. 2020 Jun 2;86(12):e00476-20. doi: 10.1128/AEM.00476-20

TABLE 1.

Validation of the LC-MS/MS-MRM method^a

Mixture and digestion Cry protein	Proteotypic peptide	Peptide mean ± SD (fmol)	Protein mean ± SD (fmol)	Relative molar composition (%)
Mixture 1
D1
Cry1Aa	APTFSWQHR	2,511 ± 410	2,226 ± 405	32
	VNITAPLSQR	1,942 ± 32	2,226 ± 405	32
Cry2Aa	ETEQFLNQR	2,558 ± 254	1,666 ± 724	24
	VNAELIGLQANIR	1,407 ± 155
	VYTVSNVNTTTNNDGVNDNGAR	1,032 ± 28
Cry6Aa	QLDSAQHDLDR	3,092 ± 51	3,126 ± 128	44
	SANDIASYGFK	3,159 ± 204	3,126 ± 128	44
D2
Cry1Aa	APTFSWQHR	2,834 ± 103	2,402 ± 511	35
	VNITAPLSQR	1,970 ± 157	2,402 ± 511	35
Cry2Aa	ETEQFLNQR	2,614 ± 458	1,754 ± 772	26
	VNAELIGLQANIR	1,567 ± 39
	VYTVSNVNTTTNNDGVNDNGAR	1,081 ± 561
Cry6Aa	QLDSAQHDLDR	2,218 ± 283	2,654 ± 576	39
	SANDIASYGFK	3,090 ± 392	2,654 ± 576	39
Mixture 2
D1
Cry1Aa	APTFSWQHR	4,510 ± 854	3,630 ± 1,172	43
	VNITAPLSQR	2,750 ± 543	3,630 ± 1,172	43
Cry2Aa	ETEQFLNQR	714 ± 210	483 ± 211	6
	VNAELIGLQANIR	430 ± 20
	VYTVSNVNTTTNNDGVNDNGAR	304 ± 55
Cry6Aa	QLDSAQHDLDR	4,258 ± 416	4,380 ± 305	51
	SANDIASYGFK	4,503 ± 213	4,380 ± 305	51
D2
Cry1Aa	APTFSWQHR	5,906 ± 1,484	4,703 ± 1,648	46
	VNITAPLSQR	3,501 ± 406	4,703 ± 1,648	46
Cry2Aa	ETEQFLNQR	732 ± 72	591 ± 155	6
	VNAELIGLQANIR	561 ± 84
	VYTVSNVNTTTNNDGVNDNGAR	480 ± 203
Cry6Aa	QLDSAQHDLDR	4,507 ± 518	4,821 ± 486	48
	SANDIASYGFK	5,134 ± 217	4,821 ± 486	48

Proteins were identified and quantified in two artificial mixtures of three Cry proteins. Cry1Aa, Cry2Aa, and Cry6Aa were mixed in a molar ratio of 1:1:1 for mixture 1 (33.3% of each Cry protein) and a molar ratio of 13:4:13 for mixture 2 (equivalent to 43.3% of Cry1Aa, 13.3% of Cry2Aa, and 43.3% of Cry6Aa). The mixtures were digested with trypsin, and proteins were identified and quantified based on proteotypic peptide abundance and by comparing the signal intensities of the endogenous and corresponding SIL peptide. Mean values represent averages of two independent analyses for each tryptic digestion (D1 and D2).