Analysis of Differentially Expressed Proteins and Modifications Induced by Formaldehyde Using LC-MS/MS
Analysis of Differentially Expressed Proteins and Modifications Induced by Formaldehyde Using...
Liu, Ranran;Han, Yue;Wu, Zhiyue;Zhang, Jianji;Zang, Yong;Shen, Lijin;Tian, Shanshan;Zhang, Kai
2022-04-29 00:00:00
separations Article Analysis of Differentially Expressed Proteins and Modifications Induced by Formaldehyde Using LC-MS/MS 1 , † 1 , † 1 1 1 2 , 1 , Ranran Liu , Yue Han , Zhiyue Wu , Jianji Zhang , Yong Zang , Lijin Shen *, Shanshan Tian * 1 , and Kai Zhang * The Province and Ministry Co-Sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Key Laboratory of Medical Epigenetics, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), Tianjin Key Laboratory of Cellular Homeostasis and Diseases, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin 300070, China; liuranr112@126.com (R.L.); yuehan@tmu.edu.cn (Y.H.); zhiyuewu@tmu.edu.cn (Z.W.); zhangjianji@tmu.edu.cn (J.Z.); zangyong1207@126.com (Y.Z.) NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital & Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin 300134, China * Correspondence: shenlj@tmu.edu.cn (L.S.); tianshanshan@tmu.edu.cn (S.T.); kzhang@tmu.edu.cn (K.Z.); Tel.: +86-22-8333-6833 (K.Z.) † These authors contributed equally to this work. Abstract: Formaldehyde (FA) is a toxic compound that is considered to have a carcinogenic effect due to its damage to biological macromolecules. However, the influence of FA at the protein level remains to be explored. Here, we used LC-MS/MS to identify the differentially expressed proteins and modifications to proteins between FA-treated and untreated HeLa cells. Among 2021 proteins identified, 196 proteins were significantly down-regulated and 152 up-regulated. The differentially expressed proteins were further analyzed using bioinformatics tools for annotating the Citation: Liu, R.; Han, Y.; Wu, Z.; Zhang, J.; Zang, Y.; Shen, L.; Tian, S.; characterization of their localizations and functions. To evaluate the interaction of FA with proteins, Zhang, K. Analysis of Differentially we performed proteomic analysis for a mass shift of 12 Da on the side chains of lysine, cysteine and Expressed Proteins and Modifications tryptophan, which are induced by FA as noticeable signals. We identified the modified proteins Induced by Formaldehyde Using and sites, suggesting direct interaction between FA and proteins. Motif analysis further showed LC-MS/MS. Separations 2022, 9, 112. the characterization of amino acid sequences that react with FA. Cluster analysis of the modified https://doi.org/10.3390/ proteins indicated that the FA-interacting networks are mostly enriched in the nuclei, ribosomes and separations9050112 metabolism. Our study presents the influence of FA on proteomes and modifications, offering a new Academic Editor: insight into the mechanisms underlying FA-induced biological effects. Victoria Samanidou Keywords: LC-MS/MS; formaldehyde; proteomics; protein modifications; high performance Received: 31 March 2022 liquid chromatography Accepted: 26 April 2022 Published: 29 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- Formaldehyde (FA) is considered a toxic substance that is used in the manufacture iations. of building materials, in household products and for illegal food preservation. Mounting evidence demonstrates that FA is toxic to the human body [1–3]. In particular, FA can cause genotoxic and carcinogenic effects due to damage to DNA or impeding of transcription [4]. Copyright: © 2022 by the authors. It is believed that tumors may be induced by long-term exposure to FA. Indeed, FA is Licensee MDPI, Basel, Switzerland. considered a nasal carcinogen through cytotoxicity and auxiliary genotoxicity [5]. FA can This article is an open access article directly damage the cells of nasal cavity, resulting in the proliferation of damaged cells, distributed under the terms and which plays a role throughout the entire process of nasal carcinogenicity. Genotoxicity conditions of the Creative Commons may be caused by FA through DNA–protein crosslinks and oxidative DNA damage [6]. Attribution (CC BY) license (https:// FA has also been reported to be associated with a risk of leukemia in some epidemiology creativecommons.org/licenses/by/ studies [7–9]. However, some studies pointed out that there is not enough evidence to 4.0/). Separations 2022, 9, 112. https://doi.org/10.3390/separations9050112 https://www.mdpi.com/journal/separations Separations 2022, 9, 112 2 of 11 support the hypothesis that FA causes myeloid leukemia [10,11]. FA has long been consid- ered to be ‘carcinogenic to humans’ in Group 1 by the International Agency for Research on Cancer (IARC, 2006), although the underlying mechanisms remain unclear. Recent works still cannot exclude the possibility that FA increases the risk