Table 1.

Mass Spectrometric Identification of RPE Proteins Labeled with 1^a

potein X corr	positions	MW (MH+)	sequence
(A) Labeled 60 kDa Region
RPE65		60945
2.2152	509–515	789.8637	DLSEVAR
1.6138	119–124	866.9948	FFSYFR
2.3747	348–355	1062.1674	ENWEEVKK
2.3770	368–376	1075.2932	YVLPLNIDK
2.5204	155–164	1144.3104	VNPETLETIK
2.4300	199–208	1169.3660	NFSIAYNIVK
3.2525	367–376	1231.4807	RYVLPLNIDK
2.7699	34–44	1269.5728	IPLWLTGSLLR
3.2833	414–425	1513.6929	QAFEFPQINYQK
4.0892	209–222	1551.7802	IPPLQADKEDPISK
4.8063	15–33	2087.3374	LFETVEELSSPLTAHVTGR
2.7135	426–446	2389.6750	YGGKPYTYAYGLGLNHFVPDR
2.3806	235–258	2771.2310	FKPSYVHSFGLTPNYIVFVETPVK
(B) Labeled 25 kDa Region
LRAT		25701
4.6637	56–69	1660.8295	THLTHYGIYLGDNR

^aRPE membranes (5–6 mg) in 1 mL of 100 mM phosphate buffer (pH 7.5) were labeled with 20 μM 1 for 1 h at 25 °C. Proteins were solubilized with 1% Triton X-100, followed by dialysis. To the neutravidin beads (0.5 mL) was added 1 mL of the solubilized, labeled RPE, and beads were resuspended at 4 °C for 3 h until complete uptake of the biotinylated proteins occurred. After the beads had been washed five times, the proteins were eluted at pH 11. The supernatant was concentrated to ~40 μL, and the proteins were subjected to SDS–PAGE. The silver-stained bands at approximately 60 (A) and 25 kDa (B) were excised and submitted for tandem mass spectrometric analysis. The sequences of the peptides were determined by electrospray MS/MS. In separate experiments, peptide molecular masses were also verified by MALDI-TOF.