Label-free proteomic comparative analysis of cow placental proteins enzymatic hydrolysis by different proteases

New found in biochemical characteristics of placenta can bring new insight for further studies on the possible markers of physiological/pathological pregnancy or function of placenta. We compared the proteome of dairy cow placenta enzymatic hydrolysis by different proteases by label-free mass spectrometry approach. In total 541, 136 and 86 proteins were identi�ed in trypsin group (TRY), pepsin group (PEP) and papain group (PAP). By comparing the proteome of PAP and TRY, 432 differentially expressed proteins (DEPs) were identi�ed. PEP vs TRY identi�ed 421 DEPs, while 136 DEPs were identi�ed in PEP and PAP. The results showed the proteins identi�ed by papain are mostly derived from extracellular matrix and collagen and enriched in relaxin signaling pathway, AGE-RAGE signaling pathway in diabetic complications; pepsin digestion can identify more muscle-related proteins, which are enriched in lysosome, platelet activation, cardiac muscle contraction, bacterial invasion of epithelial cell and small cell lung cancer; trypsin mainly enzymatically degrades extracellular matrix, blood particles, and cell surface proteins which enriched in arginine and proline metabolism, olfactory transduction proteasome, protein processing in endoplasmic reticulum, pyruvate metabolism and arrhythmogenic right ventricular cardiomyopathy (ARVC). In summary, these results provide insights into the selection of protease in dairy cow placenta proteomics.


Introduction
The placenta is an organ that connects the mother and the fetus to maintain a stable environment for the growth and development of the fetus.It regulates the growth and development of the fetus by regulating the supply of nutrients, gases, hormones, etc and has substance exchange, hormone secretion and barrier effects 1 .The placenta changes the mother's endocrine system and immune system, establishing a blood vessel link between the mother and the embryo, which is the key to maintaining the growth of the fetus 2 .
Abnormal placental function is one of the principal causes of fetal death.Proteomic technology is a common tool for studying placental abnormalities.It can study the changes of placental protein during the disease process, clarify the pathogenesis and search for differentially expressed proteins related to the pathogenesis, providing an effective means for clinical diagnosis of the disease.Placental proteomics can diagnose mare peptides and nd related biomarkers 3 ; through placental proteomics, it has been identi ed that differential proteins in the placenta of human preeclampsia are closely related to mitochondrial function, indicating that mitochondrial dysfunction is a precursor One of the pathogenesis of epilepsy 4 ; through placental proteomics, it is found that the occurrence of human recurrent miscarriage is closely related to the core factors of early embryonic development such as angiotensinogen (AGT), MAPK14 and prothrombin (F2) 5 .The digestion and extraction of protein by protease is an important factor affecting the results of proteomics.At present, trypsin is often used in placental proteomics for protein extraction.Trypsin has the advantage of high speci city, only cutting arginine (R) and lysine (K) residues 6 , but some proteins lacking arginine and lysine residues cannot be digested by trypsin.In addition, when the trypsin cleavage site is located after the glycosylated asparagine, the attached carbohydrates may sterically prevent trypsin cleavage 7 .In order to overcome the limitations of trypsin, use non-speci c enzymes, such as papain and pronase to digest the protein, which can completely digest the protein.Trypsin is most effective in a neutral or slightly alkaline environment, but some proteins have low solubility in this environment and cannot be digested.Pepsin has the strongest activity in an acidic environment and can enzymatically hydrolyze proteins with greater solubility in acidic environment.In this experiment, based on the Box-Behenken central response method, the optimal enzymatic hydrolysis of cow placental peptides trypsin, pepsin and papain with high reducing activity and extraction rate has been established, and the label-free technology is used to hydrolyze the above protease.Qualitative and quantitative analysis of the protein and peptides obtained from cow placenta is to compare the biological information of cow placenta hydrolyzed by different proteases, which provides theoretical support for the study of protease selection in cow placenta proteomics.

Ethics statement
Sample collection was performed in strict accordance with the guidelines of the Care and Use of Laboratory Animals of China, and all procedures were approved by the Animal Care and Use Committee of Sichuan Agricultural University.

ARRIVE statement
This study was carried out in compliance with the ARRIVE guidelines.

