Studies with Site-Specific Labeled HAMLET Figure 4. Site-specific labeling with aminooxy-Alexa Fluor 488 of hLA-ext. The hLA-ext protein (A) is glycosylated by the ppGalNAc-T2 enzyme in the presence of Mn2+ and UDP-C2-keto-galactose. The lower panel shows the MALDI-TOF analysis of the C-terminal extension peptide released upon thrombin cleavage. (B) The glycosylated hLA-ext protein with a single C2-keto-Gal molecule (blue circle) is conjugated with the aminooxy-Alexa Fluor 488 molecule. The lower panel shows the MALDI-TOF spectrum of the glycosylated C-terminal extension peptide released after thrombin treatment of the glycosylated hLA-ext protein. The increased molecular weight of 201 Da corresponds to a single C2-keto-gal molecule. Since the Alexa Fluor 488 conjugated C-terminal peptide released from the Alexa Fluor 488 conjugated hLA-ext protein could not be observed on a MALDI-TOF spectrum, the protein with and without thrombin treatment was analyzed on a SDS-PAGE gel (C) for the fluorescence emission detection (lower panel). Fluorescence emission from the protein band is only observed when the protein was not treated with the thrombin, suggesting that the Alexa Fluor 488 molecule is conjugated to the C-terminal extension peptide. doi:10.1371/journal.pone.0026093.g004 hLA-ext protein was conjugated with aminooxy-Alexa Fluor 488 at pH 3.9 and purified by ammonium sulfate precipitation. Since the thrombin cleaved Alexa Fluor 488 conjugated C-terminal extension peptide could not be analyzed by MALDI-TOF, a fluorescence emission at 488 nM from the Alexa Fluor 488 conjugated hLA-ext protein samples were analyzed on SDS- PAGE gel, with and without thrombin treatment. Only a fluorescence emission from the protein band that was not treated with thrombin is observed, suggesting that the Alexa Fluor 488 molecule is conjugated to the C-terminal extension peptide of the glycosylated hLA-ext protein (Figure 4C). Similarly, the aD-hLA- ext protein is also conjugated with aminooxy-Alexa Fluor 488 molecule after glycosylating with C2-keto-Gal molecule. Glycosylated hLA-ext and aD-hLA-ext proteins coupled to aminooxy-biotin molecule were analyzed on non-reduced SDS- PAGE (Figure 5A and B). Various amounts of biotin conjugated protein samples with and without thrombin treatment were run on non-reduced SDS-PAGE gel and transferred to a membrane. The biotin conjugated proteins were detected by streptavidin- HRP and chemiluminescence substrate. Only samples that were not treated with thrombin showed a chemiluminescence band at the expected molecular weight, indicating that these proteins could also be site-specifically biotinylated in the C-terminal extension peptide. HAMLET preparation and characterization HAMLET has been traditionally prepared by passing the apoprotein through an OA-conditioned anion exchange chro- matographic column [16]. Recently, Kamijima et al. have used a method for preparing HAMLET that involves mixing and heating the OA with the native protein [30] and confirmed by others [31]. We followed the heating method for the complexation with OA but used the Ca2+-depleted hLA (apoprotein) instead of Ca2+- bound hLA (holo form) during heating for 10 min at 60oC. Protein was estimated by the Bradford method, and fatty acid content was determined using liquid chromatography coupled to a mass spectrometer (Figure 6). Two forms were tested: the molten globule hLA (mg-hLA) and the aD-hLA complexed with OA. The protein to OA molar ratios on various samples were determined by LC-MS studies and listed in Table 1. Previously, Petterson- Kastberg et al. [32] investigated the ratio of protein to OA in HAMLET, and, although the method for preparing HAMLET was the conditioned column, their ratio seems to agree with the results reported here. Using 1H NMR spectroscopy, a-LA to OA ratio of 1:5.4 was observed in HAMLET, and for rhLAall-Ala to OA, ratios were 1:7.3 (using chemical analysis) and 1:9.5 (using 1H NMR spectroscopy) [32]. In our current study all the protein-OA complexes are made with 1:10 ratio and the protein to OA ratio determined by the LC-MS method is comparable to the values PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26093