of lung cancer and other cancers [12,13]. In addition, FA is thought to induce the occurrence of neurodegenerative diseases [14]. Recent work further demonstrates that FA results in the suppression of effector T cell activities, thereby contributing to an immunosuppressive environment and the progression of cancer [15]. To understand the molecular mechanism of FA-induced damage to biological macro- molecules, a variety of methods have been employed to determine the levels and effects of FA, including spectrophotometry, fluorescence, liquid chromatography (LC) and mass spectrometry (MS) [16–21]. At present, liquid chromatography tandem mass spectrometry (LC-MS/MS) is a powerful tool for proteomics analysis and protein modification identifica- tion [22,23], and thus can provide more detailed information regarding protein alterations induced by FA compared to traditional methods [24–27]. However, the effect of FA at the protein level remains to be explored. Tayri-Wilk et al. recently reported the mechanism underlying FA-induced cross-linking in proteins [16]. The FA-induced cross-linking reaction can be adopted to analyze protein structure [21,28]. Thus, we reasonably assumed that FA-induced reactions in proteins could be used as signals to decipher the effects of FA on proteomes and to determine the proteins that interact with FA. In this study, we employed LC-MS/MS for the detection of protein alterations between HeLa cells treated using FA (experimental group) and HeLa cells without FA treatment (control group). Pearson’s correlation coefficient (PCC) and principal component analysis (PCA) both demonstrated the reliability of our data. Among a total of 2021 proteins identified, 196 were significantly down-regulated and 152 were obviously up-regulated. The differentially expressed proteins were further analyzed using bioinformatics tools to characterize their localization and functions. To evaluate the effect of FA on proteins, we comprehensively analyzed a mass shift of +12 Da on lysine (K), cysteine (C) and tryptophan (W) as FA-induced signals. Thus, we identified the modified sites, suggesting direct interaction between FA and proteins. Cluster analysis of the modified proteins showed that the networks with FA interactions were mainly enriched in the nucleus, ribosomes, and metabolism. Our study presents the influence of FA on proteomes and modifications, and it is useful for understanding the mechanism underlying FA-induced biological effects. 2. Materials and Methods 2.1. Chemicals and Reagents Cell culture medium was supplied by Invitrogen (Grand Island, NY, USA). Fetal bovine serum (FBS) was purchased from VivaCell Biosciences (Shanghai, China). C18 ZipTips were obtained from Millipore (Bedford, MA, USA). Sequencing-grade modified trypsin was purchased from Promega (Madison, WI, USA). BCA protein assay kit and formic acid were bought from Thermo Fisher Scientific Inc. (Rockford, IL, USA). Formaldehyde solution (36.5–38% in water), dithiothreitol, iodoacetamide, cysteine, and ammonium bicarbonate were obtained from Sigma-Aldrich (Shanghai, China). 2.2. Cell Culture, Protein Extraction and Digestion HeLa cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin at 37 C in a humidified incubator at 5% CO . When reaching a confluence of approximately 80%, cells were treated with 1% FA (final concentration) for 15 min (for proteomics analysis) or 24 h (for modification analysis), respectively, while untreated cells were used as controls. After treatment, cells were washed twice in ice- cold phosphate-buffered saline (PBS). Plates were put on ice and 1 mL RIPA lysis buffer (1% TritonX-100, 0.1% SDS, 50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 2 mM sodium pyrophosphate,1 mM EDTA, with 1 protease inhibitor cocktail and 1 deacety- Separations 2022, 9, 112 3 of 11 lase inhibitor cocktail) was added. After 30 min of incubation, cells were scraped and supernatants were collected after centrifuging at 12,000 g for 3 min. Protein concen- trations were measured using a BCA protein assay kit, and equal total protein amounts (100 ug) in each sample were precipitated by adding trichloroacetic acid (TCA) up to 25% final concentration (v/v). After washing twice with 20 C acetone, the protein pellets were dissolved in 100 mM NH HCO (pH 8.0) for digestion. Protein solutions were subjected 4 3 to tryptic digestion at 37 C for 16 h. Dithiothreitol was added to a final concentration of 5 mM followed by incubation at 56 C for 1 h. Iodoacetamide was added to alkylate proteins with a final concentration of 15 mM followed by incubation at room temperature in the dark for 45 min. The alkylation reaction was quenched with 30 mM of cysteine (final concentration) at room temperature for another 30 min. Trypsin was then added again with a ratio of trypsin to protein of 1:100 (w/w) for digestion at 37 C for 4 h to complete the process. Three technical replicates and two biological replicates were analyzed. 