Bacterial Protein Profiling——Comparison of Three Mass Spectrometry Methodologies

Profiling the protein composition of bacteria is essential for understanding their biology,physiology and interaction with environment.Mass spectrometry has become a pivotal tool for protein analysis,facilitating the examination of expression levels,molecular masses and structural modifications.In this study,we compared the performance of three widely-used mass spectrometry methods,i.e.,matrix-assisted laser desorption/ionization(MALDI)protein fingerprinting,top-down proteomics and bottom-up proteomics,in the profiling of bacterial protein composition.It was revealed that bottom-up proteomics provided the highest protein coverage and exhibited the greatest protein profile overlap between bacterial species.In contrast,MALDI protein fingerprinting demonstrated superior detection reproducibility and effectiveness in distinguishing between bacterial species.Although top-down proteomics identified fewer proteins than bottom-up approach,it complemented MALDI fingerprinting in the discovery of bacterial protein markers,both favoring abundant,stable,and hydrophilic bacterial ribosomal proteins.This study represents the most systematic and comprehensive comparison of mass spectrometry-based protein profiling methodologies to date.It provides valuable guidelines for the selection of appropriate profiling strategies for specific analytical purposes.This will facilitate studies across various fields,including infection diagnosis,antimicrobial resistance detection and pharmaceutical target discovery.

Altered Protein Profiling in Tears from Patients in a Traumatic Vegetative State

Following severe traumatic brain injury .（sTBI）, patients may remain in a coma, vegetative state （VS）, or minimally conscious state （MCS）, all of which are also clinically termed disorders of consciousness. Patients in a coma show complete disability in the arousal system and fail to achieve awareness spontaneously; yet true coma represents a transient state and rarely lasts longer than a month [1]. The VS （also known as unresponsive wakefulness syndrome）, characterized as a state with spontaneous or stimulus-induced eye-opening but the patient appears totally unaware of self and environment, may persist for months or years [2]. In contrast, the MCS is evidenced by preserved and reproducible signs of awareness as well as sleep-wake cycles, suggesting better recovery than coma and VS. Considering that no signs of consciousness are detectable, patients in a VS suffer from a high rate of misdiagnosis [3].

Celastrol promotes apoptosis of breast cancer MDA-MB-231 cells by targeting HSDL2

Objective:Celastrol is a pentacyclic triterpenoid extracted from the traditional Chinese medicinal herb,Tripterygium wilfordii.This study aims to provide a scientific basis for the rational development and use of celastrol in breast cancer.Method:A quantitative chemical biology approach was used to investigate the protein targets and molecular mechanisms of celastrol in breast cancer cells.Results:Low-concentration celastrol exerted an anti-tumor effect by directly binding to hydroxysteroid dehydrogenase-like 2(HSDL2)and inhibiting its expression.Moreover,the expression of the pro-apoptotic protein,Bcl-2-associated X(BaX),increased,the level of the anti-apoptotic protein,B-cell lymphoma-2(Bcl-2),decreased,and the rate of apoptosis increased.After the transfection of cells with si-HSDL2,the apoptosis rate was similar to that observed after the administration of celastrol.However,apoptosis was reversed by the overexpression of HSDL2.Furthermore,our mass spectrometry(MS)data indicated a relationship between HSDL2 and the mitogen-activated protein kinase(MAPK)signaling pathway.We also found that the expression of HSDL2 was directly related to the degree of extracellular signal-regulated kinase(ERK)phosphorylation.Conclusion:Celastrol may promote apoptosis by suppressing the HSDL2/MAPK/ERK signaling pathway.

Analysis of Genetic Variation of Seed Proteins in the Genus Vigna and among Its Relatives Cultivated in China

The genetic variation of seed proteins was assayed by SDSPAGE for 24 cultivars belonging to 5 species in Vigna and 7 species in its 7 relative genera cultivated in China. There were 48 polymorphic subunit bands discriminated from electrophoretic profiles. Two dendrograms were constructed by UPGMA cluster analyses using PHYLIP3.6 respectively. Variation among genera or species was larger than that among lower taxonomic categories level. Little variation among cuhivars of yardlong bean （Vigna sesquipedalis ） and small variation of lablab （ Lablab purpureus）, pea （Pisum sativum）, or sword bean （Canavalia gladiata）, but large variation of soybean or rice bean in their origin of China were all revealed. The seed proteins profiles of traditionally regarded as typical species in Vigna such as yardlong bean, rice bean and small bean were more similar than mungbean （Vigna radiata） and black gram （Vigna mungo） were. Mungbean and black gram had distinct seed proteins pattern, they should be of two species.

