Table 1.

Mutant GFAP as a percent of total GFAP in R79C, R416W, and R239H Alexander disease patients

Total GFAP is the sum of the amount of patient GFAP and ¹⁵N standard GFAP analyzed. The control peptides for trypsin digestion were FADLTDAAAR (1), LADVYQAELR (2), ALAAELNQLR (3), and LLEGEENR (4). The control peptides for chymotrypsin digestion were ARQQVHVEL (1), RQEADEATL (2), and IEKVRF (3). IEKVRF is also present in desmin and peripherin, but these proteins are not expected to be present in the brain at significant levels compared to GFAP. Were significant levels indeed present, their inclusion would lead to overestimation of the amount of WT GFAP present and, thus, underestimation of the percent mutant GFAP. With inclusion of IEKVRF, the calculated percent mutant GFAP was actually slightly higher than that obtained if it were excluded (29% versus 27%). ND, not detected in the patient sample, the ¹⁵N standard, or both; Amt, amount; Dig, digestion; MUT, mutant.

Amt 15N Std	Total GFAP	Dig time	% Mutant		Peptide Ratios (Patient/15N Std) Intact GFAP Band					Peptide Ratios (Patient/15N-Std) Degraded GFAP Band
ng	ng	h	Intact	Degraded	Diagnostic	Controls				Diagnostic	Controls
R79C patient, trypsin digestion					WT	1	2	3	4	WT	1	2	3	4
50	120	40	21%	25%	0.76	0.99	1.00	0.96	0.88	1.41	1.90	2.09	1.83	1.72
100	170	18	22%	ND	0.46	0.63	0.59	0.53	0.59	ND	ND	ND	0.95	ND
100	170	40	21%	23%	0.48	0.65	0.61	0.63	0.56	0.85	1.15	1.18	1.07	0.99
150	220	40	19%	22%	0.38	0.51	0.47	0.47	0.42	0.57	0.77	0.78	0.71	0.66
Average ± SD			21% ± 1%	23% ± 2%
R416W patient, trypsin digestion					MUT	1	2	3	4	MUT	1	2	3	4
13.5	90.5	40	45%	19%	2.81	6.13	6.45	5.89	6.70	0.25	1.35	1.53	1.32	0.96
36	113	18	47%	20%	1.11	2.34	2.55	2.28	2.32	0.11	0.54	0.66	0.56	0.35
36	113	40	45%	22%	1.04	2.27	2.45	2.16	2.43	0.11	0.55	0.56	0.52	0.38
58.5	135.5	40	45%	22%	0.67	1.53	1.54	1.43	1.41	0.06	0.32	0.34	0.30	0.20
Average ± SD			46% ± 1%	21% ± 2%
R239H patient, chymotrypsin digestion					MUT	1	2	3		MUT	1	2	3
10	190	72	30%	ND	10.43	ND	34.99	ND		ND	ND	ND	ND
50	230	48	33%	ND	1.97	ND	7.17	4.71		ND	ND	7.07	ND
50	230	72	22%	ND	1.55	ND	6.56	7.28		ND	ND	13.97	ND
100	280	72	33%	ND	0.88	3.21	2.91	2.00		ND	ND	4.30	ND
Average ± SD			29% ± 5%