2.3. LC-MS/MS Analysis Tryptic peptides were desalted using C18 ZipTips according to the manufacturer ’s instructions before LC-MS/MS analysis. Peptides were dissolved in HPLC buffer A (0.1% (v/v) formic acid in water) before being injected into a Nano-LC system (EASY-nLC 1000, Thermo Fisher Scientific, Waltham, MA, USA). Peptide separation was performed usig a reversed-phase C18 analytical column (75 m inner-diameter 15 cm, 3 m C18) with a 75 min HPLC gradient. The gradient comprised increases from 5% to 13% solvent B (0.1% (v/v) formic acid in 100% ACN) for 16 min, from 13% to 28% for 35 min, from 28% to 40% for 15 min, and from 40% to 100% in 1 min followed by holding at 100% for the last 8 min, all at a constant flow rate of 300 nl/min. The HPLC elutes were electrosprayed directly into an Orbitrap Q-Exactive mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Mass spectrometric analysis was carried out in data-dependent mode with an automatic switch between a full MS scan and an MS/MS scan in the orbitrap. For the full MS survey scan, the automatic gain control (AGC) target was 3e and scan range was from 350 to 1750 at a resolution of 70,000. The 15 most intense peaks with charge state 2 and above were selected for fragmentation by higher-energy collision dissociation (HCD) with a normalized collision energy of 27%. MS2 spectra were acquired at a resolution of 17,500. The exclusion duration for the data-dependent scan was 20 s, and the isolation window was set at 2.0 Da. 2.4. Database Search The resulting MS/MS data were searched against the UniProt database using Maxquant software (v.1.5.5.1). Label-free quantitation (LFQ) was performed to determine the relative quantification of the proteins. Peptide sequences were searched using trypsin specificity and allowing a maximum of five missed cleavages. Mass error was set to 10 ppm for pre- cursor ions and 0.02 Da for fragment ions. Mass shifts (+12 Da) on lysine (K), cysteine (C), tryptophan (W), oxidation on methionine (M), and acetylation on protein N-terminal were set as variable modifications. False discovery rate (FDR) thresholds for protein, peptide, and modification site were specified at 1%. Minimum peptide length was set at 7. All other parameters in MaxQuant were set to the default values. MaxQuant search results were exported, and reverse matches and possible contaminants were deleted. Furthermore, modified peptides with scores less than 40 and localization probability scores less than 0.75 were removed. The relative quantification of the proteins was determined by LFQ intensity. The threshold for up- or down-regulated proteins was set as a twofold change. 2.5. Bioinformatic Analysis The majority of bioinformatics analysis was accomplished using R (v.4.1.3) and Mi- crosoft Excel. We performed correlation analysis by using the R package corrplot (v.0.92) to identify highly correlated variables. Principal component analysis (PCA) was performed through the R packages FactoMineR (v.2.0) and factoextra (v.1.0.7). The volcano plot was Separations 2022, 9, 112 4 of 11 created using the R package ggplot2 (v.3.3.5). Gene Ontology (GO) enrichment analysis was performed using a hypergeometric test in R package clusterProfiler (v.4.1.4) for which the p.adjust threshold was specified at 0.05. The String database (v.11.5) was read into Cy- toscape (v.3.9.0) for visualization of protein–protein interactions and the minimum required interaction score was set to 0.4. Motif analysis of modified sequences was visualized using iceLogo (v.1.3.8). Protein domain and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was analyzed using the String database. Structure visualization was presented with PyMOL (v.1.8.4.0). The histogram was obtained using Prism Software 9.0 (GraphPad). For all statistical analyses, a two-tailed p value less than 0.05 was considered to be statistically significant. 3. Results and Discussion 3.1. The Design of Analysis Method As a reactive compound, FA has long been considered to produce serious influences on survival, growth, and differentiation of cells [1]. In particular, FA causes the abnormal expression of proteins [27]. Recent studies showed that FA can induce the occurrence of chemical modifications on proteins [21]. For example, a mass shift of +12 Da at lysine residues (K) has been revealed as an important signal of FA adduction. Similar reactions are observed at tryptophan (W) and cysteine (C) [28]. Thus, we rationally hypothesized that these signals could be used as protein modifications for understanding the effect of FA at the protein level. A brief description of our strategy is illustrated in Figure 1. We chose two groups of cells, those treated with FA being the experimental sample while those not so treated being the control sample. Proteins were extracted and separated from cells and subjected to in-solution tryptic digestion; subsequently, the resulting peptides were analyzed using LC-MS/MS. The MS raw data were further analyzed for proteome quantification and FA-induced modifications. The differentially expressed proteins and Separations 2022, 9, x FOR PEER REVIEW 5 of 12 modifications were finally analyzed using bioinformatics tools to investigate the impact of FA. Figure 1. Schematic overview of the experimental and data analysis workflow. Figure 1. Schematic overview of the experimental and data analysis workflow. 