Placenta collection and preparation
Placental samples were collected from 9 healthy pregnant Chinese Holstein cows in a large-scale semiclosed uni ed farm in Sichuan province.The cows were selected according to the following criteria, aged between 3-5 years, with body weight above 600 kg, 2-4 parity and around 40 weeks of pregnancy.The placenta was collected after nature delivery.Only similar parts of the placentome were used for further analysis.Wash placenta in cold saline, then portion, freeze and store it at -20°C.

Liquid chromatography (LC)-electrospray ionization (ESI) tandem MS (MS/MS) analysis
Each fraction was injected for nano LC-MS/MS analysis.The peptide mixture was loaded onto a reverse phase trap column (Thermo Scienti c Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) connected to the C18-reverse phase analytical column (Thermo Scienti c Easy Column, 10cm, ID75μm, 3μm, C18-A2) in buffer A (0.1% formic acid) and separated with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a ow rate of 300nL/min controlled by IntelliFlow technology.A two-hour gradient procedure was used as follows: 0 -55% buffer B for 110 min, 55%-100% buffer B for 5 min, 100% buffer B for 5 min.Then, use Q Exactive mass spectrometer (Thermo Scienti c) for LC-MS/MS analysis.The mass spectrometer was operated in positive ion mode.The scanning range of precursor ion is 300-1800m/z.Automatic gain control (AGC) target was set to 1e6, and maximum inject time to 50ms.Dynamic exclusion duration was 60 s.Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200, and isolation window was 2 m/z.Normalized Collision Energy was 30eV, Under ll was 0.1%.

Bioinformatics analysis
Functional annotation and Network analysis were performed using STRING (http://string-db.org/) and Cytoscape platform version 3.8.2(https://cytoscape.org)based on bos-taurus genes.In particular, the two plugins of Cytoscape, namely ClueGo (version 2.5.7) and CluPedia (version 1.5.7)were used to integrate the Gene Ontology (GO) categories (Biological Process(BP), Molecular Function (MF), Cellular Component (CC)), Reactome Pathways, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Wiki Pathways annotation 9 .The κ score level was set at ≥0.4 while minimum and maximum levels were set at 3 and 8, respectively.

Results
Comparative evaluation of protein extraction e ciency in cow placenta hydrolyzed by three proteases The number of proteins identi ed from cow placenta with different digestion protocols differ signi cantly, 426, 115 and 57 in TRY, PEP and PAP, respectively (Table 1).Comparison of digestion e ciencies of three proteases showed that TRY produced signi cantly higher number of protein identi cations than PEP and PAP.TRY extracted 3.7 times (t-test, p = 0.00132) and 7.5 times (t-test, p = 0.00015) more of proteins than PEP and PAP.Meanwhile PEP extracted 2 times more of proteins than PAP (t-test, p = 0.00678).
Comparison of the common proteins among biological replicates of TRY and PEP revealed an overlap of 72-82%, while that of PAP ranged 53-77% (Fig. 1).TRY and PEP had higher reproducibility of acquisitions than PAP, with coe cient of variation (CV) from 2 to 4% for proteins and 8-10% for peptides, respectively, lower than 15% of proteins and 20% of peptides in PAP (Table 1).TRY, PEP and PAP extraction resulted in 541, 134 and 86 quanti able proteins (proteins with LFQ intensity > 0) from cow placenta, respectively.Comparison of the quanti able proteins of cow placenta among three proteases, common proteins were 80 (12.3%), 22 (3.4%) and 27 (4.1%) in TRY vs PEP, PEP vs PAP and PAP vs TRY, respectively.There were 449 (69.2%), 49 (7.6%) and 52 (8%) unique proteins in TRY, PEP and PAP, respectively (Fig. S1).Common proteins only constitute the minority of total quanti able proteins, while unique proteins of TRY are the largest proportion.Note: The table lists numbers of identi ed peptides and proteins in each biological replicate, together with the average number of identi cation (Mean ± SD) and coe cient of variation (CV%).

