Analysis of the urinary peptidome associated with Helicobacter pylori infection

AIM： To investigate the relationship between urinary peptide changes and Helicobacter pylori （H. pylorl） infection using urinary peptidome profiling. METHODS： The study was performed in volunteers （n = 137） who gave informed consent. Urinary peptides were enriched by magnetic beads based weak cation exchange chromatography＇and spectrums acquired by matrix-assisted laser desorption/ionization time-of-flight （MALDI-TOF） mass spectrometry （MS）. ClinProTools bioinformatics software was used for statistical analysis and the recognition of peptide patterns. The marker peptides were identified by LTQ Obitrap XL tandem MS. RESULTS： Approximately 50 proteins or peptides which loaded onto the magnetic beads were detected by MAL-DI-TOF MS. By optimizing the parameters of the model, the Genetic Algorithm model had good recognition capability （97%） and positive predictive value （94%）. Based on the model, 2 markers with molecular masses of 6788 and 1912 Da were found that differentiated between H. pylori positive and negative volunteers. The m/z 1912 sequence was parsed as SKQFTSSTSYN- RGDSTF. The peptide was identified as isoform 1 of the fibrinogen a chain precursor, whose concentration in urine was markedly higher in H. pylori infected volunteers than in H. pylori non-infected ones. CONCLUSION： The appearance of urinary fibrinogen degradation products is caused by an active H. pyloriinduced process.

Dynamic changes of peptidome and release of polysaccharide in sea cucumber(Apostichopus japonicus) hydrolysates depending on enzymatic hydrolysis approaches

Enzymatic hydrolysis has been widely used to produce bioactive hydrolysates from sea cucumber body wall.Here,inspired by the clarification of Apostichopus japonicus genome,we investigated the enzymatic hydrolysis of sea cucumber body wall by using the omics strategy.Shared proteins,including major yolk proteins,collagens,extracellular matrix glycoproteins and muscle proteins,were released from the body wall by different hydrolysis condition.A portfolio of 216 shared peptides were detected in the peptidome by papain with different hydrolysis time,while 32 shared peptides were detected in the peptidome by differing proteases.Unshared peptides and the relative abundance distribution profiles of shared peptides changed depending on hydrolysis approaches,indicating dynamic changes of peptidome during hydrolysis.Moreover,release of sulfated fucan and fucosylated chondroitin sulfate changed with the hydrolysis condition.The monitoring of dynamic enzymatic hydrolysis process at a molecular scale would contribute to production and quality control of sea cucumber hydrolysates.

Peptidome data-driven comprehensive individualized monitoring of membranous nephropathy with machine learning

As the most common pathological type of nephrotic syndrome,membranous nephropathy(MN)presents diversity in progression trends,facing severe complications.The precise discrimination of MN from healthy people,other types of nephrotic syndrome or those with therapeutic remission has always been huge challenge in clinics,not to mention comprehensive individualized monitoring relied on minimally invasive molecular detection means.Herein,we construct a functionalized pore architecture to couple with machine learning to aid all-round peptidome enrichment and data profiling from hundreds of human serum samples,and finally establish a set of defined peptide panel consisting of 12 specific feature signals.In addition to the realization of above-mentioned precise discrimination with more than 97%of sensitivity,88%of accuracy and f1 score,the simultaneously comprehensive individualized monitoring for MN can also be achieved,including conventionally screening diagnosis,congeneric distinction and prognostic evaluation.This work greatly advances the development of peptidome data-driven individualized monitoring means for complex diseases and undoubtedly inspire more devotion into molecular detection field.

A strategy with label-free quantification of the targeted peptides for quantitative peptidome analysis of human serum

Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.

Characterization of natural peptides in Pheretima by integrating proteogenomics and label-free peptidomics

Pheretima,also called“earthworms”,is a well-known animal-derived traditional Chinese medicine that is extensively used in over 50 Chinese patent medicines(CPMs)in Chinese Pharmacopoeia(2020 edition).However,its zoological origin is unclear,both in the herbal market and CPMs.In this study,a strategy for integrating in-house annotated protein databases constructed from close evolutionary relationship-sourced RNA sequencing data from public archival resources and various sequencing algorithms(restricted search,open search,and de novo)was developed to characterize the phenotype of natural peptides of three major commercial species of Pheretima,including Pheretima aspergillum(PA),Pheretima vulgaris(PV),and Metaphire magna(MM).We identified 10,477 natural peptides in the PA,7,451 in PV,and 5,896 in MM samples.Five specific signature peptides were screened and then validated using synthetic peptides;these demonstrated robust specificity for the authentication of PA,PV,and MM.Finally,all marker peptides were successfully applied to identify the zoological origins of Brain Heart capsules and Xiaohuoluo pills,revealing the inconsistent Pheretima species used in these CPMs.In conclusion,our integrated strategy could be used for the in-depth characterization of natural peptides of other animal-derived traditional Chinese medicines,especially non-model species with poorly annotated protein databases.