3.2. Evaluation of Analysis Data Quality It has been reported that a 15 min treatment with FA has a measurable effect on cell outcomes [29], and thus we chose that condition as the experimental group for prote- omics analysis. The control group was tested without FA treatment in parallel. To obtain accurate quantification of proteomes, we performed two biological replicate experiments and three technical replicates for each sample. To evaluate the robustness and reliability of these results, we performed Pearson’s correlation coefficient analysis (PCC) and prin- cipal component analysis (PCA). As shown in Figure 2A, all correlation coefficients were over 0.90, indicating strong correlations. Excellent correlations among replicates show that our data are reliable. Through PCA analysis of proteomic data, we could easily dis- tinguish the FA-treated samples from control samples, and no batch effects were ob- served. The two groups of samples are represented by different shapes, and the ellipse shape represents the 0.95 confidence intervals for each type. This result demonstrates that there were significant differences in protein expression between the experimental group and the control group, suggesting changes to proteomes from FA treatment. Separations 2022, 9, 112 5 of 11 3.2. Evaluation of Analysis Data Quality It has been reported that a 15 min treatment with FA has a measurable effect on cell outcomes [29], and thus we chose that condition as the experimental group for proteomics analysis. The control group was tested without FA treatment in parallel. To obtain accu- rate quantification of proteomes, we performed two biological replicate experiments and three technical replicates for each sample. To evaluate the robustness and reliability of these results, we performed Pearson’s correlation coefficient analysis (PCC) and principal component analysis (PCA). As shown in Figure 2A, all correlation coefficients were over 0.90, indicating strong correlations. Excellent correlations among replicates show that our data are reliable. Through PCA analysis of proteomic data, we could easily distinguish the FA-treated samples from control samples, and no batch effects were observed. The two groups of samples are represented by different shapes, and the ellipse shape represents the 0.95 confidence intervals for each type. This result demonstrates that there were significant Separations 2022, 9, x FOR PEER REVIEW 6 of 12 differences in protein expression between the experimental group and the control group, suggesting changes to proteomes from FA treatment. Figure 2. Evaluation of analysis data quality. (A) Pearson’s correlation coefficient (PCC). The Figure 2. Evaluation of analysis data quality. (A) Pearson’s correlation coefficient (PCC). The thickness thickness of the line represents the value of the PCC. The thinner the line, the closer it is to 1. (B). of the line represents the value of the PCC. The thinner the line, the closer it is to 1. (B). Principal Principal component analysis (PCA). The ellipse represents the 0.95 confidence intervals. component analysis (PCA). The ellipse represents the 0.95 confidence intervals. 3.3. Analysis of Proteins Differentially Induced by FA 3.3. Analysis of Proteins Differentially Induced by FA To determine the differential inducement of proteins by FA, we used A label-free To determine the differential inducement of proteins by FA, we used A label-free strategy for quantification of proteomes. The resulting normal distribution of the relative strategy for quantification of proteomes. The resulting normal distribution of the rela- protein abundance between the experimental group and the control group indicated that tive protein abundance between the experimental group and the control group indicated the data were reasonable and comparable (Figure 3A). Among a total of 2021 proteins that the data were reasonable and comparable (Figure 3A). Among a total of 2021 pro- identified, 348 proteins were significantly distinguished, including 152 up-regulated teins identified, 348 proteins were significantly distinguished, including 152 up-regulated proteins (>2-fold increase, p < 0.05) and 196 down-regulated proteins (>2-fold decrease, p proteins (>2-fold increase, p < 0.05) and 196 