Study on Relationship Between Differential Proteins of Bacillus cereus LBR-4 and Its Salt Tolerance Mechanism

In order to explore the salt tolerance mechanism of Bacillus cereus LBR-4 with salinity of 14%NaCl,differential proteomic analysis of the whole protein of LBR-4 strain expressed under 14%NaCl high salinity condition and normalculture condition(1%NaCl)was studied by two-dimensional electrophoresis and mass spectrometry.The isoelectric point of most detected proteins was between pH 4-7 and the molecular weight distribution was 10-70 ku.Compared with the normal culture condition,the expression level of 118 protein spots in the whole protein expression map changed significantly(accounting for 25.2%of the total protein spots).The expression level of 78 protein spots increased significantly,including 22 new protein spots that appeared under high salt stress.The expression levels of 40 protein spots decreased significantly,including 18 protein spots that disappeared under high salt stress.By mass spectrometry,six distinct differentially expressed protein spotswere dihydroxy acid dehydratase,cell division protein FtsZ,iron sulfur cluster synthesis protein SufD,unknown carboxylase YngE,hypothetical acetaldehyde dehydrogenase DhaS and phenylalanine acid tRNA ligase alpha subunit.It was speculated that under high salt stress,the cells had protective measures and the secretion of intracellular compatible solutes increased.The iron and sulfur clusters involved in various physiological reactions also activated the stressful suf synthesis pathway,and therate of cell division and reproduction was also slowed down and ensured the normal progress of physiological reactions inthe cells.

The circ_0002538/miR-138-5p/plasmolipin axis regulates Schwann cell migration and myelination in diabetic peripheral neuropathy

Circular RNAs(circRNAs)play a vital role in diabetic peripheral neuropathy.However,their expression and function in Schwann cells in individuals with diabetic peripheral neuropathy remain poorly understood.Here,we performed protein profiling and circRNA sequencing of sural nerves in patients with diabetic peripheral neuropathy and controls.Protein profiling revealed 265 differentially expressed proteins in the diabetic peripheral neuropathy group.Gene Ontology indicated that differentially expressed proteins were mainly enriched in myelination and mitochondrial oxidative phosphorylation.A real-time polymerase chain reaction assay performed to validate the circRNA sequencing results yielded 11 differentially expressed circRNAs.circ_0002538 was markedly downregulated in patients with diabetic peripheral neuropathy.Further in vitro experiments showed that overexpression of circ_0002538 promoted the migration of Schwann cells by upregulating plasmolipin(PLLP)expression.Moreover,overexpression of circ_0002538 in the sciatic nerve in a streptozotocin-induced mouse model of diabetic peripheral neuropathy alleviated demyelination and improved sciatic nerve function.The results of a mechanistic experiment showed that circ_0002538 promotes PLLP expression by sponging miR-138-5p,while a lack of circ_0002538 led to a PLLP deficiency that further suppressed Schwann cell migration.These findings suggest that the circ_0002538/miR-138-5p/PLLP axis can promote the migration of Schwann cells in diabetic peripheral neuropathy patients,improving myelin sheath structure and nerve function.Thus,this axis is a potential target for therapeutic treatment of diabetic peripheral neuropathy.

Identification of antimalarial targets of chloroquine by a combined deconvolution strategy of ABPP and MS-CETSA

Background: Malaria is a devastating infectious disease that disproportionally threatens hundreds of millions of people in developing countries. In the history of anti-malaria campaign, chloroquine(CQ) has played an indispensable role, however, its mechanism of action(MoA) is not fully understood.Methods: We used the principle of photo-affinity labeling and click chemistry-based functionalization in the design of a CQ probe and developed a combined deconvolution strategy of activity-based protein profiling(ABPP) and mass spectrometry-coupled cellular thermal shift assay(MS-CETSA) that identified the protein targets of CQ in an unbiased manner in this study. The interactions between CQ and these identified potential protein hits were confirmed by biophysical and enzymatic assays.Results: We developed a novel clickable, photo-affinity chloroquine analog probe(CQP) which retains the antimalarial activity in the nanomole range, and identified a total of 40 proteins that specifically interacted and photocrosslinked with CQP which was inhibited in the presence of excess CQ. Using MS-CETSA, we identified 83 candidate interacting proteins out of a total of 3375 measured parasite proteins. At the same time, we identified 8 proteins as the most potential hits which were commonly identified by both methods.Conclusions: We found that CQ could disrupt glycolysis and energy metabolism of malarial parasites through direct binding with some of the key enzymes, a new mechanism that is different from its well-known inhibitory effect of hemozoin formation. This is the first report of identifying CQ antimalarial targets by a parallel usage of labeled(ABPP)and label-free(MS-CETSA) methods.