质谱成像方法及其在临床研究中的应用

Imaging mass spectrometry (IMS) methodology for biological tissue component distribution map using MALDI-TOF MS and MALDI-TOF/TOF MS was established.Peak density distribution was probed to be quite useful for MS image classification.More than 40 000 spectra from 200 tissue sections were acquired and reproducibility between various of species groups was great than 80%.Tens of differentiately expressed components were detected by t-test (P<0.01).Classification modeling was created based on the differentiate components,blind species were analyzed for model validation,accuracy was above 90%.

多肽电泳结合质谱技术在血清多肽组学研究中的应用

A protocol for serum peptidomics using peptide electrophoresis combining with mass spectrometry was reported in this paper.Results showed that the improved peptide electrophoresis and MALDI-TOF MS could evaluate the efficiency of serum peptide processing methods.Our protocol was verified with a high reproducibility.

Peptidomic analysis of Chinese shrimp(Fenneropenaeus chinensis) hemolymph by magnetic bead-based MALDITOF MS

Peptides in shrimp hemolymph play an important role in the innate immune response.Analysis of hemolymph will help to detect and identify potential novel biomarkers of microbial infection.We used magnetic bead-based purification(ClinProt system) and matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS) to characterize shrimp hemolymph peptides.Shrimp serum and plasma were used as the source of samples for comparative analysis,and it was found that serum was more suitable for shrimp hemolymph peptidomic analysis.To screen potential specific biomarkers in serum of immune-challenged shrimps,we applied magnetic bead-based MALDI-TOF MS to serum samples from 10 immune-challenged and 10 healthy shrimps.The spectra were analyzed using FlexAnalysis 3.0 and ClinProTools 2.1 software.Thirteen peptide peaks significantly different between the two groups were selected as candidate biomarkers of lipopolysaccharide(LPS)-infection.The diagnostic model established by genetic algorithm using five of these peaks was able to discriminate LPS-challenged shrimps from healthy control shrimps with a sensitivity of 90% and a specificity of 100%.Our approach in MALDITOF MS-based peptidomics is a powerful tool for screening bioactive peptides or biomarkers derived from hemolymph,and will help to enable a better understanding of the innate immune response of shrimps.

Peptidomic analysis of whey protein hydrolysates and prediction of their antioxidant peptides

Enzymatic hydrolysis of proteins is a breakdown process of peptide bond in proteins,releasing some peptides with potential biological functions.Previous studies on enzymatic hydrolysis of whey proteins have not identified the complete peptide profiles after hydrolysis.In this study,we reconstructed a profile of peptides from whey hydrolysates with two enzymes and different processing conditions.We also developed an ensemble machine learning predictor to classify peptides obtained from whey hydrolysis.A total of 2572 peptides were identified over three process conditions with two enzymes in duplicate.499 peptides were classified and chosen as potential antioxidant peptides from whey proteins.The peptides classified as antioxidants in the hydrolysates had a proportion of 13.1%-24.5%regarding all peptides identified.These results facilitate the selection of promising peptides involved in the antioxidant properties during the enzymatic hydrolysis of whey proteins,aiding the discovery of novel antioxidant peptides.

