Untargeted metabolomics and quantitative label-free proteomics analysis of whole milk protein from Chinese native buffaloes

Figures & data

Table 1. Milk composition comparison of BBM and DBM.

Milk Source	Fat (%)	Protein (%)	Lactose (%)	Total Solids (%)	A2 β-casein (ng/ml)
BBM	8.23 ± 0.13^a	4.25 ± 0.06^a	5.21 ± 0.06^a	18.22 ± 0.05^a	18.81 ± 0.28^a
DBM	8.16 ± 0.07 ^a	4.18 ± 0.04^b	5.17 ± 0.05^a	18.28 ± 0.05^a	18.68 ± 0.37^a

Different superscript letters between the groups indicate significantly different values (p < .05).

Table 2. Determination of major differential metabolite compounds of BBM by UPLC-MS/MS.

Metabolite	Chemical Formula	m/z	rt (min)	Exact Mass	Concentration (ppm)	KEGG	Ion Source	Peak Area
Glycerophosphocholine^a	C8H21NO6P	258.11	4.30	258.11	1.63	C00670	[M]+	4.07E + 10
L-Carnitine^a	C7H15NO3	162.11	2.70	161.11	.07	C00318	[M+H]+	3.30E + 10
Catechol^a	C6H6O2	111.02	1.85	110.04	7.59	C00090	[M+H]+	3.29E + 10
γ-Aminobutyric acid^a	C4H9NO2	104.11	1.74	103.06	7.03	C00334	[M+H]+	1.84E + 10
Acetoacetic acid^a	C4H6O3	85.03	1.42	102.03	1.12	C00164	[M+H – H2O]+	1.62E + 10
2-Furancarboxaldehyde^a	C5H4O2	97.03	1.62	96.02	.30	C14279	[M+H]+	1.51E + 10
Betaine^a	C5H11NO2	118.09	1.97	117.08	4.47	C00719	[M+H]+	9.30E + 09
2-Keto-glutaramic acid^a	C5H7NO4	145.05	2.42	145.04	3.49	C00940	[M]+	9.21E + 09
L-Glutamic acid^a	C5H9NO4	148.06	2.47	147.05	.01	C00025	[M+H]+	6.65E + 09
2-Ketobutyric acid^a	C4H6O3	102.04	1.70	102.03	9.66	C00109	[M]+	6.64E + 09
Quinolinic acid^a	C7H5NO4	167.01	2.78	167.02	.31	C03722	[M]+	5.72E + 09
Butyryl-L-carnitine^a	C11H21NO4	232.15	3.87	231.15	.0007	C02862	[M+H]+	5.05E + 09
D-Fructose^a	C6H12O6	163.06	2.72	180.06	2.51	C00095	[M+H – H2O]+	4.50E + 09
Creatinine^a	C4H7N3O	114.07	1.90	113.06	5.27	C00791	[M+H]+	4.36E + 09
Hippuric acid^a	C9H9NO3	178.05	2.97	179.06	5.54	C01586	[M – H]–	3.91E + 09
L-Malic acid^a	C4H6O5	133.01	2.22	134.02	1.22	C00149	[M – H]–	3.75E + 09
Taurine^a	C2H7NO3S	125.99	2.10	125.01	1.07	C00245	[M+H]+	3.08E + 09
Acetylcholine^a	C7H16NO2	146.12	2.44	146.12	.31	C01996	[M+H]+	2.79E + 09
1,2,3-Trihydroxybenzene^b	C6H6O3	127.04	2.12	126.03	15.21	C01108	[M+H]+	2.78E + 09
cis-Aconitate^b	C6H6O6	173.01	2.88	174.02	3.94	C00417	[M – H]–	2.56E + 09
myo-Inositol^a	C6H12O6	181.02	3.02	180.06	6.01	C00137	[M+H]+	2.43E + 09
N-Methyl-2-pyrrolidinone^b	C5H9NO	100.08	1.67	99.07	1.12	C11118	[M+H]+	2.32E + 09
Phloroglucinol^b	C6H6O3	127.04	2.12	126.03	11.92	C02183	[M+H]+	2.30E + 09
Maltol^a	C6H6O3	127.04	2.12	126.03	13.05	C11918	[M+H]+	1.93E + 09
Fomepizole^c	C4H6N2	83.06	1.38	82.05	16.09	C07837	[M+H]+	1.91E + 09
Pantothenic acid^a	C9H17NO5	220.12	3.67	219.11	2.95	C00864	[M+H]+	1.90E + 09
Cytosine^a	C4H5N3O	112.05	1.87	111.04	.83	C00380	[M+H]+	1.87E + 09
Hydroquinone^a	C6H6O2	110.02	1.83	110.04	6.95	C00530	[M]+	1.82E + 09
Piperidine^c	C5H11N	86.10	1.43	85.09	7.35	C01746	[M+H]+	1.47E + 09
Pipecolic acid^a	C6H11NO2	130.05	2.17	129.08	22.18	C00408	[M+H]+	1.46E + 09
5-Hydroxyindoleacetic acid^a	C10H9NO3	192.06	3.20	191.06	16.04	C05635	[M+H]+	1.43E + 09
Glucose 6-phosphate^b	C6H13O9P	259.02	4.32	260.03	2.22	C00092	[M – H]–	1.41E + 09
L-Proline^a	C5H9NO2	116.07	1.93	115.06	7.20	C00148	[M+H]+	1.05E + 09
Mesaconate^b	C5H6O4	129.02	2.15	130.03	1.77	C01732	[M – H]–	1.04E + 09
Uracil^a	C4H4N2O2	113.03	1.88	112.03	.03	C00106	[M+H]+	9.98E + 08