down-regulated proteins (>2-fold decrease, < 0.05), as shown in Figure 3B; the results are summarized in Supplementary Material p < 0.05), as shown in Figure 3B; the results are summarized in Supplementary Material Table S1–S3. We further characterized the significantly changed proteins via GO analysis. Tables S1–S3. We further characterized the significantly changed proteins via GO analysis. ItIt ca can n be be se seen en frfr om om FiFigur gure e 3C 3C,D ,D th that at th the e do down-r wn-reg egulated ulated proteins w proteins wer ere enriche e enriched d in DNA- in DNA- and chrom and chromatin-r atin-related prot elated proteins, eins, while th while the e up-r up-r eg egulated ulated proteins w proteins wer ere mostly e mostly enr enriched iched in in met metabolic abolic pat pathways hways ssuch uch a as s ffatt atty y aci acids ds and lip and lipids. ids. T These hese r results esults suggest suggest that that alterations alterations to to proteins proteins arar e e ind induced uced b by y F A. FA. Separations 2022, 9, x FOR PEER REVIEW 7 of 12 Separations 2022, 9, 112 6 of 11 Figure 3. Identification of differentially expressed proteins and GO enrichment analysis. (A) His- Figure 3. Identification of differentially expressed proteins and GO enrichment analysis. (A) His- togram showing distribution of relative protein abundance between FA-treated and control cells. togram showing distribution of relative protein abundance between FA-treated and control cells. (B) Volcano plot of differential protein expression in FA-treated group compared to control group. (B) Volcano plot of differential protein expression in FA-treated group compared to control group. Differentially expressed proteins were deemed significant if p < 0.05 and absolute fold change > 2. Differentially expressed proteins were deemed significant if p < 0.05 and absolute fold change >2. Blue dots represent down-regulated proteins; red dots represent up-regulated proteins; grey dots Blue dots represent down-regulated proteins; red dots represent up-regulated proteins; grey dots represent other identified proteins that were not significantly changed. (C,D) GO enrichment represent other analysis o identifiedfp up-regulated or down-regulat roteins that were not significantly ed proteins. B changed.P(: Cbiol ,D)ogica GO enrichment l process; CC analysis : cellular compo- nent; MF: molecular function. of up-regulated or down-regulated proteins. BP: biological process; CC: cellular component; MF: molecular function. 3.4. The Analysis of Protein Modifications Induced by FA 3.4. The Analysis of Protein Modifications Induced by FA It is known that FA can induce chemical reactions involving proteins. However, the It is known that FA can induce chemical reactions involving proteins. However, the detailed chemical modifications and mechanisms underlying FA-induced reactions are detailed chemical not v modifications ery clear. Receand nt stmechanisms udies revealed underlying that +12 Da F o A-induced n the side c reactions hain of th ar ee ly not sine residue very clear. Recent is an ob studies vious s revealed ignal of FA that react +12ion Da s w on ith the prot side eins chain in MS of an the alys lysine is [16,r 2esidue 1,30]. The is reaction mechanism is shown in Supplementary Material Figure S1. FA is first added to the amino an obvious signal of FA reactions with proteins in MS analysis [16,21,30]. The reaction group on the side chain of the lysine residue (+30 Da), and then is turned into an imine mechanism is shown in Supplementary Material Figure S1. FA is first added to the amino group via dehydration (+12 Da). Similar adduction occurs on other amino acids such as group on the side chain of the lysine residue (+30 Da), and then is turned into an imine cysteine and tryptophan [31]. Thus, we analyzed these modifications at the proteomics group via dehydration (+12 Da). Similar adduction occurs on other amino acids such as level in the experimental group and the control group. We found increases in number of cysteine and tryptophan [31]. Thus, we analyzed these modifications at the proteomics level modified sites in the experimental group, compared to the control group (Figure 4A), in the experimental group and the control group. We found increases in number of modified suggesting that these modified sites were induced by FA. Next, we analyzed the fre- sites in the experimental group, compared to the control group (Figure 4A), suggesting quency of the modifications in proteins, including 52 proteins that had one modified site, that these modified sites were induced by FA. Next, we analyzed the frequency of the 42 proteins that had two modified sites, and 25 proteins that had three or more modified modifications in proteins, including 52 proteins that had one modified site, 42 proteins that sites (Figure 4B), as shown in Supplementary Material