The hard tissues of animals, such as skeletons and teeth, are constructed by a biologically controlled process called biomineralization. In invertebrate animals, biominerals are considered important for their evolutionary success. These biominerals are hieratical biocomposites with excellent mechanical properties, and their formation has intrigued researchers for decades. Although proteins account for ~5 wt% of biominerals, they are critical players in biomineralization. With the development of high-throughput analysis methods, such as proteomics, biomineral protein data are rapidly accumulating, thus necessitating a refined model for biomineralization. This review focuses on biomineral proteomics in invertebrate animals to highlight the diversity of biomineral proteins (generally 40-80 proteins), and the results indicate that biomineralization includes thermodynamic crystal growth as well as intense extracellular matrix activity and/or vesicle transport. Biominerals have multiple functions linked to biological immunity and antipathogen activity. A comparison of proteomes across species and biomineral types showed that von Willebrand factor type A and epidermal growth factor, which frequently couple with other extracellular domains, are the most common domains. Combined with species-specific repetitive low complexity domains, shell matrix proteins can be employed to predict biomineral types. Furthermore, this review discusses the applications of biomineral proteomics in diverse fields, such as tissue regeneration, developmental biology, archeology, environmental science, and material science.

The increasing burden of respiratory disease is a rising concern in India. Although chronic colonisation is primarily caused by pathogenic fungi, the common environmental fungi also play an important role in developing sensitisation. This study aims to examine the allergenic potency of mycelial proteins of a common indoor fungus Aspergillus ochraceus to a selected atopic patient cohort as well as to identify the novel IgE-binding proteins through an immunoproteomic approach. 1-D and 2-D IgE specific western blot detected the IgE reactive proteins which were identified through MALDI-TOF/TOF and manual de novo peptide sequencing. The results revealed the detection of 10 cross-reactive IgE-binding proteins. Cluster analysis of 1-D immunoblot with individual patient sera identified NADP(+)-dependent glycerol dehydrogenase (GldB) homologous protein as a major allergen, which was further purified and the allergenicity was assessed. Other IgE-binding proteins showed homology with allergens like short-chain dehydrogenase, NAD-dependent mannitol dehydrogenase, and subtilisin-like serine protease. GldB purified under native conditions showed IgE reactivity amongst the selected patient cohort, which is reported for the first time in this study. The identified IgE-binding proteins can act as candidate molecules for developing hypoallergenic vaccines for designing specific immunotherapeutic techniques to fungal allergy. THE SIGNIFICANCE OF THE STUDY: Exposure to environmental fungal allergens is directly associated with promoting allergic response as well as complicating existing respiratory disease, leading to poor respiratory health. Amongst others, Aspergillus spp. contributes to the majority of the fungal derived atopic diseases. Aspergillus ochraceus is a common indoor mould in India, however, its allergenic potency was not explored till date. In this study, we establish A. ochraceus responsible to cause an allergic response to susceptible individuals and identified 10 IgE-binding proteins using an immunoproteomics approach for the first time. A. ochraceus being unsequenced, a homology-driven proteomics approach was used to identify the IgE-binding proteins which can be extended to identify proteins from other unsequenced species. The information on the IgE-binding proteins could be used as a step towards characterising them by molecular and structural methods to investigate the molecular basis of allergenicity. This will also help to enrich the existing database of allergenic proteins and pave a way towards developing therapeutic avenues.

Early detection of prostate cancer may lead to the overdiagnosis and overtreatment of patients as well as missing significant cancers. The current diagnostic approach uses elevated serum concentrations of prostate-specific antigen (PSA) as an indicator of risk. However, this test has been widely criticized as it shows poor specificity and sensitivity. In order to improve early detection and diagnosis, several studies have investigated whether different PSA proteoforms are correlated to prostate cancer. Until now, studies and methodologies for the comprehensive characterization of PSA proteoforms from biofluids are scarce. For this purpose, we developed an intact protein assay to analyze PSA by capillary electrophoresis-electrospray ionization-mass spectrometry after affinity purification from patients' urine. Here, we determined six proteolytic cleavage variants. In regard to glycosylation, tri-, di-, mono- and non-sialylated complex-type N-glycans were found on non-cleaved PSA, as well as the non-glycosylated variant. The performance of the intact protein assay was assessed using a pooled sample, obtaining an inter-day variability of 15%. Furthermore, urinary patient samples were analyzed by intact protein analysis and a bottom-up approach (glycopeptide analysis). This combined approach revealed complimentary information on both levels, demonstrating the benefit of using two orthogonal techniques to provide a thorough profile of urinary PSA. SIGNIFICANCE: The detection of clinically relevant prostate cancer requires a more specific and sensitive biomarker and, in this case, several PSA proteoforms may be able to aid or improve the current PSA test. However, a comprehensive analysis of the intact PSA proteoform profile is still lacking. This study investigated the PSA proteoforms present in urine and, in particular, determined the relative contribution of cleaved PSA and non-cleaved PSA forms to the total glycosylation profile. Importantly, intact protein analysis did not require further sample treatment before being measured by CE-ESI-MS. Furthermore, its glycosylation was also assessed in a bottom-up approach to provide complementary information. Overall, these results represent an important basis for future characterization and biomarker studies.