Analysis of cow placenta quanti able proteins whit three proteases
The quanti able proteins of cow placenta whit trypsin, pepsin and papain were further analyzed in terms of distribution of proteins' molecular weight (Mw), sequence coverage, peptides length and unique peptides ratio.Mw distribution is important in evaluating proteins size.In this study, Mw of quanti able proteins extracted with three proteases was relative wide, and showed almost no difference.Mw results demonstrated that 71%, 76% and 67% proteins in TRY, PEP and PAP, respectively, were lower than 70kDa.
Meanwhile, approximately 20% of the quanti able proteins exceeded 100kDa (Fig. 2A).Sequence coverage determined the overall accuracy of detected proteins, while that was relatively high in the present study.There were 46%, 63% and 42% proteins with more than 10% sequence coverage distributions of quanti able proteins (Fig. 2B).Peptides length revealed the characteristic of proteases.In this study, 92%,76% and 88% detected peptides were lower than 20 in TRY, PEP and PAP, respectively (Fig. 2C).Additionally, each group consists its unique set of peptides endowing it with speci c properties.
Protein detection reliability tended to improve with the number of unique peptides in a protein group 10 .In the present study, the distribution curve of the number of unique peptides gradually increased, which means the number of both unique peptides and reliable proteins was relatively large (Fig. 2D).
GO and KEGG analysis of cow placenta quanti able proteins whit three proteases GO analysis of cow placenta quanti able proteins with three proteases showed highly similar distribution in biological progress, cellular component and molecular function (Fig. S2).The highest percentage are metabolic progress and biological regulation, followed by cellular component organization, response to stimulus and developmental process in biological progress.The quanti able proteins mainly distributed in membrane, nucleus, protein-containing complex, cytosol and cytoskeleton in cellular component.Molecular function-based analysis showed that a majority of the proteins were involved in processes such as protein binding, ion binding, hydrolase activity, nucleotide binding, structural molecule activity and nucleic acid binding.
KEGG pathway analysis was carried out to understand the biological functions and the speci c pathways related to cow placenta.Top 10 KEGG pathways in TRY, PEP and PAP are presented in Fig. S3.Focal adhesion, PI3K-Akt signaling pathway, Human papillomavirus infection and ECM-receptor interaction are common pathway in RY, PEP and PAP.

Identi cation of differentially expressed proteins (DEPs)
The DEPs were de ned based on a 2.0-fold change threshold (with a fold change > 2.0 or < 0.50, p < 0.05) (Fig. 3) or speci cally expressed (Table 2) in comparisons between groups according to mass spectrum data.PAP vs TRY detected 432 DEPs, including 34 up-regulated and 398 down-regulated (Fig. 3A, Table 2); PEP vs TRY detected 421 DEPs, including 56 up-regulated and 365 down-regulated (Fig. 3B, Table 2).PEP vs PAP detected 136 DEPs, including 100 up-regulated and down-regulated (Fig. 3C, Table 2).Note:" Presence " refers to proteins which consistent presence in the rst group and absence in the other group; " Absence " refers to proteins which consistent presence in the second group and absence of the other group.

Cluster analysis of DEPs
The hierarchical clustering algorithm (Hierarchical Cluster) is used to perform cluster analysis on each group of DEPs, and the data is displayed as a heat map (Heatmap).Figure .S4 shows that the DEPs screened by the standard of 2.0-fold change threshold (with a fold change > 2.0 or < 0.50, p < 0.05) can effectively separate the comparison groups, showing that they screened differentially expressed proteins can represent the difference between the two groups.

GO and KEGG enrichment analysis of the DEPs
Perform GO enrichment analysis on the up-regulated proteins and down-regulated proteins of each comparison group to compare the difference in bioinformatics of dairy cow placenta with different proteases.GO enrichment results show differences in biological information caused by proteases.The statistically signi cant (p < 0.05) network analysis performed using Cytoscape is shown in Fig. 4. DEPs enzymatic hydrolysis by papain enriched in collagen trimer, extracellular region part, extracellular exosome, chroma n granule membrane and protein heterodimerization (Fig. 4A-B).DEPs enzymatic hydrolysis by trypsin enriched in blood microparticle, membrane region, complex of collagen trimer, extracellular organelle, extracellular matrix, extracellular matrix component, cell surface, viral nucleocapsid, regulation of locomotion, maintenance of location, syncytium formation and anatomical structure formation involved in morphogenesis (Fig. 4C-D).DEPs enzymatic hydrolysis by pepsin enriched in I band, immunological synapse, sarcomere, costamere, contractile ber, structural molecule activity conferring elasticity, extracellular matrix binding, coagulation, structural constituent of muscle, NAD metabolic process, extracellular matrix structural constitution, collagen metabolic process, platelet activation, regulation plasma lipoprotein particle levels and cell death response oxidative stress (Fig. 4E-F).
In order to compare the difference in bioinformatics of dairy cow placenta with different proteases, KEGG enrichment analysis was performed on the up-regulated protein and down-regulated protein of each comparison group.