Table S4. had two modified sites, and 25 proteins that had three or more modified sites (Figure 4B), as shown in Supplementary Material Table S4. Separations 2022, 9, x FOR PEER REVIEW 8 of 12 Separations 2022, 9, 112 7 of 11 Figure 4. Characterization of domains and sequence recognition of motifs. (A) Comparison of the number of modifications between FA-treated group and control group. (B) Frequency of FA-induced Figure 4. Characterization of domains and sequence recognition of motifs. (A) Comparison of the number of mod modification on pr ific oteins. ations between FA-treated (C) The consensus sequence group and logos show control group. enrichment of ( amino B) Frequenc acid residues y of FA-induced modification on proteins. (C) The consensus sequence logos show enrichment of among the K+12 Da using icelogo software. Motifs with significance of p < 0.05 are shown. The motifs amino acid residues among the K+12 Da using icelogo software. Motifs with significance of p < 0.05 take lysine as center and show the distribution of seven amino acids on both sides. (D) Statistics of are shown. The motifs take lysine as center and show the distribution of seven amino acids on both the various domains of FA-induced modification proteins in HeLa. sides. (D) Statistics of the various domains of FA-induced modification proteins in HeLa. We also analyzed the position-specific amino acid frequency of the surrounding We also analyzed the position-specific amino acid frequency of the surrounding sequences (15 amino acids to both termini) of modification using icelogo software, as sequences (15 amino acids to both termini) of modification using icelogo software, as shown in Figure 4C. To understand the selectivity of the FA-induced reaction to proteins, shown in Figure 4C. To understand the selectivity of the FA-induced reaction to proteins, we further studied the structural properties of the modified proteins through the String we further studied the structural properties of the modified proteins through the String database. We found that GroEL-like apical domain superfamily, Chaperone tailless complex database. We found that GroEL-like apical domain superfamily, Chaperone tailless polypeptide 1 (TCP-1), NAD (P)-binding domain superfamily and ATPase and nucleotide complex polypeptide 1 (TCP-1), NAD (P)-binding domain superfamily and ATPase and binding domains were the main domains of these modified proteins (Figure 4D). Next, nucleotide b we selectedifive nding typical domains were proteins fr the om main the Gr do oEL-like mains of these apical domain modified superfamily proteins (Fig andur the e 4D) Chaper . Next one , wtailless e selectcomplex ed five ty polypeptide pical proteins 1 (TCP-1) from th domain. e GroEL- The like pr ap otein ical dom structur ain supe es wer r-e family and the Chaperone tailless complex polypeptide 1 (TCP-1) domain. The protein obtained from the RCSB PDB database (PDB ID: 6NR8). The five protein subunits were structure TCP1, CCT2, s were obtained CCT4, CCT5, from the and CCT8, RCSB PDB and they databa belonged se (PDB ID: 6N to the homologou R8). The five protei s proteins. n subunits were TCP1 As shown in Figure , CCT 5, we 2, CCT4 annotated , CCT5 the , an site d CCT of the 8, and modification they belonged on the to th protein, e homolo- with lysine (K) in yellow and cysteine (C) in green. The identified modification sites of the gous proteins. As shown in Figure 5, we annotated the site of the modification on the proteins observed were as follows: K406 and K400 of TCP1 protein, K402 of CCT2, K414 protein, with lysine (K) in yellow and cysteine (C) in green. The identified modification and K418 of CCT4, C407 of CCT5, and K400 and K406 of CCT8. It can be seen that the sites of the proteins observed were as follows: K406 and K400 of TCP1 protein, K402 of five proteins are structurally similar, and the modification sites are clustered in the same CCT2, K414 and K418 of CCT4, C407 of CCT5, and K400 and K406 of CCT8. It can be seen region of the protein, suggesting that specific domains and sites prefer to interact with Separations 2022, 9, x FOR PEER REVIEW 9 of 12 Separations 2022, 9, 112 8 of 11 that the five proteins are structurally similar, and the modification sites are clustered in the same region of the protein, suggesting that specific domains and sites prefer to in- teract with FA. To further characterize the proteins interacting with FA, we analyzed the FA. To further characterize the proteins interacting with FA, we analyzed the modified modified proteins using KEGG. As shown in Figure 6A, the modified proteins were en- proteins using KEGG. As shown in Figure 6A, the modified proteins were enriched in riched in ribosome proteins, nucleoproteins and metabolic enzymes. To investigate cor- relations ribosome am pr ong the mod oteins, nucleopr ified protein oteins and s, we metabolic