Extracellular vesicles (EVs) are important for the transport of biomolecular materials and intercellular communication in eukaryotes. Recent research has revealed that they are involved in plant-pathogen interaction and pathogenesis of infected cells. Phytophthora capsici is a highly devastating oomycete pathogen with a broad host range. To increase infection and facilitate colonization, it secretes effector proteins during interaction with plants. In this study, we characterize for the first time the EVs from pathogen P. capsici through transmission electron microscopy. For the biological study of EVs, results showed that mixing high concentrations of EVs with zoospores could enhance the virulence of P. capsici. By sequencing the protein composition of EVs by liquid chromatography in tandem with mass spectrometry we found that there are many proteins related to metabolism, oxidation/reduction, and transport in EVs, indicating that they have important roles in pathogenesis and immunological processes within the host. SIGNIFICANCE: Extracellular vesicles (EVs) are important both at normal physiological processes as well as pathological progression during pathogen and host interaction. In this paper we first establish the extraction method of EVs from the important oomycete pathogen Phytophthora capsici. Bioinformatics analysis of EV proteomics revealed a variety of pathogenic-related proteins, like oxidation/reduction-related proteins, stress response proteins as well as elicitors. Our results will help better understanding the biological function of the EVs during plant and P. capsici interaction and providing the evidence for the role of EVs in pathogenesis of the P. capsici.

Mass-spectrometry (MS) based phosphoproteomics is increasingly used to explore aberrant cellular signaling and kinase driver activity, aiming to improve kinase inhibitor (KI) treatment selection in malignancies. Phosphorylation is a dynamic, highly regulated post-translational modification that may be affected by variation in pre-analytical sample handling, hampering the translational value of phosphoproteomics-based analyses. Here, we investigate the effect of delay in mononuclear cell isolation on acute myeloid leukemia (AML) phosphorylation profiles. We performed MS on immuno-precipitated phosphotyrosine (pY)-containing peptides isolated from AML samples after seven pre-defined delays before sample processing (direct processing, thirty minutes, one hour, two hours, three hours, four hours and 24 h delay). Up to four hours, pY phosphoproteomics profiles show limited variation. However, in samples processed with a delay of 24 h, we observed significant change in these phosphorylation profiles, with differential phosphorylation of 22 pY phosphopeptides (p < 0.01). This includes increased phosphorylation of pY phosphopeptides of JNK and p38 kinases indicative of stress response activation. Based on these results, we conclude that processing of AML samples should be standardized at all times and should occur within four hours after sample collection. SIGNIFICANCE: Our study provides a practical time-frame in which fresh peripheral blood samples from acute myeloid patients should be processed for phosphoproteomics, in order to warrant correct interpretation of in vivo biology. We show that up to four hours of delayed processing after sample collection, pY phosphoproteomic profiles remain stable. Extended delays are associated with perturbation of phosphorylation profiles. After a delay of 24 h, JNK activation loop phosphorylation is markedly increased and may serve as a biomarker for delayed processing. These findings are relevant for biomedical acute myeloid leukemia research, as phosphoproteomic techniques are of particular interest to investigate aberrant kinase signaling in relation to disease emergence and kinase inhibitor response. With these data, we aim to contribute to reproducible research with meaningful outcomes.