Analysis of Nutrient Profile of Finger Millet(Eleusine coracana(L.)Gaertn.)for Baby Food Formulation Using Pigeon Pea(Cajanus cajan(L.)Millsp.)as Protein Source

Finger millet(Eleusine coracana(L.)Gaertn.)is a drought resistant crop with potentially tremendous but under-explored source of nutraceutical properties as compared to other regularly consumed cereals in the era of drawback of nutritional security,these characteristics must be harnessed to develop finger millet as a novel functional food.Under-nutrition caused by inadequate diets,and other factors that influence nutritional status,is the underlying factor in 45%child deaths.In Kenya only 25%of young children are fed adequately diverse diets.The main objective of this study was to prepare baby food formulas using finger millets with pigeon peas as protein source and to analyze their nutritional profiles.Two finger millets varieties(i)Snapping Green Early,low altitude and medium altitude varieties and(ii)U-15)were studied to determine effects of environment on nutrient profiles.This study showed that Snapping Green Early had better nutrient profiles(12.13%protein and is high in Ca,Mg,Fe,Zn and P)than U-15(11.69%protein and lower nutrients(Ca,Mg,Fe,Zn and P)),and hence was selected for use in the malting process as best variety.As expected,the pigeon peas had the highest protein value(21%).The samples malted for 72 h resulted in reduction of tannin concentration from 0.091%to 0.03%and the amount of nutrients(Ca,Mg,Fe and Zn)doubled and in fact the protein profile increased by 8.31%.The appropriate ratio for the formulation of the baby food was 70:30.The composting resulted in 18.5%increase in protein.

胰腺癌血浆蛋白质组学研究

目的利用双向电泳(2-DE)的方法进行胰腺癌的比较蛋白质组学研究,寻找和鉴定与胰腺癌相关或特异的生物标志分子。方法选择12例胰腺癌患者以及与之性别年龄匹配的12例正常人和10例慢性胰腺炎患者的血浆标本进行研究。对去除ALB和IgG等预分离方法进行探索,确定最佳的血浆2-DE方案。通过比较胰腺癌(术前、术后)、正常人和慢性胰腺炎患者的血浆2-DE图谱,寻找差异蛋白并进行质谱鉴定。结果去除ALB和IgG以及超滤的预处理仅使血浆2-DE图谱的局部分辨率提高。共发现并鉴定了5种与胰腺癌或慢性胰腺炎相关的差异蛋白:Haptoglobin(Hp),Hemoglobin(Hb),α1-antitrypsin(α1AT),Immunoglobin J chain(Ig J) and Leucine-rich α2-glycoprotein(LRG)。Hp β链在胰腺癌患者中轻度升高,胰腺癌患者的Hp2-2表型的比例高于正常人,而缺少Hp1-1表型。胰腺癌和慢性胰腺炎患者的血浆Hb高于正常人。α1AT的变化趋势为慢性胰腺炎>胰腺癌>正常人。慢性胰腺炎患者的IgJ链高于胰腺癌患者和正常人,可能来源于血分泌型IgA(sIgA)。胰腺癌患者的LRG水平较正常人和慢性胰腺炎轻度升高。术后Hp、α1AT和LRG均明显升高,未发现术后明显下降的蛋白。结论去除ALB和IgG以及超滤去除大分子的预处理方法对于血浆蛋白的差异比较并无太大优势,但在质谱鉴定方面可以与未处理的方法互相补充。在发现的5种差异蛋白中,Hp的表型、Hb、IgJ链和LRG与胰腺癌或慢性胰腺炎的关系在以往的文献中未见报道,这些差异蛋白对于胰腺癌或慢性胰腺炎的意义及诊断价值还需要进一步的验证。

Current status and prospects of clinical proteomics studies on detection of colorectal cancer:Hopes and fears

