remission after QSBLE treatment were randomly selected for proteomics. Isobaric tags for relative and absolute quantification（iTRAQ） combined with liquid chromatography tandem mass spectrometry（LC-MS/MS） was used to analyze the proteins in serum of HSP patients before and after treatment， and differential proteins were analyzed bioinformatically and the protein-protein interaction（PPI） networks were constructed. ResultA total of 378 proteins were identified from serum， including 18 differentially expressed proteins， of which 15 proteins were up-regulated and 3 proteins were down regulated. Bioinformatics showed that the differential proteins were mainly involved in biological processes such as immune response， immunoglobulin production， phagocytosis， adaptive immune response before and after treatment. Biological processes， pathways and proteins were used to construct the PPI network， the proteins represented by immunoglobulin heavy constant γ1（IGHG1）， immunoglobulin λ-chain 7-43（IGLV7-43）， gelsolin（GSN） and 60 kDa heat shock protein（HSPD1） were involved in biological processes and related pathways such as adaptive immune response， immunoglobulin production， leukocyte-mediated immunity， regulation of stress response， regulation of immune system processes， regulation of trauma response， and these proteins were at the center of the PPI network. ConclusionQSBLE may play a role in the treatment of HSP by regulating the expression of IGHG1， IGLV7-43， GSN， HSPD1 and other key proteins to affect immune-related biological processes.

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INTRODUCTION: Gestational diabetes mellitus (GDM) refers to abnormal glucose tolerance that occurs or is firstly diagnosed during pregnancy. GDM is related to various adverse pregnancy outcomes, but GDM pathogeny has not been fully elucidated. Nevertheless, previous studies have observed that many proteins in the placentas of patients with GDM are dysregulated. The present study aimed to establish a novel differentially expressed protein (DEP) landscape of GDM and normal maternal placentas and to explore the possible connection between DEPs and GDM pathogenesis. This study provides new insights into the mechanism of GDM and should make an important contribution to the development of biomarkers. METHODS: The morphological characteristics of the placenta were observed on 30 GDM and normal maternal placental tissues stained with haematoxylin and eosin. Isobaric tags for relative and absolute quantitation (iTRAQ) was used in the proteomics screening of the DEPs of the normal and GDM maternal placentas. Bioinformatics analysis was performed on the DEPs, and parallel reaction monitoring (PRM) was performed to verify the DEPs. Finally, the quantitative analysis of iTRAQ and PRM was verified by immunohistochemical assay. RESULTS: A total of 68 DEPs in the GDM placenta were identified with iTRAQ proteomics experiment, comprising 21 up-regulated and 47 down-regulated DEPs. Bioinformatics analysis showed that the regulation of transport, catabolic process of non-coding RNA, cytoskeleton and cell binding were the most abundant Gene Ontology terms, and RNA degradation was an important pathway for significant enrichment. Protein-protein interaction network analysis showed that heterogeneous nuclear ribonucleoproteins A2/B1 (HNRNPA2B1), heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB), heterogeneous nuclear ribonucleoprotein L (HNRNPL) and heterogeneous nuclear ribonucleoprotein A3 (HNRNPA3) were the cores of the up-regulated proteins. Band 3 anion transport protein (SLC4A1), spectrin beta chain erythrocytic (SPTB), ankyrin-1 (ANK1), spectrin beta chain non-erythrocytic 2 (SPTBN2), D-3-phosphoglycerate dehydrogenase (PHGDH) and exosome complex component RRP42 (EXOSC7) were the cores of the down-regulated proteins. These proteins are involved in the binding, splicing, processing, transport and degradation of RNA and in the formation and maintenance of the cytoskeleton. PRM verification results showed that seven proteins, namely, epiplakin (EPPK1), cold-inducible RNA-binding protein (CIRBP), HNRNPA2B1, HNRNPAB, HNRNPL, Ras-related protein Rab-21 (RAB21) and Ras-related protein Rab-3B (RAB3B), were up-regulated, whereas SPTB and SLC4A1 were down-regulated. The results of immunohistochemical assay also showed that the expression of five proteins, namely EPPK1, HNRNPA2B1, HNRNPAB, CIRBP and RAB21, were significantly higher in GDM placental tissues (P < 0.01). The GDM placentas showed changes in the morphological evaluation, including poor villous maturation, obvious increase in the number of syncytiotrophoblast nodules, thickening of the wall of dry villous arterioles with lumen stenosis, increased fibrinous exudation and excessive filling of villous interstitial vessels. DISCUSSION: Differentially expressed proteins related to a variety of biological processes in the GDM placenta were found. Fourteen proteins, namely, HNRNPA2B1, HNRNPAB, HNRNPL, HNRNPA3, EPPK1, CIRBP, RAB21, RAB3B, SLC4A1, SPTB, ANK1, SPTBN2, PHGDH and EXOSC7, which were differentially expressed in the placenta, may play an important role in regulating the occurrence and development of gestational diabetes through multi-channel and multi-link regulation.