Proteomics is increasingly used for exploring disease biomarkers and therapeutic targets. The data-independent acquisition (DIA) method collects all peptide signals in a sample, and provides a convenient way to archive disease-related molecular features for further exploration. In this study, we first established a high-coverage human hepatocellular carcinoma (HCC) spectral library containing 9393 protein groups, 119,903 peptides. Furthermore, we optimised the DIA method with respect to four key parameters: settings for mass spectrometry acquisition, gradient length, amount of sample loading, and length of analytical column. More than 6000 proteins from HepG2 cells could be stably quantified using the optimised one-shot DIA approach with a 2 h gradient time. One-shot DIA identified a similar number of proteins as did multi-fraction data-dependent acquisition (DDA) from the same group of HCC samples, but at a quarter of the total acquisition time. DIA data could recapture the classification results obtained from DDA data, thus paving the way for large-scale, multi-centre proteomics analysis of clinical samples. SIGNIFICANCE: The organ-specific spectral library for HCC and the optimised 2 h DIA approach met the urgent demands for large-scale quantitative proteomics analysis of HCC clinical samples. Compared with multi-fraction-DDA, the optimised one-shot DIA could reach a similar identification while consuming shorter acquisition time, thus making it possible to analyse thousands of clinical samples.

Salivary secretions play critical roles in interactions among insects, insect-vectored pathogens, and host plants. The Asian citrus psyllid Diaphorina citri is a sap-sucking Hemipteran that serves as a vector for Candidatus Liberibacter asiaticus, the causal agent of citrus greening disease ("Huanglongbing" or HLB). D. citri continuously injects saliva into host plants using specialized stylets so as to feed and transmit the HLB pathogen. Knowledge on the composition and function of salivary proteins of this pest is very limited. In this study, proteomic and transcriptomic approaches were adopted to characterize the protein composition of the saliva and salivary glands in D. citri. A total of 246 and 483 proteins were identified in saliva and dissected salivary glands, respectively, via LC-MS/MS analyses. Comparative analyses of the identified proteins were performed between D. citri and other reported Hemipteran insect species. Transcription levels of the genes coding for the identified proteins were determined via RNA-sequencing among different tissues including salivary glands and other digestive tissues. Identification of putative effectors that are expressed exclusively or abundantly in salivary glands provides the foundation for future functional studies towards the understanding of their roles in interactions among D. citri, HLB pathogen, and their citrus host. BIOLOGICAL SIGNIFICANCE: This is a systematic analysis on proteins in saliva and dissected salivary glands. A high percentage of novel proteins have been identified due to the large amounts of samples collected. This report gives a more comprehensive repertoire of potential effector proteins that may be possibly involved in modulating host defense, altering nutrient metabolism, and facilitating Ca. L. asiaticus transmission.

A wide variety of factors prior to slaughter may affect the stress status of beef cattle, giving rise to well-known 'dark-cutting' defective meats characterised by a high ultimate pH (pHu). To understand the underlying mechanisms of pHu fluctuations in beef cattle there was studied the proteome changes caused by pre-slaughter stress through a gel-free proteomic approach. Comparative peptidomic analysis was carried out on 12 loin samples at 24 h post-mortem from Longissimus thoracis et lumborum bovine muscle of crossbred animals, previously sorted into two different groups according to their pHu values: normal (pHu < 6.0) and high (pHu ≥ 6.0). Tryptic peptides from direct protein extracts were approached by combining untargeted (intact mass, MS1) and targeted (Selected Reaction Monitoring, SRM) quantitative LC-MS assays followed by chemometric analysis. Seventeen peptide biomarkers belonging to 10 different proteins appropriately discriminated sample groups assayed. Results may promote the use of this simple and effective methodology towards the creation of new insights in meat quality research. SIGNIFICANCE: The significance of this study was the optimization of an affordable straightforward gel-free proteomic approach addressing the differentiation of the muscle sub-proteome of normal and high pHu meat samples. This strategy allowed the study of tryptic peptides from direct meat protein extracts by combining untargeted MS1 and targeted SRM quantitative assays performed by conventional LC-MS detection. Affordability, simplicity and robustness of this methodology can facilitate its readily implementation in routine protocols for quality assessment of meat.