analyzenzymes. ed protein– To prot investigate ein interaction net- correlations among the modified proteins, we analyzed protein–protein interaction networks via String works via String and Cytoscape, and the interaction network indicated that these modi- and Cytoscape, and the interaction network indicated that these modified proteins are fied proteins are closely related (Figure 6B). The results illustrated the proteins that in- closely related (Figure 6B). The results illustrated the proteins that interacted with FA in teracted with FA in cells and presented the characterization and relationships of these cells and presented the characterization and relationships of these modified proteins. modified proteins. Figure 5. Analysis of different modified protein structures induced by FA. (A) Superposition dia- Figure 5. Analysis of different modified protein structures induced by FA. (A) Superposition diagram gram of structural comparison of the five proteins. (B) TCP1 protein; the identified modification of structural comparison of the five proteins. (B) TCP1 protein; the identified modification sites are sites are K406 and K400. (C) CCT2 protein; the identified modification site is K402. (D) CCT4 pro- K406 and K400. (C) CCT2 protein; the identified modification site is K402. (D) CCT4 protein; the tein; the identified modification sites are C414 and K418. (E) CCT5 protein; the identified modifi- identified modification sites are C414 and K418. (E) CCT5 protein; the identified modification site is cation site is C407. (F) CCT8 protein; the identified modification sites are K400, K406. Yellow and C407. (F) CCT8 protein; the identified modification sites are K400, K406. Yellow and green represent green represent modification sites lysine (K) and cysteine (C), respectively. modification sites lysine (K) and cysteine (C), respectively. As a As atoxic toxic co compound, mpound, FA is FA is associated associated with with a a v variety ariety of d of diseases, iseases, inc including luding tumors tumors and and ne neurodegenerative urodegenerat and ive and immune immune diseases dise [1,3ase ,14s, [1 15].,3Although ,14,15]. Al the though the underl underlying mechanisms ying mecha remain nisms unclear rema , cytotoxicity in unclear, cy and totoxi genotoxicity city and genotoxi induced by city FA inis duc consider ed by F ed A is co to be nsider damaging ed to be damaging to human to human health. For example, health. For ex FA can ampl cause e, FA c degenerat an cause degeneration ion and necrosis and of respiratory necrosis epithelial cells in the nasal cavity, resulting in cell proliferation and squamous metaplasia, of respiratory epithelial cells in the nasal cavity, resulting in cell proliferation and squa- and these changes are associated with tumor progression [6]. In addition, FA has displayed mous metaplasia, and these changes are associated with tumor progression [6]. In addi- mutagenic and genotoxic activities by crosslinking DNA and protein, finally resulting in tion, FA has displayed mutagenic and genotoxic activities by crosslinking DNA and nasal cavity tumorigenicity [6]. However, the pathway of nasal cavity tumor development protein, finally resulting in nasal cavity tumorigenicity [6]. However, the pathway of is currently not very clear. FA had been considered to be associated with risk of leukemia nasal cavity tumor development is currently not very clear. FA had been considered to be in some epidemiology studies [8]; however, recent studies demonstrated that not enough associated with risk of leukemia in some epidemiology studies [8]; however, recent evidence could support the relationship between FA exposure and cancer [11]. Therefore, studies demonstrated that not enough evidence could support the relationship between the mechanism of FA-induced carcinogenicity remains to be explored further. Separations 2022, 9, x FOR PEER REVIEW 10 of 12 FA exposure and cancer [11]. Therefore, the mechanism of FA-induced carcinogenicity remains to be explored further. In this work, we provide insight into the alterations to proteomics and the modifi- cations induced by FA. From a total of 2021 proteins identified, we found effects of FA on protein levels, in particular revealing that down-regulated proteins were enriched in DNA and chromatin, while up-regulated proteins were mostly localized in metabolic pathways. The identification of protein modifications induced by FA provides evidence that supports the interactions between proteins and FA, suggesting a FA-interaction network. This information may be a vital clue to revealing the mechanism of FA-induced Separations 2022, 9, 112 9 of 11 biological effects in future works. Figure 6. Functional annotation of FA-induced modification to proteins. (A) Interaction networks Figure 6. Functional annotation of FA-induced modification to proteins. (A) Interaction