The superscript letter after the metabolite compound name corresponds to the bioinformatics database used in this identification, where (a): BioDeepDB (b): mzCloud, and (c): MoNA; rt: retention time; KEGG refers to metabolic pathway mapping in KEGG mapper; ion source is associated to the sensitive atmospheric pressure ESI, both in ESI⁺ and ESI^– mode; and peak area represents the intensity measure of the metabolite compound.

Table 3. Determination of major differential metabolite compounds of DBM by UPLC-MS/MS.

Metabolite	Chemical Formula	m/z	rt (min)	Exact Mass	Concentration (ppm)	KEGG	Ion Source	Peak Area
Glycerophosphocholine^a	C8H21NO6P	258.11	4.30	258.11	7.10	C00670	[M]+	6.22E + 10
Creatine^a	C4H9N3O2	132.08	2.20	131.07	1.34	C00300	[M+H]+	4.14E + 10
Catechol^a	C6H6O2	111.02	1.85	110.04	8.68	C00090	[M+H]+	3.27E + 10
Phosphorylcholine^a	C5H15NO4P	184.07	3.07	184.07	.19	C00588	[M+H]+	1.51E + 10
Acetoacetic acid^a	C4H6O3	85.03	1.42	102.03	7.79	C00164	[M+H – H2O]+	1.50E + 10
2-Furancarboxaldehyde^a	C5H4O2	97.03	1.62	96.02	3.70	C14279	[M+H]+	1.37E + 10
L-Carnitine^a	C7H15NO3	162.11	2.70	161.11	.001	C00318	[M+H]+	1.20E + 10
γ-Aminobutyric acid^a	C4H9NO2	104.11	1.74	103.06	8.27	C00334	[M+H]+	1.18E + 10
2-Ketobutyric acid^a	C4H6O3	102.03	1.70	102.03	3.43	C00109	[M]+	6.83E + 09
D-Fructose^a	C6H12O6	163.06	2.72	180.06	2.66	C00095	[M+H – H2O]+	4.38E + 09
Cellobiose^a	C12H22O11	341.10	5.68	342.12	29.68	C00185	[M – H]–	3.78E + 09
Butyryl-L-carnitine^a	C11H21NO4	232.15	3.87	231.15	.004	C02862	[M+H]+	3.28E + 09
Taurine^a	C2H7NO3S	125.99	2.10	125.01	.96	C00245	[M+H]+	3.19E + 09
(S)-4-Hydroxymandelate^a	C8H8O4	169.05	2.82	168.04	.48	C03198	[M+H]+	2.89E + 09
Betaine^a	C5H11NO2	118.09	1.97	117.08	.61	C00719	[M+H]+	2.89E + 09
6-Acetyl-D-glucose^d	C8H14O7	221.07	3.68	222.07	5.64	C02655	[M – H]–	2.72E + 09
Creatinine^a	C4H7N3O	114.07	1.90	113.06	5.66	C00791	[M+H]+	2.68E + 09
1,2,3-Trihydroxybenzene^b	C6H6O3	127.04	2.12	126.03	12.22	C01108	[M+H]+	2.62E + 09
Fomepizole^c	C4H6N2	83.06	1.38	82.05	1.04	C07837	[M + 2ACN +2 H]2+	2.27E + 09
Stachyose^a	C24H42O21	667.23	11.12	666.22	3.17	C01613	[M+H]+	1.47E + 09
Hippuric acid^a	C9H9NO3	178.05	2.97	179.06	5.35	C01586	[M – H]–	1.41E + 09