Wing discs of Bombyx mori (B. mori) are transformed into wings during metamorphosis via dramatic morphological and structural changes. Mutations in genes related to the wings cause the adults to have altered wing shapes or abnormal wing colour. At present, there are more than 20 wing mutants recorded in the silkworm. However, the key factors that influence B. mori wing development are still unclear. Here, we used the strains +Wes/+Wes and Wes/+Wes that are typical for the normal wing and shriveled wing phenotypes, respectively, to identify differentially expressed proteins by label-free data-independent acquisition (DIA). Ten enriched GO terms and 9 KEGG pathways were identified based on the 3993 proteins in the wings. Among the identified and quantified proteins, 370 differentially expressed proteins (DEPs) were detected (P-value <0.01, |log2FC| > 0.58). Mapping of the DEPs to the reference canonical pathways in KEGG showed that the top 20% of the pathways were related to fatty acid, cutin, suberin and wax biosynthesis, protein processing in endoplasmic reticulum, protein export, etc. Of the 370 DEPs, 238 were down-regulated, and 132 were up-regulated of Wes/+Wes compared with +Wes/+Wes. Numerous cuticular proteins were down-regulated, and fatty metabolism enzymes were up-regulated, in Wes/+Wes compared with +Wes/+Wes. SIGNIFICANCE: The comparative analysis of proteomes suggested that cuticular proteins and fatty metabolism enzymes are the main abnormally expressed proteins in the pupal wings of Wes/+Wes, leading to curly and shrunken wings after moth transformation. Our results also identify the substances affecting the development of silkworm wings from the perspective of proteins. The information from this study is important for further research on the molecular mechanisms of wing development in lepidopteran insects, and these differentially expressed genes may be targets for Lepidoptera pest control.

In the study on fermented acid rice soup (rice-acid) inoculated with L. paracasei H4-11 and K. marxianus L1-1, the concentrations of main flavor components on the third day of fermentation were significantly higher than those on the first day. Transcriptome analysis and proteome analysis based on RNA sequencing and 4D label-free proteomic techniques were combined to provide new insights into the molecular mechanisms of flavor characteristics and antioxidant activity of the two strains during the development of rice-acid. The key up-regulated genes and proteins in L. paracasei and K. marxianus L1-1, which were involved in flavor formation and antioxidant activity in rice-acid development, were different. The KEGG pathways involving the up-regulated genes and proteins in L. paracasei included starch and sucrose metabolism, pyruvate metabolism, amino sugar, and nucleotide sugar metabolism, and glycolysis/guconeogenesis. The KEGG pathways involving the up-regulated genes and proteins in K. marxianus L1-1 mainly included glycolysis/gluconeogenesis, TCA cycle, pyruvate metabolism, and other pathways related to antioxidant capacity. We successfully identified key genes and proteins associated with the metabolism and accumulation of flavor components and antioxidant activity. These findings provide new insights into the molecular mechanisms of flavor formation in co-cultivation with L. paracasei and K. marxianus. SIGNIFICANCE: It is anticipated that this study would provide us an insight into the mechanisms of flavor components accumulation and antioxidant activity of acid rice soup in China's minority areas. Importantly, this research provides the foundation of biological and chemical analysis for the application of the co-culture of L. paracasei H4-11 and K. marxianus in non-dairy products.

This study was aimed to explore the metabolomical mechanisms for the potentially ameliorative effect of cyst(e)ine (Cys) fortification on growth performance of broilers fed low crude protein (CP) diet. A total of 432 1-d-old broilers were randomly divided into 6 groups, each of which received one of the following diets: normal-CP diet (positive control, PC), low-CP diet (negative control, NC), NC diet fortified with 0.05%, 0.1%, 0.15% or 0.2% of Cys. Samples were collected on d 42. Results showed that increasing Cys fortification quadratically elevated (P < 0.05) the accumulative growth performance and leg muscle yield of broilers fed NC diet, with 0.1% being the optimal dose. Thus, samples from PC, NC and NC plus 0.1% Cys (NCC) groups were selected for further analysis. Both dietary CP reduction and fortification of 0.1% Cys in NC diet caused complex changes (P < 0.05) in serum amino acids and some other metabolites primarily involved in lipid metabolism. Multiple lipogenesis-related pathways were regulated (P < 0.05) following Cys fortification in NC diet, which could at least partially interpret the benefit of Cys fortification in NC diet on broiler performance. In conclusion, fortifying low-CP diet with 0.1% Cys promoted the growth performance of broilers probably through modulating serum metabolite profile.