Modern trends in animal venom research-omics and nanomaterials

Animal venom research is a specialized investigation field, in which a number of different methods are used and this array is constantly expanding. Thus, recently emerged omics and nanotechnologies have already been successfully applied to venom research. Animal venoms have been studied for quite a long time. The traditional reductionist approach has been to isolate individual toxins and then study their structure and function. Unfortunately, the characterization of the venom as a whole system and its multiple effects on an entire organism were not possible until recent times. The development of new methods in mass spectrometry and sequencing have allowed such characterizations of venom, encompassing the identification of new toxins present in venoms at extremely low concentrations to changes in metabolism of prey organisms after envenomation. In particular, this type of comprehensive research has become possible due to the development of the various omics technologies: Proteomics, peptidomics, transcriptomics, genomics and metabolomics. As in other research fields, these omics technologies ushered in a revolution for venom studies, which is now entering the era of big data. Nanotechnology is a very new branch of technology and developing at an extremely rapid pace. It has found application in many spheres and has not bypassed the venom studies. Nanomaterials are quite promising in medicine, and most studies combining venoms and nanomaterials are dedicated to medical applications. Conjugates of nanoparticles with venom components have been proposed for use as drugs or diagnostics. For example, nanoparticles conjugated with chlorotoxin- a toxin in scorpion venom, which has been shown to bind specifically to glioma cells- are considered as potential glioma-targeted drugs, and conjugates of neurotoxins with fluorescent semiconductor nanoparticles or quantum dots may be used to detect endogenous targets expressed in live cells. The data on application of omics and nanotechnologies in venom research are systematized concisely in this paper.

Peptides and Peptidomics: A Tool with Potential in Control of Plant Viral Diseases

Plant viruses are the most infectious agents in commercially important crops worldwide. Plant viral diseases are important because both decreased yielding and quality of fruits, flowers or vegetables lead to million-dollar losses in production. At present there are no reports which suggest a direct control of plant virus. A new strategy for plant virus control has been raised since 13 years ago—the use of peptides. Peptides could offer a direct interaction by affinity selection against viral proteins involved in infection cycle, like capsid or movement protein (e.g.) and affect viral replication. Peptidomics, as a new tool to study peptides, led us screening and selecting the best peptide with antiviral activity, and re-designing it to enhance the biological effect as well as the potential of bioactivity of those peptides secreted by microbes present in soil. In this paper we review current aspects in the use of peptides and peptidomics as a strategy to study new methods that lead a direct control against plant viral diseases.

Genomics,transcriptomics,and peptidomics of the greater wax moth Galleria mellonella neuropeptides and their expression in response to lead stress

Neuropeptides are crucial in regulation of a rich variety of developmental,physiological,and behavioral functions throughout the life cycle of insects.Using an integrated approach of multiomics,we identified neuropeptide precursors in the greater wax moth Galleria mellonella,which is a harmful pest of honeybee hives with a worldwide distribution.Here,a total of 63 and 67 neuropeptide precursors were predicted and annotated in the G.mellonella genome and transcriptome,in which 40 neuropeptide precursors were confirmed in the G.mellonella peptidome.Interestingly,we identified 12 neuropeptide precursor genes present in G.mellonella but absent in honeybees,which may be potential novel pesticide target sites.Honeybee hives were contaminated with heavy metals such as lead,enabling its bioaccumulation in G.mellonella bodies through the food chain,we performed transcriptome sequencing to analyze the effects of Pb stress on the mRNA expression level of G.mellonella neuropeptide precursors.After treatment by Pb,the expression of neuropeptide F1 was found to be significantly downregulated,implying that this neuropeptide might be associated with responding to the heavy metal stress in G.mellonella.This study comprehensively identified neuropeptide precursors in G.mellonella,and discussed the effects of heavy metals on insect neuropeptides,with the example of G.mellonella.The results are valuable for future elucidation of how neuropeptides regulate physiological functions in G.mellonella and contribute to our understanding of the insect's environmental plasticity and identify potential new biomarkers to assess heavy metal toxicity in insects.

Peptidomic Analysis Reveals that Novel Peptide LDP2 Protects Against Hepatic Ischemia/Reperfusion Injury

Background and Aims:Hepatic ischemia/reperfusion(I/R)injury has become an inevitable issue during liver transplantation,with no effective treatments available.However,peptide drugs provide promising regimens for the treatment of this injury and peptidomics has gradually attracted increasing attention.This study was designed to analyze the spectrum of peptides in injured livers and explore the potential beneficial peptides involved in I/R injury.Methods:C57BL/6 mice were used to establish a liver I/R injury animal model.Changes in peptide profiles in I/R-injured livers were analyzed by mass spectrometry,and the functions of the identified peptides were predicted by bioinformatics.AML12 cells were used to simulate hepatic I/R injury in vitro.After treatment with candidate liver-derived peptides(LDPs)1–10,the cells were collected at various reperfusion times for further study.Results:Our preliminary study demonstrated that 6 h of reperfusion caused the most liver I/R injury.Peptidomic results suggested that 10 down-regulated peptides were most likely to alleviate I/R injury by supporting mitochondrial function.Most importantly,a novel peptide,LDP2,was identified that alleviated I/R injury of AML12 cells.It increased cell viability and reduced the expression of inflammation-and apoptosis-related proteins.In addition,LDP2 inhibited the expression of proteins related to autophagy.Conclusions:Investigation of changes in the profiles of peptides in I/R-injured livers led to identification of a novel peptide,LDP2 with potential function in liver protection by inhibiting inflammation,apoptosis,and autophagy.