Colorectal adenocarcinoma （CRC） is the third most common type of cancer and the fourth most frequent cause of death due to cancer worldwide. Given the natural history of CRC, early diagnosis appears to be the most appropriate tool to reduce disease-related mortality. A field of recent interest is clinical proteomics, which was reported to lead to high sensitivity and specificities for early detection of several solid tumors. This emerging field uses mass spectrometry-based protein profiles/patterns of easy accessible body fluids to distinguish cancer from non-cancer patients. These discrepancies may be a result of： （i） proteins being abnormally produced or shed and added to the serum proteome, （2） proteins clipped or modified as a consequence of the disease process, or （3） proteins subtracted from the proteome owing to disease-related proteolytic degradation pathways. Therefore, protein pattern diagnostics would provide easy and reliable tools for detection of cancer. This paper focuses on the current status of clinical proteomics research in oncology and in colorectal cancer especially, and will reflect on pitfalls and fears in this relatively new area of clinical medicine, which are reproducibility issues and pre-analytical factors, statistical issues, and identification and nature of discriminating proteins/peptides.

Physiological Evaluation of Wheat (<i>Triticum aestivum</i>L.) Genotypes at Pre-Anthesis Stage under Heat Stress Conditions

Experiments were conducted to analyze effects of high temperature stress on wheat at pre-anthesis growth stage. Twenty four wheat genotypes exposed to a sub optimal temperature (>35°C) which showed altered physiological, biochemical and agronomic characteristics. Accumulation of proline, presence of new protein bands and higher antioxidant enzyme activity in leaves of G.7 and G.17 reflects their better adaptive response under heat stress conditions. G.17 and G.19 showed least reduction in number of spikelets per spike, biological yield and 100 grain weight. It was inferred that the genotypes G.7, G.17 and G.19 exhibited greater heat tolerance and could be recommended for cultivation under heat stress conditions.

A fluorogenic-inhibitor-based probe for profiling and imaging of monoamine oxidase A in live human glioma cells and clinical tissues

Monoamine oxidase A(MAO-A)plays a critical role in the development of glioma and other neurological disorders.Specific analysis of MAO-A activities and its drug interactions in intact tissue is important for biological and pharmacological research,but highly challenging with current chemical tools.Fluorogenic-inhibitor-based probes offer improved selectivity,sensitivity,and effectiveness to image and profile endogenous targets in an activity-based manner from mammalian cells,which are however rare.Herein,we report HD1 as the first fluorogenic-inhibitor-based probe that can selectively label endogenous MAO-A from various mammalian cells and clinical tissues.The probe was delicately designed based on N-propargyl tetrahydropyridine,a small MAO-A-specific fluorogenic and inhibitory war-head,so that the probe becomes fluorescent upon in situ enzymatic oxidation and covalent labeling of MAO-A.With the excellent binding affinity(in vitro K_(i)=285 n M)and fluorogenic properties,HD1 offers a promising approach to simultaneously image endogenous MAO-A activities by super-resolution fluorescence microscopy and study its drug interactions by subsequent activity-based protein profiling,in both live cells and human glioma tissues.

Analysis of Two-Dimensional Gel Electrophoresis Map of Methicillin-Resistant <i>Staphylococcus aureus</i>Treated with Acetone Extract from <i>Canarium odontophyllum</i>Miq. Leaves

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a global health concern that has caused severe health threats over the past decade. Leaves extract of C. odontophyllum has been proven previously as an anti MRSA agent. Proteomics provide a technique that used to analyze the differential of protein expression profile between untreated and treated MRSA with subinhibitory concentrations of acetone extract from C. odontophyllum leaves. This study aims to determine the optimum parameter for analysis of protein expression profile using two-dimension gels electrophoresis (2-DE) for MRSA protein after treatment with acetone extract from C. odontophyllum leaves. Comparison of the Protein Expression Profile (PEP) between the untreated and treated MRSA was analyzed using PDQuest software. The optimum condition for MRSA protein treated with acetone extract from C. odontophyllum leaves to produce the best resolution with greater spot distribution was as follows: 100 μg volume of MRSA protein that loaded after passive rehydration then was run until reaching 25 kVrhs during IEF using 17 cm IPG strip within ranges of pH 4 - 7. Analysis of protein expression from the 2-DE gel map shows that 9 protein spots up-regulated and 41 protein spots were down-regulated with more than 2-fold differences (p < 0.05). This preliminary study on the PEP of MRSA treated with acetone extract of C. odontophyllum leave may provide an insight into the antimicrobial mechanism, which could lead to the identification of target protein for future novel therapeutic development against MRSA infections.

