The Same Proteomics Analysis of Jersey Yak Crossbreeds and Yak Milk

doi:10.7668/hbnxb.20193976

Abstract

Abstract:

To analyze the same protein of whey and milk fat globule membrane (MFGM)of jersey yak crossbreeds milk and yak milk by proteomics method. The milk yield of jersey yak crossbreeds was higher than that of yak. If the function of jersey yak crossbreeds milk is like that of yak milk,it will improve the economic level of local herdsmen. Whey and milk fat were separated by centrifugation,and proteins were extracted respectively. The same protein was obtained by qualitative and quantitative analysis of protein by tandem mass tag (TMT)technique. GO functional annotation,KEGG metabolic pathway,protein interaction and other bioinformatics analysis were performed on the same protein. Result analysis indicated that 651 whey proteins and 990 MFGM proteins were identified in the peak lactation period of jersey yak crossbreeds milk and yak milk by label quantitative proteomics,and 298 whey proteins and 283 MFGM proteins were screened. GO annotation analysis showed that the same proteins of jersey yak crossbreeds milk and yak milk whey were mainly involved in the negative regulation of endopeptidase activity,immune response and immunoglobulin production. The same proteins of MFGM were mainly involved in the negative regulation of endopeptidase activity and complement activation. The cell components of the same proteins of whey and MFGM in the classical pathway were mainly extracellular space and extracellular region. The molecular functions involved were mainly guanosine 5'-triphosphate (GTP)enzyme activity and GTP binding. KEGG enrichment analysis showed that the main enrichment pathways of the same proteins in jersey yak crossbreeds milk and yak milk whey and MFGM included complement and coagulation cascades,phage and protein processing in endoplasmic reticulum. During the peak lactation period,the proteins with higher abundance in the milk of jersey yak crossbreeds and yak showed excellent performance in immunity and promoting nutrient absorption. However,the milk yield of jersey yak crossbreeds was higher than that of yak,which would increase the economic income of local herdsmen and provide more valuable plateau dairy products for people.

Key words: Jersey yak crossbreeds milk, Yak milk, Whey protein, Milk fat globule membrane protein, Proteomics

GAO Na, XU Gengquan, WANG Meng, WANG Libin, QIU Shantong, YU Zhen, YU Sijiu, PAN Yangyang. The Same Proteomics Analysis of Jersey Yak Crossbreeds and Yak Milk[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(S1): 381-392. doi: 10.7668/hbnxb.20193976.

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URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20193976

http://www.hbnxb.net/EN/Y2023/V38/IS1/381

Figures/Tables 20

Tab.1 Information of experimental animal(

x ¯

±s)

物种 Species	采样日期 Sampling date	分组 Group number	样本数 Sample number	泌乳天数 Lactation days
娟犏牛奶	2021-08-14—2021-08-20	Ⅰ	10	180.80±0.11
Jersey yak crossbreeds milk		Ⅱ	10	184.40±0.18
		Ⅲ	10	180.30±0.24
牦牛奶	2021-08-23—2021-08-29	Ⅰ	10	182.80±0.15
Yak milk		Ⅱ	10	180.10±0.24
		Ⅲ	10	180.60±0.20

Tab.1 Information of experimental animal(

x ¯

±s)

物种 Species	采样日期 Sampling date	分组 Group number	样本数 Sample number	泌乳天数 Lactation days
娟犏牛奶	2021-08-14—2021-08-20	Ⅰ	10	180.80±0.11
Jersey yak crossbreeds milk		Ⅱ	10	184.40±0.18
		Ⅲ	10	180.30±0.24
牦牛奶	2021-08-23—2021-08-29	Ⅰ	10	182.80±0.15
Yak milk		Ⅱ	10	180.10±0.24
		Ⅲ	10	180.60±0.20

Tab.2 Database retrieval parameters

项目Items	设置Settings
固定修饰Static modification	TMT (N-term, K);Carbamidomethyl(C)
可变修饰Dynamic modification	Oxidation(M),Acetyl(N-term)
酶切模式Digestion	Trypsin
仪器Instrument	Q Exactive HF
一级质量冗余MS1 tolerance	10 ppm
二级质量冗余MS2 tolerance	0.02 Da
漏切位点Missed Cleavages	2
数据库Databa	uniprot-taxonomy_9913.fasta