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The Golgi is the central organelle in the secretory pathway, essential for post-translational modifications, sorting and trafficking of secretory and membrane proteins and lipids in all eukaryotic cells. During mitosis, the mammalian Golgi membranes undergo continuous disassembly and reassembly processes which are critical for Golgi biogenesis during the cell division. To better understand the underlying molecular mechanism of this highly dynamic process, we analyzed the proteins that are in or associated with interphase and mitotic Golgi membranes using an in vitro Golgi assembly assay and quantitative proteomics. In this study, by combining an isobaric mass tag labeling strategy with OFFGEL peptide fractionation, LC-MS/MS analyses identified and quantified a total of 1193 Golgi-resident or -associated proteins. These proteins included Golgi structural proteins, Golgi-resident enzymes, Rab GTPases, and SNARE proteins. This systematic quantitative proteomic study revealed the comprehensive molecular machinery of the Golgi and the dynamic protein changes in its disassembly and reassembly processes. Here we describe the detailed procedures and protocols for this analysis.

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A growing body of evidence has linked the gut microbiota to liver metabolism. The manipulation of intestinal microflora has been considered as a promising avenue to promote liver health. However, the effects of Lactobacillus gasseri LA39, a potential probiotic, on liver metabolism remain unclear. Accumulating studies have investigated the proteomic profile for mining the host biological events affected by microbes, and used the germ-free (GF) mouse model to evaluate host-microbe interaction. Here, we explored the effects of L. gasseri LA39 gavage on the protein expression profiles of the liver of GF mice. Our results showed that a total of 128 proteins were upregulated, whereas a total of 123 proteins were downregulated by treatment with L. gasseri LA39. Further bioinformatics analyses suggested that the primary bile acid (BA) biosynthesis pathway in the liver was activated by L. gasseri LA39. Three differentially expressed proteins (cytochrome P450 family 27 subfamily A member 1 (CYP27A1), cytochrome P450 family 7 subfamily B member 1 (CYP7B1), and cytochrome P450 family 8 subfamily B member 1 (CYP8B1)) involved in the primary BA biosynthesis pathway were further validated by western blot assay. In addition, targeted metabolomic analyses demonstrated that serum and fecal β‍-muricholic acid (a primary BA), dehydrolithocholic acid (a secondary BA), and glycolithocholic acid-3-sulfate (a secondary BA) were significantly increased by L. gasseri LA39. Thus, our data revealed that L. gasseri LA39 activates the hepatic primary BA biosynthesis and promotes the intestinal secondary BA biotransformation. Based on these findings, we suggest that L. gasseri LA39 confers an important function in the gut‒liver axis through regulating BA metabolism.