We lack a predictive understanding of the environmental drivers determining the structure and function of archaeal communities as well as the proteome associated with these important soil organisms. Here, we characterized the structure (by 16S rRNA gene sequencing) and function (by metaproteomics) of archaea from 32 soil samples across terrestrial ecosystems with contrasting climate and vegetation types. Our multi-"omics" approach unveiled that genes from Nitrosophaerales and Thermoplasmata dominated soils collected from four continents, and that archaea comprise 2.3 ± 0.3% of microbial proteins in these soils. Aridity positively correlated with the proportion of Nitrosophaerales genes and the number of archaeal proteins. The interaction of climate x vegetation shaped the functional profile of the archaeal community. Our study provides novel insights into the structure and function of soil archaea across climates, and highlights that these communities may be influenced by increasing global aridity.

Toxoplasma gondii is one of the most successful intracellular parasites in the world. The dynamic, adhesion, invasion, and even replication capabilities of Toxoplasma are based on dynamic machinery located in the pellicle, a three membrane complex that surrounds the parasite. Among the proteins that carry out these processes are inner membrane complex (IMC) proteins, gliding-associated proteins (GAP), diverse myosins, actin, tubulin, and SRS proteins. Despite the importance of the pellicle, the knowledge of its composition is limited. Broad protein identification from an enriched pellicle fraction was obtained by independent digestion with trypsin and chymotrypsin and quantified by mass spectrometry. By trypsin digestion, 548 proteins were identified, while by chymotrypsin digestion, additional 22 proteins were identified. Besides, a group of "sequences related to SAG1" proteins (SRS) were detected together with unidentified new proteins. From identified SRS proteins, SRS51 was chosen for analysis and modeling as its similarities with crystallized adhesion proteins, exhibiting the presence of a spatial groove that is apparently involved in adhesion and cell invasion. As SRS proteins have been reported to be involved in the activation of the host's immune response, further studies could consider them as targets in the design of vaccines or of drugs against Toxoplasma. SIGNIFICANCE: To date, the proteomic composition of the pellicle of Toxoplasma is unknown. Most proteins reported in Toxoplasma pellicle have been poorly studied, and many others remain unidentified. Herein, a group of new SRS proteins is described. Some SRS proteins previously described from pellicle fraction have adhesion properties to the host cell membrane, so their study would provide data related to invasion mechanism and to open possibilities for considering them as targets in the design of immunoprotective strategies or the design of new pharmacological treatments.

Citrus junos is a widely used citrus grafting rootstock in china because of its excellent tolerance to cold stress. However, the physiological and molecular mechanisms underlying this process remain unknown. In this study, physiological and tandem mass tag-based proteomic analyses were performed to elucidate the mechanism of the Citrus junos response to cold stress. Physiological data showed that severe cold stress decreased photosynthetic parameters and caused cell membrane damage and membrane lipid peroxidation in Citrus junos leaves compared to the control. A total of 6, 678 distinct proteins species were identified, and 413 proteins species were significantly differentially accumulated in the leaves of Citrus junos seedling after cold stress. Bioinformatics analysis revealed that the differentially abundance protein species mainly related to the starch and sucrose metabolism, secondary metabolites biosynthesis and phenylpropanoid biosynthesis in the leaves of Citrus junos seedling after cold stress. Further physiological assays showed that the contents of soluble starch, fructose, glucose and phenols were significantly increased in Citrus junos leaves after cold stress. Collectively, our data reveals that sugar and secondary metabolism could play important roles in Citrus junos in response to cold stress.

Scorpion venoms are formed by toxins harmful to various organisms, including humans. Several techniques have been developed to understand the role of proteins in animal venoms, including proteomics approach. Rhopalurus agamemnon (Koch, 1839) is the largest scorpion in the Buthidae family in the Brazilian Cerrado, measuring up to 110 mm in total length. The accident with R. agamemnon is painful and causes some systemic reactions, but the specie's venom remains uninvestigated. We explore the venom protein composition using a proteomic and a biological-directed approach identifying 230 protein compounds including enzymes like Hyaluronidase, metalloproteinase, L-amino acid oxidase and amylase, the last two are first reported for scorpion venoms. Some of those new reports are important to demonstrate how distant we are from a total comprehension of the diversity about venoms in general, due to their diversity in composition and function. BIOLOGICAL SIGNIFICANCE: In this study, we explored the composition of venom proteins from the scorpion Rhopalurus agamemnon. We identified 230 proteins from the venom including new enzyme reports. These data highlight the unique diversity of the venom proteins from the scorpion R. agamemnon, provide insights into new mechanisms of envenomation and enlarge the protein database of scorpion venoms. The discovery of new proteins provides a new scenario for the development of new drugs and suggests molecular targets to venom components.

