Table 2. Hi3 peptide quantitative analysis of abundant plasma proteins.

(50:1 substrate:enzyme, 20 hours digestion, 0.21 ug on column injection).

No	Protein name	median (ng/mL)	IRV	IAV	AV	LIN	ST	LOQ	n	FN
1	Albumin	7.4 x107	12	15	11	0.992	0.955	6.0 x103	31	1
2	Ig gamma	4.8 x106	30	17	5	0.985	0.998	8.5 x104	31	2
3	Serotransferrin	2.7 x106	19	19	9	0.991	0.976	2.6 x105	31	3
4	Fibrinogen	2.6 x106	14	15	9	0.995	0.991	7.0 x104	30	4
5	Complement C3	2.3 x106	17	16	7	0.987	0.974	5.6 x104	31	5
6	Alpha-2-macroglobulin	1.8 x106	16	11	4	0.990	0.993	5.2 x104	31	6
7	Apolipoprotein A-I	1.7 x106	29	14	8	0.991	0.976	2.2 x104	31	7
8	Alpha-1-antitrypsin	1.6 x106	14	16	15	0.985	0.964	2.7 x104	31	8
9	Haptoglobin	1.3 x106	26	20	9	0.993	0.991	3.2 x104	31	9
10	Ig kappa chain C region	1.0 x106	25	14	8	0.989	0.985	1.7 x104	31	10
11	Apolipoprotein B-100	9.7 x105	14	15	9	0.966	0.999	2.5 x105	31	11
12	Hemopexin	9.1 x105	12	12	4	0.999	0.960	2.1 x104	31	12
13	Ig alpha	7.3 x105	18	17	8	0.998	0.982	2.3 x104	31	13
14	Complement C4	6.8 x105	16	16	14	0.979	-	1.3 x105	31	14
15	Fibronectin	5.0 x105	-	-	32	-	-	-	22	15
16	Ig lambda chain C region	4.0 x105	22	17	7	0.989	0.976	1.5 x104	31	16
17	Ceruloplasmin	3.5 x105	22	16	8	0.990	0.992	1.8 x104	31	17
18	Inter-alpha-trypsin inhibitor hc H2	3.5 x105	14	18	14	0.991	0.965	2.4 x104	31	18
19	Complement factor H	2.7 x105	20	20	5	0.991	0.990	3.3 x104	31	19
20	Vitamin D-binding protein	2.7 x105	15	15	9	0.994	0.993	3.3 x104	31	20
21	Kininogen-1	2.6 x105	17	14	10	0.989	0.963	3.6 x104	31	21
22	Alpha-2-HS-glycoprotein	2.4 x105	16	13	12	0.990	0.961	4.0 x103	31	22
23	Plasminogen	2.2 x105	22	20	11	0.991	0.983	4.7 x104	31	23
24	Ig mu chain C region	2.2 x105	26	14	17	0.993	0.997	1.4 x104	31	24
25	Apolipoprotein A-II	2.0 x105	27	17	6	0.995	0.988	2.4 x104	31	25
26	Alpha-1-antichymotrypsin	1.8 x105	16	6	6	0.989	0.961	6.0 x103	31	26
27	Inter-alpha-trypsin inhibitor hc H1	1.8 x105	16	-	18	0.993	-	4.4 x104	31	27
28	Alpha-1-acid glycoprotein 1	1.7 x105	41	16	10	0.990	0.971	6.0 x103	31	28
29	Beta-2-glycoprotein 1	1.7 x105	11	31	7	0.993	0.966	7.2 x104	31	29
30	Inter-alpha-trypsin inhibitor hc H4	1.7 x105	43	10	7	0.993	0.995	2.0 x104	31	30
31	Complement factor B	1.5 x105	25	52	11	0.995	0.974	2.5 x104	31	31
32	Clusterin	1.5 x105	10	17	10	0.990	0.958	1.7 x104	31	32
33	Alpha-1B-glycoprotein	1.4 x105	18	12	9	0.989	0.955	1.0 x104	31	33
34	Prothrombin	1.4 x105	16	23	51	0.993	0.974	1.7 x104	31	34
35	Antithrombin-III	1.4 x105	20	19	7	0.984	0.962	5.0 x103	31	35
36	Plasma protease C1 inhibitor	1.3 x105	19	18	10	0.964	0.966	1.1 x104	31	36
37	Vitronectin	1.1 x105	13	13	10	0.995	0.982	1.4 x104	28	37
38	Apolipoprotein A-IV	1.0 x105	18	16	9	0.994	0.972	9.0 x103	31	38