Biochemical reactions in metabolite-protein interaction

Active endogenous metabolites regulate the viability of cells. This process is controlled by a series ofinteractions between small metabolites and large proteins. Previously, several studies had reported thatmetabolite regulates the protein functions, such as diacylglycerol to protein kinase C, lactose regulationof the lac repressor, and HIF-1α stabilization by 2-hydroxyglutarate. However, decades old traditionalbiochemical methods are insufficient to systematically investigate the bio-molecular reactions for a high-throughput discovery. Here, we have reviewed an update on the recently developed chemical proteomicscalled activity-based protein profiling （ABPP）. ABPP is able to identify proteins interacted eithercovalently or non-covalently with metabolites significantly. Thus, ABPP will facilitate the characteriza-tion of specific metabolite regulating; proteins in human disease progression.

Interactome Mapping: Using Protein Microarray Technology to Reconstruct Diverse Protein Networks

A major focus of systems biology is to characterize interactions between cellular compo- nents, in order to develop an accurate picture of the intricate networks within biological systems. Over the past decade, protein microarrays have greatly contributed to advances in proteomics and are becoming an important platform for systems biology. Protein microarrays are highly flex- ible, ranging from large-scale proteome microarrays to smaller customizable microarrays, making the technology amenable for detection of a broad spectrum of biochemical properties of proteins. In this article, we will focus on the numerous studies that have utilized protein microarrays to recon- struct biological networks including protein-DNA interactions, posttranslational protein modifica- tions （PTMs）, lectin glycan recognition, pathogen-host interactions and hierarchical signaling cascades. The diversity in applications allows for integration of interaction data from numerous molecular classes and cellular states, providing insight into the structure of complex biological sys- tems. We will also discuss emerging applications and future directions of protein microarray tech- nology in the global frontier.

Prognosis of the short-term efficacy of thymectomy for myasthenia gravis with serum protein profiles

Background The extended thymectomy for myasthenia gravis （MG） is currently available, but in 20%-40% of the patients the results were not satisfactory. There are no ideal indicators forecasting surgical results before operation. The surface enhanced laser desorption ionization-time of flight-mass spectroscopy （SELDI-TOF-MS） is a currently new technique for detection of protein profiles, and some progresses have been made in cancer diagnosis and efficacy evaluation, but there is no report on efficacy forecasting of MG surgery. This study aimed to establish an efficacy prognosis model for forecasting the efficacy of surgery for MG by analysis of serum protein profiles of MG patients before surgery. Methods Fifty-six MG patients 6 months after extended thymectomy were enrolled in the study. They were classified into effective or non-effective groups according to symptoms and medication. Their pre-operative blood samples were analyzed for protein profiles by the SELDI-TOF MS technique, and protein peaks were identified for establishment of the efficacy prognosis model of MG surgery. Additional 100 MG patients were subjected to model validation and their pre-operation protein profiles reviewed for post-operative results. The results were compared with those of the post-operative follow-up so as to validate the prognosis model. Results For the model establishment, symptoms were improved in 33 patients and not improved in 18 patients, with an effective rate of 64.7%. Five （8.9%） patients were lost to follow-up. Within the molecular weight range of 1 000 to 20 000, 3 specific protein peaks were found to be significantly different between the effective and non-effective groups, ie M4110-76, M3394-58, and M1258-55. Using the efficacy prognosis model constructed with these data, the accuracy rate of classification was 87.9% for the effective group, and 83.3% for the non-effective group, with a total accuracy rate of 86.3%. For the model evaluation, 2 （8.9%） patients were lost to follow-up, 62 patients were effective and 36 were non- effective. By comparing with the real results of follow-up with 65 effective and 33 non-effective patients with an effective rate of 66.3%, the accuracy rate of prediction by the prognosis model was 86.2% for the effective group, and was 81.8% for the non-effective group with a total accuracy rate of 84.5%. Conclusions By protein profiles analysis of pre-operative blood samples taken from MG patients with the SELDI-TOF MS technique, protein peaks correlated with surgery efficacy in MG patients can be found for primary forecasting short-term efficacy of surgery for MG patients.