Discussions
Proteomics has been widely applied to study the placenta related disease of human, but there are few studies related to placenta in dairy cow.Trypsin and pepsin are widely used in protein extraction and digestion in proteomics research [11][12][13][14] , while papain is mostly used in the extraction of natural biologically active peptides 15,16 .
In this study we carried out a comparative proteomic analysis of dairy cow placenta enzymatic hydrolysis by three proteases using a label-free MS approach.The main aim of this study was to give an objective, critical assessment on the performance of three proteases for digestion in proteomics of cow placenta.This study was based on the optimal enzymatic hydrolysis conditions of cow placenta with trypsin, pepsin and papain 8 .
Comparison of the overall digestion e ciency between three proteases showed that trypsin was superior to pepsin and papain in number of protein identi cations for cow placenta.We presume that trypsin superior e ciency is due to improved protein solubility and proteolytic e ciency of trypsin.Trypsin has the advantage of high speci city which cleaves exclusively at arginine (R) and lysine (K) residue 17 , optimal pH closes to neutral 18 , meanwhile the pH of amniotic uid closes to 7.0.Therefore, we assumed that placental proteins are soluble in a neutral environment, which may be the primary reason of why trypsin enzyme solution e ciency is better than pepsin and papain.Jiao's study reported that a total of 788 proteins were identi ed in placenta of healthy pregnant mice by label free proteomic with trypsin digestion 19 .Wawrzykowski reported 886 proteins were identi ed in cow placenta by 2D separation with trypsin digestion 20 .Compared with the above study, the amount of protein extracted by trypsin in this study is relatively small, which may be due to the direct enzymatic hydrolysis of placenta homogenate by trypsin, lack of detergents and denaturants such as RapiGest.In this study, there is room for improvement in protein digestion.However, the biological repetition rate of trypsin was more than 70%, the molecular weight and sequence coverage were relatively wide, the peptides length conformed to the characteristics of trypsin digestion, and the unique peptides distribution curve increases gradually, all the above results notarized the reliability of proteome data.
Pepsin also has strong speci city, and the hydrolytic sites are aromatic and hydrophobic amino acids.A total of 110 proteins were identi ed by pepsin hydrolyzed mealworm in Bouklis' research 14 , which was closed to the result of this study, and the quality control data of pepsin was better than that of trypsin and papain, indicating that pepsin is reliable in proteomics.However, the number of identi ed proteins by pepsin is signi cantly less than that of trypsin, which may be caused by pH.Pepsin optimal pH rang 1.5 to 2.0 21 , however some placental proteins have poor solubility in this acidic environment, which caused the low enzymatic hydrolysis e ciency of pepsin.
Papain were proteases that can release relatively larger quantity of bioactive peptides 22 .Therefore, papain was mainly used for the extraction and digestion of natural active peptides in the current research.Papain hydrolyzed oat proteins had high ability to quench ABTS%+ radicals and to chelate ferrous ions while displaying the second strongest activity for ROO% radicals 23 .Antioxidant activity of porcine liver hydrolysates using papain were relatively high 24 .Papain can release high number of potential angiotensin converting enzyme (ACE)-inhibitory peptides from tilapia (Oreochromis spp.) processing co-products, frame and skin 15 .In the above research, proteomic techniques were used analyze molecular characteristics of proteins.However, peptides generated with less than 5 amino acids (due to their high MWs and electrical charge) were not effectively detected by the MS/MS spectra 25 .Thus, we speculate that most of the polypeptides hydrolysis by papain with less than 5 amino acids, which leads to a great number of potential bioactive peptides and a small number of protein identi cation, and the quality control data con rm this view, with a coe cient of variation of more than 15%, low protein repetition rate and molecular weight, narrow sequence coverage and peptide length distribution and few unique peptides.
Our results indicated that quanti able proteins of cow placenta with trypsin, pepsin and papain were associated with almost same biological progress, molecular function and cellular component.The biological progress mainly included biological regulation and metabolic process, similar to the protein patterns in bovine placenta at early-mid pregnancy 26 .The cellular component included protein-containing complex, membrane, cytosol nucleus and extracellular space.The main cellular component of identi ed proteins in retained and released bovine placenta were cytoplasm, nucleus and membrane 20 .This result showed that the proteins that cause placental retention may be mainly distributed in cytoplasm.
Classi cation of the quanti able proteins, in accordance to molecular functions, revealed that the majority of proteins showed binding and catalytic activities, similar to the study of Ner-Kluza 26 .In conclusion, the GO annotation of this study was almost similar to the current research.Further, the KEGG analysis indicated focal adhesion, PI3K-Akt signaling pathway, human papillomavirus infection and ECMreceptor interaction were common pathway in cow placenta with three proteases.Proteins important for adhesive processes were detected in retain bovine placenta and disturbances in the metabolism of extracellular matrix proteins what may lead to improper placental detachmen.Our study con rmed focal adhesion and ECM-receptor interaction pathway were important in normal released placenta.
Different enzyme digestion sites leaded to different protein identi cation further induced the biological information differences.In order to explore these differences, up-regulated and down-regulated DEPs were analyzed in each comparison group.Predictively, the results were completely different.
Trypsin had been evidenced in previous studies can be used in proteomics such as pork, beef, chicken, sh, milk, and shrimp 18,20,26−28 .Trypsin mainly enzymatically decomposes extracellular matrix, blood particles, and cell surface proteins.These proteins mainly perform positioning functions and participate in biological processes such as syncytium formation.Trypsin can be selected for the study of amino acid metabolism, proteasome and endoplasmic reticulum, dysosmia and arrhythmogenic right ventricular cardiomyopathy.
Pepsin mainly enzymatically degrades myo brils, muscle ber I-bands, actin ribs and muscle contraction bers, has the function of activating elastic bers and linking extracellular matrix, and is involved in blood coagulation, muscle structure composition, NAD metabolism, and extracellular matrix composition.Biological processes such as collagen metabolism and cell apoptosis caused by oxidative stress.The above results indicate that pepsin can signi cant hydrolysis effect on muscle tissue in the placenta, and more biological information related to muscle tissue can be identi ed.When studying muscle tissuerelated proteomics, pepsin digestion may be used to obtain more comprehensive biological information.Pepsin can be selected to study diseases such as lysosome, platelet aggregation and myocardial contraction, bacterial invasiveness and small cell lung cancer.
Papain has a strong ability to decompose extracellular matrix and collagen and can be used in Relaxin signaling pathway and AGE-RAGE signaling pathway in diabetic complications research according to GO and KEGG results