39	C4b-binding protein alpha chain	1.0 x105	18	13	35	0.996	0.997	1.5 x104	31	39
40	Histidine-rich glycoprotein	9.0 x104	21	13	10	0.976	0.958	8.0 x103	31	40
No	Protein name	median (ng/mL)	IRV	IAV	AV	LIN	ST	LOQ	n	FN
41	Gelsolin	7.0 x104	23	36	11	0.990	-	3.5 x104	22	41
42	Heparin cofactor 2	6.6 x104	17	-	100	0.991	-	1.1 x104	11	42
43	Afamin	5.7 x104	16	12	15	0.998	0.954	1.0 x105	18	43
44	Angiotensinogen	5.7 x104	19	16	5	0.989	0.992	1.1 x104	30	44
45	Paraoxonase/arylesterase 1	5.1 x104	22	-	22	0.982	0.999	7.0 x103	23	45
46	Alpha-1-acid glycoprotein 2	5.0 x104	44	15	13	0.999	0.958	4.0 x103	31	46
47	Hemoglobin subunit alpha	4.1 x104	40	-	7	0.991	-	3.0 x103	31	47
48	Apolipoprotein C-III	3.9 x104	32	27	20	-	0.978	1.4 x104	31	48
49	Protein AMBP	3.9 x104	26	10	16	0.825	0.949	7.0 x103	22	49
50	Hemoglobin subunit beta	3.8 x104	17	42	8	0.992	0.983	4.0 x103	31	50
51	Pregnancy zone protein	3.6 x104	64	64	75	0.959	0.998	7.0 x103	15	-
52	Zinc alpha 2 glycoprotein	3.3 x104	24	12	212	0.772	0.971	4.0 x103	20	51
53	Apolipoprotein E	3.1 x104	19	6	11	0.996	0.964	1.7 x104	27	52
54	CD5 antigen like protein	2.5 x104	8	7	15	-	0.997	1.3 x104	19	-
55	Haptoglobin-related protein	2.3 x104	24	24	21	0.968	0.995	3.0 x103	16	53
56	Retinol-binding protein 4	2.2 x104	20	-	27	-	-	-	14	54
57	Apolipoprotein D	2.1 x104	-	-	13	-	-	-	20	55
58	Apolipoprotein C-I	1.6 x104	-	-	11	-	-	-	26	56
59	Apolipoprotein C-II	1.6 x104	-	23	7	-	0.922	-	19	57

Median (ng/mL): median of protein concentrations determined from number of samples shown in column n. IRV: inter-assay variation, the coefficients of variation obtained from 7 aliquots of a pooled sample separately digested and measured over the course of 3 months of normal operation of the instrument. IAV: intra-assay variation, coefficient of variation obtained from 6 aliquots of a pooled sample separately digested and subsequently measured during 1 day. AV: analytical variability, determined from 10 replicate injections of a single digested sample throughout 9 days of measurements. LIN: linearity of measurements, the Pearson’s correlation coefficient shows linearity between total protein load on the analytical column and nanogram protein quantified by HI3 peptide quantitation using the ADH1 digest standard; only determined when at least 4 points were available for a protein. See also S1 Fig. ST: Pearson’s correlation of protein quantitation using two different digest standards (ADH1 and ENO1 from yeast). LOQ: limit of quantitation (ng/mL) estimated by dilution of a plasma sample in a constant background of a digest standard. n: number of samples (out of 31) in which the protein was quantified, FN: protein number on the x-axis of Fig 1c. hc: heavy chain.