Tab.3 Standard absorbance and concentration

编号Number	1	2	3	4	5	6	7	8
平均吸光度Average absorbance	0.0	0.032	0.072	0.141	0.275	0.368	0.480	0.618
浓度/(μg/μL)Concentration	0.0	0.025	0.050	0.100	0.200	0.300	0.400	0.500

Fig.1 Quantitative standard curve of jersey yak crossbreeds milk and yak milk whey protein and MFGM protein

Tab.4 Absorbance and concentration of samples to be measured

编号 Number	吸光度1 Absorbance 1	吸光度2 Absorbance 2	吸光度3 Absorbance 3	平均吸光度 Average absorbance	测定浓度/(μg/μL) Measured concentration	真实浓度/(μg/μL) True concentration
MQ1	0.521	0.435	0.503	0.486	0.393	15.73
MQ2	0.485	0.498	0.580	0.521	0.422z	16.87
MQ3	0.396	0.438	0.432	0.422	0.340	17.01
MZ1	0.108	0.122	0.136	0.122	0.093	0.93
MZ2	0.135	0.168	0.162	0.155	0.120	1.20
MZ3	0.152	0.142	0.156	0.150	0.116	1.16
JQ1	0.427	0.440	0.417	0.428	0.345	13.81
JQ2	0.345	0.379	0.389	0.371	0.298	14.91
JQ3	0.411	0.397	0.408	0.405	0.326	16.32
JZ1	0.153	0.148	0.167	0.156	0.121	1.21
JZ2	0.125	0.158	0.165	0.149	0.115	1.15
JZ3	0.147	0.175	0.176	0.166	0.129	1.29

Fig.2 SDS-PAGE electrophoresis of jersey yak crossbreeds milk and yak milk whey protein and MFGM protein M.Yak milk;J.Jersey yak crossbreeds milk;Q.Whey protein;Z. MFGM protein. The same as Fig.3,4,11,12.

Fig.3 Wern diagram of whey protein and MFGM protein of jersey yak crossbreeds milk and yak milk

Fig.4 Box diagram of whey protein and MFGM protein abundance in jersey yak crossbreeds milk and yak milk samples

Tab.5 Identification information of the same protein in main whey of jersey yak crossbreeds milk and yak milk

登录号 Accession	蛋白质名称 Protein name	基因名称 Gene names	M分子质量/ku M molecular mass	J分子质量/ku J molecular mass	P值 P value	差异倍数 Fold change
Q2KJ32	甲硫醇氧化酶	MTO	181.60	157.90	0.048 9	1.15
Q5GN72	α-1-酸性糖蛋白	AGP	115.70	138.57	0.009 9	0.83
P02663	α-S2-酪蛋白	CSN1S2	130.27	122.67	0.001 1	1.06
P07688	组织蛋白酶B	CTSB	109.87	119.30	0.170 0	0.92
A0A140T897	白蛋白	ALB	117.83	109.17	0.048 9	1.08
O46375	转甲状腺素	TTR	110.47	116.13	0.361 0	1.01
Q29443	血清铁传递蛋白	TF	103.70	105.30	0.341 0	0.98
Q28078	上皮粘蛋白	MUC1	73.50	66.57	0.555 0	1.10
P30932	CD9抗原	CD9	75.42	69.25	0.762 0	1.09
Q3ZCD0	CD81抗原	CD81	105.63	112.07	0.005 9	0.94
P26201	血小板糖蛋白4	CD36	102.70	88.40	0.070 1	1.16
Q3T0E0	铜伴侣蛋白	ATOX1	93.77	99.33	0.268 0	0.94

Tab.6 Identification information of the same protein in main MFGM of jersey yak crossbreeds milk and yak milk

