Comparative evaluation of protein extraction efficiency in cow placenta hydrolyzed by three proteases
The number of proteins identified from cow placenta with different digestion protocols differ significantly, 426, 115 and 57 in TRY, PEP and PAP, respectively (Table 1). Comparison of digestion efficiencies of three proteases showed that TRY produced significantly higher number of protein identifications 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 coefficient 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 quantifiable proteins (proteins with LFQ intensity > 0) from cow placenta, respectively. Comparison of the quantifiable 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 quantifiable proteins, while unique proteins of TRY are the largest proportion.
Table 1
Protein and peptide identifications from cow placenta by trypsin, pepsin and papain.
Sample name
|
Proteins
|
Peptides
|
BR1
|
BR2
|
BR3
|
Mean
|
SD
|
CV (%)
|
BR1
|
BR2
|
BR3
|
Mean
|
SD
|
CV (%)
|
TRY
|
401
|
443
|
430
|
425
|
18
|
4
|
2022
|
2489
|
2002
|
2171
|
225
|
10
|
PEP
|
117
|
111
|
113
|
114
|
2
|
2
|
993
|
939
|
816
|
916
|
74
|
8
|
PAP
|
64
|
59
|
44
|
56
|
8
|
15
|
99
|
98
|
62
|
86
|
17
|
20
|
Note: The table lists numbers of identified peptides and proteins in each biological replicate, together with the average number of identification (Mean ± SD) and coefficient of variation (CV%). |
Analysis of cow placenta quantifiable proteins whit three proteases
The quantifiable 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 quantifiable 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 quantifiable 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 quantifiable 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 specific properties. Protein detection reliability tended to improve with the number of unique peptides in a protein group10. 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 quantifiable proteins whit three proteases
GO analysis of cow placenta quantifiable 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 quantifiable 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 specific 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.
Identification of differentially expressed proteins (DEPs)
The DEPs were defined based on a 2.0-fold change threshold (with a fold change > 2.0 or < 0.50, p < 0.05) (Fig. 3) or specifically 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).
Table 2
Specially expressed protein in each group。
Comparisons
|
Consistent presence /absence expression profile
|
Presence
|
Absence
|
Papain group vs Trypsin group
|
31
|
391
|
Trypsin group vs Pepsin group
|
47
|
340
|
Pepsin group vs Papain group
|
96
|
34
|
Note:" Presence " refers to proteins which consistent presence in the first 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 significant (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, chromaffin 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 fiber, 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. 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).