Development of Diagnostic Biomarkers for Detecting Diabetic Retinopathy at Early Stages Using Quantitative Proteomics

Table 2

The linearity, sensitivity, and analytical reproducibility of the MRM assay.

	Name	Peptide sequence		Average relative response (L/H)^a	Correlation coefficient ()^b	Dynamic range	RT (min)^c	RT CV%^d			LLOD (fmol/µL)^e	LLOQ (fmol/µL)^f	Normal con. (ng/mL)^g	Average con. ng/µL			Conc. reference
		(heavy and light)						No DR	Mi NPDR	Mo NPDR				No DR	Mi NPDR	Mo NPDR
1	APLP2	Light	WEPDPTGTK	0.01	0.967	1,000	20.8	7	3.84	3.8	5.88	17.66	⋅	0.61	0.73	0.45
		Heavy	WEPDPTGT[13C-15N-K]					7.28	3.8	2.44
2	APOA4	Light	LAPLAEDVR	2.99	0.995	2,000	25.1	5.3	3.82	2.47	0.13	0.41	160,000	125.28	149.92	166.04	[29]
		Heavy	LAPLAEDV[13C-15N-R]					5.38	3.87	2.46
3	APOH	Light	ATVVYQGER	5.39	0.999	2,000	16	7.58	4.39	4.3	1.1	3.57	258,000	254.86	269.92	190.82	[30]
		Heavy	ATVVYQGE[13C-15N-R]					7.53	4.47	4.21
4	B3GNT1	Light	QYGFNR	0.95	0.941	1,000	18.4	6.77	4.14	3.72	2.57	7.71	⋅	51.98	47.74	34.24
		Heavy	QYGFN[13C-15N-R]					6.72	4.03	3.78
5	C4B	Light	VDFTLSSER	4.78	0.999	2,000	29	4.86	2.99	1.73	0.32	1.01	95,000	266.04	239.1	193.78	[30]
		Heavy	VDFTLSSE[13C-15N-R]					4.85	2.91	1.7
6	C5	Light	TDAPDLPEENQAR	2.49	0.998	2,000	22.1	7.03	3.62	1.71	0.38	1.14	69,000	189.66	124.84	15.74	[30]
		Heavy	TDAPDLPEENQA[13C-15N-R]					7.04	3.56	1.77
7	C7	Light	AASGTQNNVLR	0.44	0.989	2,000	13.9	8.24	4.8	4.92	0.52	1.57	60,000	24.27	22.49	13.76	[9, 30]
		Heavy	AASGTQNNVL[13C-15N-R]					8.25	4.81	4.84
8	CD14	Light	FPAIQNLALR	0.085	0.993	1,000	41.3	2.81	1.72	1.27	5.96	17.88	5,000	4.26	4.28	3.03	[32]
		Heavy	FPAIQNLAL[13C-15N-R]					2.91	1.72	1.26
9	CLU	Light	ASSIIDELFQDR	3.3	0.998	1,000	46.3	0.94	0.23	0.11	0.06	0.19	100,000	115.98	165.18	143.37	[30, 33]
		Heavy	ASSIIDELFQD[13C-15N-R]					0.95	0.26	0.12
10	FN1	Light	FLATTPNSLLVSWQPPR	0.3	0.994	2,000	45.8	0.95	0.25	0.13	1.31	3.93	275,000	19.75	15.16	22.93	[9, 29–31]
		Heavy	FLATTPNSLLVSWQPP[13C-15N-R]					0.94	0.28	0.12
11	GSN	Light	GGVASGFK	0.59	0.998	2,000	15.4	7.78	4.43	4.46	4.15	12.46	240,000	29.64	29.64	19.87	[30]
		Heavy	GGVASGF[13C-15N-K]					7.94	4.34	4.55
12	ITIH2	Light	VQSTITSR	0.9	0.996	2,000	9.41	7.2	3.07	3.91	0.19	0.59	140,000	39.32	45.44	17.41	[30]
		Heavy	VQSTITS[13C-15N-R]					7.25	3.09	3.96
13	KRT1	Light	AQYEDIAQK	0.26	0.987	2,000	19.6	8.19	4.14	3.37	1.28	3.85	⋅	11.56	13.24	8.04
		Heavy	AQYEDIAQ[13C-15N-K]					8.35	4.35	3.35
14	SERPINF1	Light	LSYEGEVTK	0.87	0.996	2,000	20.2	6.88	3.79	2.44	0.8	2.41	4,600	47.11	43.8	41.28	[30]
		Heavy	LSYEGEVT[13C-15N-K]					6.95	4.52	3.47
15	VTN	Light	DVWGIEGPIDAAFTR	5.67	0.987	1,000	46.4	0.87	0.23	0.17	0.32	0.96	100,000	223.49	283.84	257.51	[30]
		Heavy	DVWGIEGPIDAAFT[13C-15N-R]					0.89	0.82	0.58

Average relative response (L.H) represents the peak area ratio of light to heavy signal. ^bThe correlation coefficient for each protein () represents the linearity of the MRM analysis. ^cRetention time (RT) represents the average retention time in 60 individual samples. ^dRT CV% represents the retention time variation in the No DR, Mi NPDR, and Mo NPDR groups. ^eLowest limit of detection (LLOD) is calculated based on the variance of the blank sample and the standard deviation (S.D.) of the sample with the lowest “spiked” in concentration. ^fThe lowest limit of quantitation (LLOQ) was calculated as follows: LLOQ = LLOD 3. ^gNormal protein concentrations in plasma were obtained by literature searches as described in the Results section.