L-Glutamine^a	C5H10N2O3	147.08	2.45	146.07	.002	C00064	[M+H]+	1.39E + 09
11-Dehydrocorticosterone^a	C21H28O4	325.18	5.42	344.20	8.86	C05490	[M – H2O – H]–	1.37E + 09
L-Leucine^a	C6H13NO2	132.10	2.20	131.09	1.15	C00123	[M+H]+	1.15E + 09
Pantothenic acid^a	C9H17NO5	220.12	3.67	219.11	.10	C00864	[M+H]+	1.11E + 09
Quinoline^a	C9H7N	130.16	2.17	129.06	1.20	C06413	[M+H]+	1.02E + 09
Phloroglucinol^b	C6H6O3	127.04	2.12	126.03	13.13	C02183	[M+H]+	1.02E + 09
L-Proline^a	C5H9NO2	116.07	1.93	115.06	2.29	C00148	[M+H]+	1.00E + 09
Gluconic acid^a	C6H12O7	195.05	3.25	196.06	.30	C00257	[M – H]–	9.49E + 08
2-Deoxystreptamine^a	C6H14N2O3	163.12	2.72	162.10	2.86	C02627	[M+H]+	9.24E + 08
Cytosine^a	C4H5N3O	112.05	1.87	111.04	.09	C00380	[M+H]+	9.07E + 08
Phosphocreatine^a	C4H10N3O5P	210.03	3.50	211.04	7.44	C02305	[M – H]–	8.99E + 08
4,5-Dihydroorotic acid^a	C5H6N2O4	158.96	2.65	158.03	7.90	C00337	[M+H]+	8.91E + 08
Arachidic acid^a	C20H40O2	311.17	5.19	312.30	.46	C06425	[M – H]–	8.62E + 08
Methyloxaloacetate^a	C5H6O5	147.03	2.60	146.02	.02	C06030	[M+H]+	8.21E + 08

The superscript letter after the metabolite compound name corresponds to the bioinformatics database used in this identification, where (a): BioDeepDB, (b): mzCloud, (c): MoNA, and (d): MetDNA.

Figure 1. Venn diagram of total identified protein numbers in BBM and DBM.

Figure 2. (a) RSD coefficient distribution before and after data correction of all the protein quantitative values in the corresponding sample. A lower CV coefficient indicated better repeatability of all samples. (b) Pearson correlation coefficient of all quantitative protein expressions between the two corresponding samples.

Figure 3. The distribution of enriched GO terms in BBM and DBM on three functional categories.

Figure 4. The distribution of enriched KEGG pathway in BBM and DBM.

Figure 5. Ppis of the identified proteins in BBM and DBM. STRING database was used to establish the protein-protein interactions of quantified proteins identified by HPLC-MS/MS. Each node in the PPI network represents an identified protein, and the type of node represents the degree of connection of the protein.