networks of FA-induced modifications to proteins in HeLa. Blue indicates 54 ribosome-associated proteins; of FA-induced modifications to proteins in HeLa. Blue indicates 54 ribosome-associated proteins; purple indicates 34 proteins associated with nucleoprotein; yellow indicates 31 proteins associated purple indicates 34 proteins associated with nucleoprotein; yellow indicates 31 proteins associated with metabolism. (B) Analysis via KEGG of protein modifications induced by FA in HeLa. with metabolism. (B) Analysis via KEGG of protein modifications induced by FA in HeLa. 4. Conclusions In this work, we provide insight into the alterations to proteomics and the modifica- tions Ininduced this stud by y, w FA. e c Fr oom mbia ne total d LC of -M 2021 S/MS pr a oteins nd bioin identified, formatics tools we found for the analysis of effects of FA on proteomes and protein modifications induced by FA. We identified 152 up-regulated protein levels, in particular revealing that down-regulated proteins were enriched in DNA proteins and chromatin, that were while main up-r ly enriched egulated in pr meta oteins bolism were mostly and 196 down localized -reg inulated pr metabolic oteins th pathways. at we The re f identification ocused on nu of clpr eaotein r-relamodifications ted processes.induced We ident by ified the FA provides 119 modified pro evidence that teins that supports add theu interactions cted a massbetween shift of 1 pr 2 Da oteins at K, W and F a A, nd C suggesting , and cha arF act A-interaction erized the m network. otifs of tThis he minfor odi- - mation may be a vital clue to revealing the mechanism of FA-induced biological effects in fications. Herein, we developed an approach towards analysis of the effect of FA at the prot futur ein l e works. evel, providing a new view for understanding the biological significance of FA addition. 4. Conclusions Supplementary Materials: The following supporting information can be downloaded at: In this study, we combined LC-MS/MS and bioinformatics tools for the analysis of www.mdpi.com/xxx/s1, Figure S1: Formaldehyde reacts with proteins to produce +30 Da or +12 Da proteomes and protein modifications induced by FA. We identified 152 up-regulated pro- mass shift on lysine, cysteine and tryptophan residues; Table S1: Information of identified proteins teins that were mainly enriched in metabolism and 196 down-regulated proteins that were of FA-treated 15 min and untreated; Table S2: Information of upregulated (fold change > 2 increase, focused on nuclear-related processes. We identified the 119 modified proteins that adducted p < 0.05) proteins induced by FA; Table S3: Information of downregulated (fold change > 2 decrease, a mass shift of 12 Da at K, W and C, and characterized the motifs of the modifications. p < 0.05) proteins induced by FA; Table S4: Information of total proteins identified with Herein, we developed an approach towards analysis of the effect of FA at the protein level, FA-modified sites. providing a new view for understanding the biological significance of FA addition. Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations9050112/s1, Figure S1: Formaldehyde reacts with proteins to produce +30 Da or +12 Da mass shift on lysine, cysteine and tryptophan residues; Table S1: Information of identified proteins of FA-treated 15 min and untreated; Table S2: Information of upregulated (fold change >2 increase, p < 0.05) proteins induced by FA; Table S3: Information of downregulated (fold change >2 decrease, p < 0.05) proteins induced by FA; Table S4: Information of total proteins identified with FA-modified sites. Author Contributions: S.T. and K.Z. conceived and designed the experiments. R.L. and Y.H. per- formed the experiments. R.L., Y.H., Z.W., J.Z., Y.Z., L.S., S.T. and K.Z. analyzed the data. R.L., S.T. and K.Z. wrote and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript. Separations 2022, 9, 112 10 of 11 Funding: This research was supported by the Funding of National Natural Science Foundation of China to K.Z. (21874100 and 22074103), Tianjin Municipal Science and Technology Commission to K.Z. (19JCZDJC35000) and to S.T. (19JCQNJC08900), and the Talent Excellence Program from Tianjin Medical University to K.Z. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The mass spectrometry proteomics data were deposited to the Pro- teomeXchange Consortium (http://proteomecentral.proteomexchange.org, accessed on 19 April 2022) via the iProX partner repository, with data set identifier PXD033286. Conflicts of Interest: The authors declare no conflict of interest. References 1. Bernardini, L.; Barbosa, E.; Charao, M.F.; Brucker, N. Formaldehyde toxicity reports fromin vitro and in vivo studies: A review and updated data. Drug Chem. Toxicol. 2020, 45, 972–984. [CrossRef] [PubMed] 2. 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