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Hyperbaric Oxygen Therapy (HBOT) has definitive therapeutic effects on spinal cord injury (SCI), but its mechanism of action is still unclear. Here, we've conducted a systemic proteomic analysis to identify differentially expressed proteins (DEPs) between SCI rats and HBOT + SCI rats. The function clustering analysis showed that the top enriched pathways of DEPs include oxygen transport activity, oxygen binding, and regulation of T cell proliferation. The results of functional and signal pathway analyses indicated that metabolic pathways, thermogenesis, LXR/RXR activation, acute phase response signaling, and the intrinsic prothrombin pathway in the SCI + HBOT group was higher than SCI group.

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Background: In China, Oldenlandia diffusa (OD) has been prescribed as a therapeutic herb for rheumatoid arthritis (RA). We previously conducted a preliminary study of the anti-inflammatory effect of OD, and the purpose of this study is to further investigate its mechanism. Methods: We performed a quantitative proteomic analysis of synovium, identified the differentially expressed proteins, and performed bioinformatics analyses. With the help of network pharmacology, we aimed to find the key synovial proteins which OD or its key compound might influence. To verify the result, liquid chromatography-mass spectrometry (LC-MS) was applied to quantify and qualify the absorbable potential compounds of OD. The anti-inflammatory effect was evaluated by morphological, histopathological, and cytokine analyses. Target proteins were observed by immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Results: MMP3 and CAV1 were identified as 2 of the differentially expressed proteins in RA synovium, and might be influenced by quercetin, the active compound of OD. MMP3 might be altered through atherosclerosis signaling, while CAV1 might be altered through caveolar-mediated endocytosis signaling. According to our verification, quercetin was identified as the absorbed and effective compound of OD, and it could exert an anti-inflammatory effect on the collagen-induced arthritis (CIA) model, including serum cytokine expression, synovial hyperplasia and lymphocyte infiltration, articular cartilage lesion. Quercetin could also down-regulate the synovial expression of MMP3 and CAV1, and could exert better effects at a high dose. Conclusions: Quercetin was the main active compound of OD in the treatment of RA. OD might alleviate inflammatory responses in CIA rats by suppressing the expression of MMP3 and CAV1 through quercetin, and at a high dose, quercetin could exert a better anti-inflammatory effect.

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Ducks and wild aquatic birds are the natural reservoirs of avian influenza viruses. However, the host proteome response that causes disease in vivo by the H5N1 HPAI virus is still unclear. This study presented a comprehensive analysis of the proteome response in Muscovy duck lung tissue during 3 days of infection with either a highly virulent DK383 or an avirulent DK212. An unbiased strategy- isobaric tags for relative and absolute quantitation (iTRAQ) in conjunction with high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) was utilized to investigate the infection mechanism. Pathways derived from analysis of 292 significantly altered proteins may contribute to the high pathogenic nature and disease progression of H5N1 viruses. Global proteome profiles indicated improved correlation with the virus titers and gene expression patterns between the two strains of the H5N1 virus. DK383 replicated more efficiently and induced a stronger response specific to severe disease. While proteins involved in the immune response of neutrophils were increased markedly by DK383, DK212 evoked a distinct response characterized by an increase in proteins involved in the maturation of dendritic cells, adhesion of phagocytes, and immune response of macrophages. The differentially activated Akt/mTOR/p70S6K pathway might involve in the host response to H5N1 viruses. Therefore, systematically integrated with datasets from primary genomic and virus titer results, proteomic analyses may help reveal the potential pathogenesis.

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Yaks (Bos mutus) live in the Qinghai-Tibet plateau. The quality of yak meat is unique due to its genetic and physiological characteristics. Identification of the proteome of yak muscle could help to reveal its meat-quality properties. The common proteome, phosphoproteome, and N-glycoproteome of yak longissimus thoracis (YLT) were analyzed by liquid chromatography-tandem mass spectrometry-based shotgun analysis. A total of 1812 common proteins, 1303 phosphoproteins (3918 phosphorylation sites), and 204 N-glycoproteins (285 N-glycosylation sites) were identified in YLT. The common proteins in YLT were involved mainly in myofibril structure and energy metabolism; phosphoproteins were associated primarily with myofibril organization, regulation of energy metabolism, and signaling; N-glycoproteins were engaged mainly in extracellular-matrix organization, cellular immunity, and organismal homeostasis. We reported, for the first time, the "panorama" of the YLT proteome, specifically the N-glycoproteome of YLT. Our results provide essential information for understanding post mortem physiology (rigor mortis and aging) and the quality of yak meat.