Collagen derived species-specific peptides for distinguishing donkey-hide gelatin(Asini Corii Colla)

Objective:As an important food therapy product with traditional Chinese medicine(TCM) applications,donkey-hide gelatin(Asini Corii Colla,ACC) has been used for thousands of years.However,till now few effective strategy had been proposed to distinguish ACC from other animal hide gelatins,especially closely related horse-and mule-hide gelatins,which was an embarrassment of ACC quality control.Methods:Combined mass spectrometry and bioinformatic methods have been applied to identify and verify two ACC-specific peptides(Pep-1 and Pep-2) capable of distinguishing ACC from other closely related animal gelatins with high selectivity.Results:It confirmed that these two peptides could be not only used for distinguishing ACC from highly homologous horse-hide and mule-hide gelatins as well as other animal hide gelatins.Conclusion:The present study provides a simple method for species-specific peptides discovery,which can be used for assessing the quality of animal gelatin products,and ensure they are authenticable and traceable.

Mass spectrometry-based proteomics and peptidomics for systems biology and biomarker discovery

The scientific community has shown great interest in the field of mass spectrometry-based proteomics and peptidomics for its applications in biology. Proteomics technologies have evolved to produce large data sets of proteins or peptides involved in various biologic and disease progression processes generating testable hypothesis for complex biologic questions. This review provides an introduction to relevant topics in proteomics and peptidomics including biologic material selection, sample preparation, separation techniques, peptide fragmentation, post-translational modifications, quantification, bioinformatics, and biomarker discovery and validation. In addition, current literature, remaining challenges, and emerging technologies for proteomics and peptidomics are presented.

Identification of zinc-binding peptides in ADAM17-inhibiting whey protein hydrolysates using IMAC-Zn^(2+) coupled with shotgun peptidomics

Food components possessing zinc ligands can be used to inhibit zinc-dependent enzymes.In this study,zinc-binding peptides were derived from whey protein hydrolysates,and their ultrafiltration(>1 and<1 kDa)fractions,produced with Esperase(WPH-Esp),Everlase and Savinase.Immobilized metal affinity chromatography(IMAC-Zn^(2+))increased the zinc-binding capacity of the peptide fraction(83%)when compared to WPH-Esp(23%)and its<1 kDa fraction(40%).The increased zinc-binding capacity of the sample increased the inhibitory activity against the zinc-dependent“a disintegrin and metalloproteinase 17”.LC-MS/MS analysis using a shotgun peptidomics approach resulted in the identification of 24 peptides originating from bovineβ-lactoglobulin,α-lactalbumin,serum albumin,β-casein,κ-casein,osteopontin-k,and folate receptor-αin the fraction.The identified peptides contained different combinations of the strong zinc-binding group of residues,His+Cys,Asp+Glu and Phe+Tyr,although Cys residues were absent in the sequences.In silico predictions showed that the IMAC-Zn^(2+)peptides were non-toxins.However,the peptides possessed poor drug-like and pharmacokinetic properties;this was possibly due to their long chain lengths(5–19 residues).Taken together,this work provided an array of food peptide-based zinc ligands for further investigation of structure-function relationships and development of nutraceuticals against inflammatory and other zinc-related diseases.

Discovery of lung squamous carcinoma biomarkers by profiling the plasma peptide with LC/MS/MS

Biomarkers can be used for the screening and clinical diagnosis of cancer,and peptidomics approach has been proven successful in the research of biomarkers.To develop better peptidomic technologies for fast,accurate,and reliable detection of peptides biomarkers for lung cancer,we have improved the procedures of blood collection to minimize the degradation of the blood proteins and optimize the extraction of peptidome peptides from plasma samples based on acetonitrile precipitation associated with size exclusion chromatography（SEC）.Studies show that squamous cell carcinomas are found to express CAGEl,SPAT9 and TEX28 genes at significantly higher rates,and the results suggest that as tumors progress,the level of CAGEl,SPAT9 and TEX28 genes are likely to increase and lead to immunization.This suggests a potentially important therapeutic method for cancer testis-based cancer vaccines.