Chemoproteomics identifies Ykt6 as the direct target of schisandrin A for neuroprotection

Schisandrin A is a natural dibenzocyclooctene lignan with potent neuroprotective activity.However,the specific mechanisms or direct target proteins have not been clarified up to now.In this study,we designed and synthesized the probes of schisandrin A with photoreactive diazirine and clickable alkyne to identify its direct target in SH-SY5Y cells by employing activity-based protein profiling(ABPP)technique.Ykt6 was prominent among the 13 proteins obtained with high confidence and we confirmed Ykt6 as the direct target of schisandrin A by CETSA,IF,SPR and knockdown assay.Functionally,schisandrin A protected the cells against the injury induced by glutamate by regulating autophagy via Ykt6.This discovery may provide a novel therapeutic option for various neuronal cell damage-mediated diseases.

Cancer:A proteomic disease

The development of cancer is a pathological process involving multiple environmental carcinogenic factors and genetic alterations.For decades,cancer researchers have focused on genomic and transcriptomic analyses.The completion of the Human Genome Project has opened the door to the post-genome era and oncoproteomics.Proteins play a critical role in tumorigenesis and influence the differences between normal cells and malignant cells.This report proposes the concept that cancer is a proteomic disease.This concept is based on examining protein expression profiles,post-translational modifications,and protein-protein interactions in carcinogenesis using recent advances in comparative,functional and structural proteomics.This approach provides a new way of viewing carcinogenesis,presents new clues in biomarker discovery for cancer diagnosis and therapy,and reveals important scientific findings and their significance to clinical applications.

Cucurbitacin B-induced G2/M cell cycle arrest of conjunctival melanoma cells mediated by GRP78-FOXM1-KIF20A pathway

Conjunctival melanoma(CM) is a rare and fatal malignant eye tumor. In this study, we deciphered a novel anti-CM mechanism of a natural tetracyclic compound named as cucurbitacin B(CuB). We found that CuB remarkably inhibited the proliferation of CM cells including CM-AS16,CRMM1, CRMM2 and CM2005.1, without toxicity to normal cells. CuB can also induce CM cells G2/M cell cycle arrest. RNA-seq screening identified KIF20A, a key downstream effector of FOXM1 pathway, was abolished by CuB treatment. Further target identification by activity-based protein profiling chemoproteomic approach revealed that GRP78 is a potential target of CuB. Several lines of evidence demonstrated that CuB interacted with GRP78 and bound with a Kdvalue of0.11 μmol/L. Furthermore, ATPase activity evaluation showed that CuB suppressed GRP78 both in human recombinant GRP78 protein and cellular lysates. Knockdown of the GRP78 gene significantly induced the downregulation of FOXM1 and related pathway proteins including KIF20A, underlying an interesting therapeutic perspective. Finally, CuB significantly inhibited tumor progression in NCG mice without causing obvious side effects in vivo. Taken together, our current work proved that GRP78-FOXM1-KIF20A as a promising pathway for CM therapy, and the traditional medicine CuB as a candidate drug to hinder this pathway.

A targeted covalent inhibitor of p97 with proteome-wide selectivity

A resurging interest in targeted covalent inhibitors(TCIs)focus on compounds capable of irreversibly reacting with nucleophilic amino acids in a druggable target.p97 is an emerging protein target for cancer therapy,viral infections and neurodegenerative diseases.Extensive efforts were devoted to the development of p97 inhibitors.The most promising inhibitor of p97 was in phase 1 clinical trials,but failed due to the off-target-induced toxicity,suggesting the selective inhibitors of p97 are highly needed.We report herein a new type of TCIs(i.e.,FL-18)that showed proteome-wide selectivity towards p97.Equipped with a Michael acceptor and a basic imidazole,FL-18 showed potent inhibition towards U87 MG tumor cells,and in proteome-wide profiling,selectively modified endogenous p97 as confirmed by in situ fluorescence scanning,label-free quantitative proteomics and functional validations.FL-18 selectively modified cysteine residues located within the D2 ATP site of p97.This covalent labeling of cysteine residue in p97 was verified by LC-MS/MS-based site-mapping and site-directed mutagenesis.Further structure-activity relationship(SAR)studies with FL-18 analogs were established.Collectively,FL-18 is the first known small-molecule TCI capable of covalent engagement of p97 with proteome-wide selectivity,thus providing a promising scaffold for cancer therapy.