Conclusion
This study identi ed the differential proteins produced by the enzymatic hydrolysis of cow placenta by different proteases.The production of these differential proteins is related to the characteristics of protease cleavage.In particular, proteins related to protein binding and ion binding protein-containing complex and membrane metabolic process and biological regulation were detected in various enzymatic hydrolysis products.All in all, these ndings explain the basic biological information of healthy dairy cow placenta and are helpful for the screening of speci cally expressed proteins and biomarkers in dairy cow pregnancy diseases.At the same time, it can provide guidance on the selection of protease for speci c tissues and speci c directions for proteomics.
Protein digestion and absorption Glycolysis / Gluconeogenesis Hypertrophic cardiomyopathy (HCM) Dilated cardiomyopathy (DCM) Focal adhesion Adherens junction Tight junction Leukocyte transendothelial migration Regulation of actin cytoskeleton PI3K-Akt signaling pathway Focal adhesion ECM-receptor interaction Regulation of actin cytoskeleton Amoebiasis are common enrichment pathway in each group (Fig. 5).The unique enrichment pathways of DEPs produced by papain are Relaxin signaling pathway and AGE-RAGE signaling pathway in diabetic complications (Figure.5A-B).The unique enrichment pathways of DEPs produced by pepsin are Lysosome, Platelet activation, Cardiac muscle contraction, Bacterial invasion of epithelial cells, and Small cell lung cancer (Figure.5C-D).The unique enrichment pathway of DEPs produced by trypsin is Arginine and proline metabolism, Olfactory transduction, Proteasome, Protein processing in endoplasmic reticulum, Pyruvate metabolism, Arrhythmogenic right ventricular cardiomyopathy (ARVC) (Figure.5E-F).

Figure 1 Protein
Figure 1

Figure 4 GO
Figure 4

Table 1
Protein and peptide identi cations from cow placenta by trypsin, pepsin and papain.