Nostoc flagelliforme is a type of terrestrial cyanobacteria that is distributed in arid or semi-arid steppes in China. To research the molecular mechanisms underlying the adaptation of N. flagelliforme to drought stress, the succinylated expression profile and changes in N. flagelliforme that resulted as a response to dehydration were analyzed by label-free proteomics. A total of 1149 succinylated sites, 1128 succinylated peptides, and 396 succinylated proteins were identified. Succinylated proteins were differentially involved in photosynthesis and energy metabolism, as well as in reactive oxygen species (ROS) scavenging. Motif-X analysis of succinylated sites determined a succinylation motif [KxxG]. N. flagelliforme adapts to dehydration by increasing glucose metabolism and pentose phosphate pathway flux, and decreasing photosynthetic rate, which some of the key proteins were succinylated. ROS scavenging was mainly involved in the regulation of the enzyme antioxidant defense system and non-enzymatic antioxidant defense system through succinylation modification, thus eliminating excessive ROS. Protein succinylation of N. flagelliforme may play an important regulatory role in response to dehydration. The results are foundational, as they can inform future research into the mechanisms involved in the succinylation regulation mechanism of N. flagelliforme in response to dehydration. SIGNIFICANCE: The global succinylation network involved in response to dehydration in N. flagelliforme has been established. We found that many succinylated proteins were involved in photosynthesis, glucose metabolism and antioxidation. The global survey of succinylated proteins and the changes of succinylated levels in response to dehydration provided effective information for the drought tolerance mechanism in N. flagelliforme.

Long-term spaceflight has always been challenging for astronauts due to the extremely complicated space environmental conditions, including microgravity, noise, confinement, and circadian rhythms disorders, which may cause adverse effects on astronauts' mental health, such as anxiety and depression. Unfortunately, so far, the underlying mechanism is not fully understood. Hence, a novel type of box and rat cage was designed and built in order to simulate complex space environment on the ground. After earth-based simulation for 21 days, the rats exhibited the depressive-like behavior according to the sucrose preference and forced swimming test. We applied label-free quantitative proteomics to explore the molecular mechanisms of depressive-like behavior through global changes in cortical protein abundance, given that the cortex is the hub of emotional management. The results revealed up-regulated spliceosome proteins in contrast to down-regulated oxidative phosphorylation (OXPHOS), glutamatergic, and GABAergic synapse related proteins in the simulated complex space environment (SCSE) group. Furthermore, PSD-95 protein was found down-regulated in mass spectrometry, reflecting its role in the psychopathology of depression, which was further validated by Western blotting. These findings provide valuable information to better understand the mechanisms of depressive-like behavior. SIGNIFICANCE: Quantitative proteomic analysis can quantify differentially abundant proteins related to a variety of potential signaling pathways in the rat cortex in the simulated complex space environment. These findings not only provide valuable information to better understand the mechanisms of depressive-like behavior, but also might offer the potential targets and develop countermeasures for the mental disorders to maintain the health of astronauts during the long-term spaceflight.

Pemetrexed (PEM), a multi-target folate antagonist, has been extensively used for the treatment of non-small cell lung cancer (NSCLC). However, the therapeutic efficacy of PEM is limited by tumor resistance. In this project, iTRAQ and parallel reaction monitoring (PRM)-based LC-MS/MS comparative proteomic analysis was performed to identify protein determinants of PEM resistance in A549/PEM cells versus A549 parental cells. A total of 567 differentially expressed proteins (DEPs) were identified by iTRAQ analysis. The function and classification of DEPs were analyzed through GO and KEGG Pathway databases. Moreover, PRM analysis further validated the expression changes of 14 DEPs identified by iTRAQ analysis. Moreover, insulin-like growth factor (IGF) 2 mRNA-binding protein 2 (IGF2BP2) or folate receptor alpha (FOLR1) knockdown weakened PEM resistance, reduced cell viability and promoted cell apoptosis in A549/PEM cells. IGF2BP2 depletion inhibited cell migration, invasion and epithelial-mesenchymal transition (EMT), while FOLR1 loss had no much effect on cell migration, invasion and EMT in A549/PEM cells. Our study can provide a deep insight into molecular mechanisms of PEM resistance in NSCLC and contribute to the development of more effective therapeutic schedules. SIGNIFICANCE: Our study can provide deeper insight into molecular mechanisms of PEM resistance in NSCLC and contribute to the development of more effective therapeutic schedules.