登录号 Accession	蛋白质名称 Protein name	基因名称 Gene names	M分子质量/ku M molecular mass	J分子质量/ku J molecular mass	P值 P value	差异倍数 Fold change
Q32PA1	CD59糖蛋白	CD59	182.93	126.43	0.050 1	1.45
F1MR22	高分子免疫球蛋白受体	PIGR	176.63	131.70	0.580 0	1.34
A0A0P0HAN3	β-乳球蛋白	LGB	106.00	116.17	0.088 1	0.91
P07688	组织蛋白酶B	CTSB	118.93	113.23	0.534 0	1.05
A0A140T897	白蛋白	ALB	118.93	121.83	0.810 0	0.98
O46375	转甲状腺素	TTR	108.37	90.07	0.095 4	1.20
Q28078	上皮粘蛋白	MUC1	92.93	87.84	0.064 6	1.06
P30932	CD9抗原	CD9	94.40	108.07	0.057 3	0.87
Q3ZCD0	CD81抗原	CD81	73.03	93.21	0.092 2	0.78
P26201	血小板糖蛋白4	CD36	85.30	89.40	0.053 0	0.95

Fig.5 Go annotation of the same protein whey between jersey yak crossbreeds milk and yak milk 1.Negative regulation of endopeptidase activity;2.Immune response;3.Immunoglobulin production;4.Acute phase response;5.Complement activation,classical pathway;6.Complement activation,alternative pathway;7.Proteolysis involved in cellular protein catabolic process;8.Receptor internalization;9.Cellular response to low density lipoprotein particle;10.Regulation of macrophage migration;11.Extracellular space;12.Extracellular region;13.Cell surface;14.Endoplasmic reticulum lumen;15.External side of plasma membrane;16.Lysosome;17.Apical plasma membrane;18.Melanosome;19.Extracellular exosome;20.Golgi lumen;21.GTPase activity;22.GTP binding;23.Serine-type endopeptidase inhibitor activity;24.GDP binding;25.Cysteine-type endopeptidase inhibitor activity;26.Endopeptidase inhibitor activity;27.Misfolded protein binding;28.Disordered domain specific binding;29.Structural constituent of skin epidermis;30.Myosin V binding.

Fig.6 Go annotation of the same protein MFGM between jersey yak crossbreeds milk and yak milk 1.Negative regulation of endopeptidase activity;2.Proteolysis involved in cellular protein catabolic process;3.Single fertilization;4.Glycolytic process;5.Complement activation,classical pathway;6.Complement activation,alternative pathway;7.Cellular response to thyroid hormone stimulus;8.Cellular response to low-density lipoprotein particle;9.Response to progesterone;10.Regulation of macrophage migration;11.Extracellular space;12.Extracellular region;13.Golgi apparatus;14.Endoplasmic reticulum lumen;15.External side of plasma membrane;16.Lysosome;17.Apical plasma membrane;18.Melanosome;19.Extracellular exosome;20.Cornified envelope;21.GTP binding;22.GTPase activity;23.Serine-type endopeptidase inhibitor activity;24.Cysteine-type endopeptidase inhibitor activity;25.GDP binding;26.Misfolded protein binding;27.Structural constituent of skin epidermis;28.Proteoglycan binding;29.Enterobactin binding;30.Adenylate cyclase activator activity.

Tab.7 The same proteins in jersey yak crossbreeds milk and yak milk whey were enriched in the immune system and digestive system

分类 Classification	途径 Pathway	蛋白 Protein	P值 P-value
免疫系统 Immune system	补体和凝血级联反应	C3、SERPINC1、SERPINA1、SERPING1、KNG1、C9、KNG1、SERPINA5、SERPINF2、ACPB2、C8B、CFD、CFH、C6、CD46	1.15E-09
	造血细胞系	QCD14、PCD36、CD9	0.055 181 337
	自然杀伤细胞介导的细胞毒性	RAC1	0.602 058 303
	B细胞受体信号通路	RAC1、CD81	0.162 131 267
	Fc-epsilon-RI信号通路	RAC1	0.201 269 725
	FcγR介导的吞噬作用	GSN、CDC42、RAC1、ARPC1B	0.034 223 458
	血小板活化	ACTB、RAP1B、GNAS、GNAI3	0.221 518 653
	白细胞经内皮细胞迁移	ACTB、EZR、CDC42、RAP1B、RAC1、GNAI3	0.044 873 234
	Toll样受体信号通路	CD14、LBP、ARAC1	0.261 884 837
	抗原处理和呈现	HSPA5、B2M、CTSB、LGMN、CALR、PDIA3、CTSS	0.003 219 575
	T细胞受体信号通路	CDC42	0.911 154 068
	趋化因子信号通路	CDC42、RAP1B、RAC1、GNAI3、GNG5	0.235 478 004
	NOD样受体信号通路	YWHAE、CTSB、TXN	0.608 079 636
	C型凝集素受体信号通路	CALM1、RRAS2	0.593 188 474
消化系统	唾液分泌	LPO、CST3、GNAS、CALM1	0.123 753 251
Digestive system	矿物质吸收	Q29443∶TF、G3X6N3∶TF、LC34A2、ATOX1、SLC5A1	0.000 773 007
	胆固醇代谢	NPC2、CD36、APOH、LIPG	0.015 534 178
	胃酸分泌	ACTB、EZR、GNAS、CALM1、GNAI3	0.021 399 560
	脂肪消化吸收	CD36、AGPAT1	0.227 596 007
	胰腺分泌物	RAP1B、CPB2、RAC1、GNAS	0.189 221 135
	蛋白质消化吸收	COL18A1、CPB2、SLC3A2	0.379 689 638
	胆汁分泌	GNAS、SLC5A1	0.572 585 960
	碳水化合物消化和吸收	SLC5A1	0.561 451 805

