Table 1. Bion-binding proteins identified by LC-MS/MS and ranked by spectral counting.

				Bions
No.	Protein identified	Accession Number	MW	Ca2+	Mg2+	Mn2+	Sr2+	Ba2+
1	Fetuin-A	FETUA_BOVIN	38	373.4	154.8	150.4	495.1	490.3
2	Albumin	ALBU_BOVIN	69	232.5	339.7	322.9	97.2	82.9
3	Apolipoprotein A-I	APOA1_BOVIN	30	16.2	21.5	50.5	7.9	9.1
4	Prothrombin	THRB_BOVIN	71	14.1	17.2	18.2	29.5	61.7
5	Complement C3	CO3_BOVIN	187	26.1	21.5	62.1	12.8	13.1
6	Hemoglobin fetal subunit beta	HBBF_BOVIN	16	10.6	–	17.7	7.9	7.1
7	Alpha-1-antiproteinase	A1AT_BOVIN	46	7.8	17.2	22.7	3.9	6.1
8	Vitamin K-dependent protein S	PROS_BOVIN	75	3.5	21.5	3.5	27.5	35.4
9	Serotransferrin	TRFE_BOVIN	78	6.3	34.4	18.7	–	–
10	Coagulation factor X	FA10_BOVIN	55	10.6	–	3.5	19.6	17.2
11	Coagulation factor IX	FA9_BOVIN	47	4.9	–	4.0	14.7	17.2
12	Apolipoprotein A-II	APOA2_BOVIN	11	4.9	–	12.6	1.0	1.0
13	Vitamin D-binding protein	VTDB_BOVIN	53	3.5	8.6	12.6	1.0	–
14	Vitamin K-dependent protein C	PROC_BOVIN	51	1.4	8.6	1.5	12.8	16.2
15	Hemoglobin subunit alpha	HBA_BOVIN	15	7.0	–	10.1	2.0	2.0
16	Adiponectin	ADIPO_BOVIN	26	3.5	8.6	7.1	9.8	3.0
17	Coagulation factor V	FA5_BOVIN	249	4.2	–	5.6	5.9	3.0
18	Alpha-2-macroglobulin	A2MG_BOVIN	168	1.4	12.9	3.5	–	2.0
19	Antithrombin-III	ANT3_BOVIN	52	2.1	–	9.1	2.9	3.0
20	Alpha-fetoprotein	FETA_BOVIN	69	2.1	–	20.7	–	–
21	Heat shock protein HSP 90-alpha	HS90A_BOVIN	85	6.3	–	1.5	11.8	12.1
22	Complement factor H	CFAH_BOVIN	140	4.2	–	12.6	–	–
23	Thrombospondin-1	TSP1_BOVIN	130	0.7	–	5.6	2.0	1.0
24	Apolipoprotein M	APOM_PIG	21	–	–	2.5	–	–
25	Secreted phosphoprotein 24	SPP24_BOVIN	23	1.4	–	–	2.9	–
26	Apolipoprotein E	APOE_BOVIN	36	–	8.6	2.0	–	–
27	Osteopontin	OSTP_BOVIN	31	1.4	–	1.0	2.0	2.0
28	Alpha-2-antiplasmin	A2AP_BOVIN	55	0.7	4.3	1.0	2.0	1.0
29	Protein disulfide-isomerase	PDIA1_BOVIN	57	1.4	–	–	2.9	–
30	Complement component C9	CO9_BOVIN	62	–	–	2.5	–	–
31	Vitamin K-dependent protein Z	PROZ_BOVIN	43	–	–	–	2.0	1.0
32	Complement C4 (Fragments)	CO4_BOVIN	102	2.8	–	2.5	–	3.0
33	Coagulation factor VII	FA7_BOVIN	49	–	–	–	2.0	2.0
34	Endoplasmin	ENPL_BOVIN (+2)	92	1.4	–	–	–	3.0
35	Apolipoprotein C-III	APOC3_BOVIN	11	1.4	–	1.0	–	–
36	Glucosidase 2 subunit beta	GLU2B_BOVIN	60	–	–	0.5	2.0	–
37	Mannose-binding protein C	MBL2_BOVIN	26	2.1	–	1.5	–	–
38	Fibrinogen alpha chain	FIBA_BOVIN	67	0.7	–	1.5	–	–
39	Protein AMBP	AMBP_BOVIN	39	1.4	8.6	2.0	–	–
40	Alpha-1-acid glycoprotein	A1AG_BOVIN	23	–	–	1.0	–	–
41	Transthyretin	TTHY_BOVIN	16	–	–	1.5	–	–
42	Clusterin	CLUS_BOVIN	51	–	–	0.5	–	–
43	Moesin	MOES_BOVIN	68	–	–	1.5	–	–
44	Complement factor B	CFAB_BOVIN	85	1.4	–	0.5	–	–
45	Adenylyl cyclase-associated protein 1	CAP1_BOVIN	51	–	–	2.0	–	–
46	Proactivator polypeptide	SAP_BOVIN	58	–	–	0.5	–	–
47	Factor XIIa inhibitor	F12AI_BOVIN	52	1.4	–	–	–	–
48	Tetranectin	TETN_BOVIN	22	–	–	1.5	–	–

Bions were prepared by adding CaCl₂, MnCl₂, SrCl₂, or BaCl₂ at a final concentration of 10 mM, or MgCl₂ at 20 mM in DMEM containing 5% FBS. Na₂HPO₄ at 10 mM was then added (in a final volume of 1 ml). Following incubation in cell culture conditions overnight, bions were washed and then treated with 0.1 mM EDTA to help release the bound proteins. Proteomic analysis was performed using LC-MS/MS as described in Materials and Methods. Spectral counting values were normalized by multiplying each number of spectra by the average of total spectra count for the five samples shown, and then dividing by the sum of spectra for the corresponding sample. MW, molecular weight in kDa.