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BACKGROUND: Preoperative neoadjuvant chemoradiation (nCRT) has been the standard treatment for locally advanced rectal cancer. Serum biomarkers to stratify patients with respect to prognosis and response to nCRT are needed due to the diverse response to the therapy. METHODS: Thirteen paired pre- and post-nCRT sera from rectal cancer patients were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) method. Twenty-five proteins were selected for validation by parallel reaction monitoring (PRM) in ninety-one patients. RESULTS: Totally, 310 proteins were identified and quantified in sera samples. Reactome pathway analysis showed that the immune activation-related pathways were enriched in response to nCRT. Twenty-five proteins were selected for further validation. PRM result showed that the level of PZP was higher in pathological complete response (pCR) patients than non-pCR patients. The Random Forest algorithm identified a prediction model composed of 10 protein markers, which allowed discrimination between pCR patients and non-pCR patients (area under the curve (AUC) = 0.886 on testing set). Higher HEP2 and GELS or lower S10A8 in baseline sera were associated with better prognosis. Higher APOA1 in post nCRT sera was associated with better disease-free survival (DFS). CONCLUSIONS: We identified and confirmed a 10-protein panel for nCRT response prediction and four potential biomarkers HEP2, GELS, S10A8 and APOA1 for prognosis of rectal cancer based on iTRAQ-based comparative proteomics screening and PRM-based targeted proteomic validation.

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BACKGROUND: Due to the development of insecticide resistance in mosquitoes, with worldwide mosquito-borne diseases resurgence in recent years, recent advances in proteome technology have facilitated a proteome-wide analysis of insecticide resistance-associated proteins in mosquitoes. Understanding the complexity of the molecular basis of insecticide resistance mechanisms employed by mosquitoes will help in designing the most effective and sustainable mosquito control methods. RESULTS: After 30 generations, insecticide-selected strains showed elevated resistance levels to the cypermethrin used for selection. Proteome data allowed the detection of 2892 proteins, of which 2885 differentially expressed proteins (DEPs) achieved quantitative significances in four stages (egg, larvae, pupae, adult) of Culex pipiens pallens cypermethrin-resistant strain as compared to the susceptible strain. Among them, a significant enrichment of proteins, including cuticular proteins, enzymes involved in the detoxification (cytochrome P450, glutathione S-transferases, esterase, ATP-binding cassette) and some biological pathways (oxidative phosphorylation, hippo signalling) that are potentially involved in cypermethrin resistance, was observed. Thirty-one representative DEPs (cytochrome P450, glutathione S-transferase, cuticle protein) during Cx. pipiens pallens developmental stages were confirmed by a parallel reaction monitoring strategy. CONCLUSIONS: The present study confirmed the power of isobaric tags for relative and absolute quantification for identifying concomitantly quantitative proteome changes associated with cypermethrin in Cx. pipiens pallens. Proteome analysis suggests that proteome modifications can be selected rapidly by cypermethrin, and multiple resistance mechanisms operate simultaneously in cypermethrin-resistance of Cx. pipiens pallens, Our results interpret that an up-regulated expression of proteins and enzymes like cytochrome P450, glutathione S-transferases, esterase etc. has an impact in insecticide resistance. Previously neglected penetration resistance (cuticular proteins) may play an important role in the adaptive response of Cx. pipiens pallens to insecticides. This information may serve as a basis for future work concerning the possible role of these proteins in cypermethrin resistance in mosquito Cx. pipiens pallens. © 2022 Society of Chemical Industry.

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