Fig.7 KEGG pathway analysis of the same protein in whey of jersey yak crossbreeds milk and yak milk (top 20)

Tab.8 The same proteins in jersey yak crossbreeds milk and yak milk MFGM were enriched in the immune system and digestive system

分类 Classification	途径 Pathway	蛋白 Protein	P值 P-value
免疫系统 Immune system	补体和凝血级联反应	C3、SERPINC1、SERPINA1、SERPING1、C9、KNG1、SERPINA5、CD59、SERPINF2、CPB2、C8B、CFD、C6、CD46	7.88E-09
	造血细胞系	CD14、CD36、CD59、CD9	0.000 941 698
	自然杀伤细胞介导的细胞毒性	RAC1	0.188 402 927
	B细胞受体信号通路	RAC1、CD81	0.017 976 739
	Fc-epsilon-RI信号通路	RAC1	0.018 083 258
	FcγR介导的吞噬作用	GSN、CDC42、RAC1	0.009 662 499
	Toll样受体信号通路	CD14、LBP、RAC1	0.252 342 925
	抗原处理和呈现	HSPA5、B2M、CTSB、HSP90AB1、CTSL、LGMN、CALR、PDIA3、CTSS	0.000 117 112
	T细胞受体信号通路	CDC42	0.906 725 910
	白细胞经内皮细胞迁移	CDC42、RAC1	0.721 406 867
	趋化因子信号通路	CDC42、RAC1、GNG12、GNG5	0.405 970 520
	IL-17信号通路	LCN2、HSP90AB1、S100A8	0.191 105 850
	NOD样受体信号通路	CTSB、HSP90AB1	0.814 552 018
	Th17细胞分化	HSP90AB1	0.916 476 674
	血小板活化	GNAS	0.908 426 184
	C型凝集素受体信号通路	CALM1	0.860 272 455
消化系统	唾液分泌	LPO、CST3、GNAS、CALM1	0.117 300 437
Digestive system	矿物质吸收	TF、SLC34A2、ATOX1、SLC5A1	0.004 296 143
	胆固醇代谢	NPC2、CD36、LIPG	0.067 677 182
	脂肪消化吸收	CD36、AGPAT1	0.220 962 895
	蛋白质消化吸收	COL14A1、COL18A1、CPB2、SLC3A2	0.171 346 351
	胰腺分泌物	CPB2、RAC1、GNAS	0.379 942 338
	胃酸分泌	GNAS、CALM1	0.460 447 816
	胆汁分泌	GNAS、SLC5A1	0.561 589 000
	碳水化合物消化和吸收	SLC5A1	0.554 128 648

Fig.8 KEGG pathway analysis of the same protein in MFGM of jersey yak crossbreeds milk and yak milk (top 20)

Fig.9 STRING interaction network analysis of the same protein whey between jersey yak crossbreeds milk and yak milk

Fig.10 STRING interaction network analysis of the same protein MFGM between jersey yak crossbreeds milk and yak milk

Fig.11 The expression of MUCI,CD9,CD81 and CD36 in the whey identical protein of jersey yak crossbreeds milk and yak milk

Fig.12 The expression of MUCI,CD9,CD81 and CD36 in MFGM identical protein of jersey yak crossbreeds milk and yak milk

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