Supplementary Materials Supporting Information supp_294_34_12567__index. can be and studies in rats. Results ProANP, proBNP, and proCNP are O-glycoproteins To explore cytosolic and secretory granular proteins) from different tissues (neonatal porcine atria, adult porcine ventricle, and human prostate cancer tissue) known to express NPs at high levels. We used a sequential enrichment strategy for (Fig. S1 and Table S1). We identified both core 1 and monosialylated core 1 structures (Table S1), suggesting that the neuraminidase pretreatment was partially incomplete. Open in a separate window Figure 1. Summary of identified of NPs with previously reported with with indicate corin or furin cleavage sites. A conserved serine in the mature NPs is in and with an and marked with a on BNP and CNP. The six amino acids upstream of the proprotein convertase activation sites are shown with previously identified in shows conservation among Rhod-2 AM all NPs in five species. The show the conservation tracks for each NP as indicated. The conserved serine in the disulfide ring of ANP is in and with an and and Fig. S2 (and and and and glycosylation assays as described previously (30). Natriuretic peptides share a characteristic disulfide bridge in the C-terminal region (Cys105CCys121 in proANP). We used both 20-mer partly overlapping synthetic peptides covering all potential Ser/Thr acceptor residues in proANP (peptides 1C9), mature ANP Rhod-2 AM with the disulfide bridge (peptide 10) (Fig. S3or the respective bands. with with proteolysis time-course assay monitoring cleavage of peptides and glycopeptides by semi-quantitative MALDI-TOF MS analysis (38). We found that neprilysin degraded ANP completely within 15 min, whereas ANP with the simplest GalNAc (Tn) monosaccharide and cleavage analysis of equal amounts of ANP and of each (Fig. 1core 1 synthase (and core 1 synthase (summary of peptides and glycopeptides used in receptor activation assays. All peptides were synthesized with a disulfide bridge. and are directly comparable, since the experiment was performed simultaneously on the same plates. The same ANP and CNP data are shown in both and for purposes of clarity and ease of comparison. and bioavailability and activity of ANP glycoforms in rats. We measured mean arterial pressure (MAP), renal actions, and peptide hormone changes in normal rats with 60-min intravenous infusion of artificial ANP, ST-ANPS117, or ST-ANPS123, accompanied by 30-min post-infusion clearance. General, ANP induced powerful blood circulation pressure (BP)-reducing effect weighed against ST-ANPS117 or ST-ANPS123 during infusion (Fig. 5and Rhod-2 AM and (Fig. S5, and activities of 0.01 ST-ANPS117; #, 0.05 ST-ANPS123; $, 0.05 ST-ANPS117; ***, 0.0001 all groups; **, 0.001 all groups (two-way ANOVA, Tukey’s post hoc tests). proteolysis assays with neprilysin and insulin-degrading enzyme is in agreement with this. We further found that the proximity of the studies with both plasma and urine. The position and size of the for 30 min. Subsequently, the supernatant was lyophilized and reconstituted in water. An aliquot corresponding to 3 mg of protein for the heart samples and 5 mg of protein for the prostate sample was adjusted to 50 mm ammonium bicarbonate, reduced by 5 mm DTT (30 min, 60 C), and alkylated by adding 10 mm iodoacetamide (30 min, room heat (RT)). The sample was then digested with 50 g of trypsin (Roche Applied Science) (37 C, overnight), purified on C18 Sep-Pak (Waters), and desialylated with 150 models of neuraminidase (P0720, New England Biolabs) in 50 mm sodium citrate (pH 6.0) (37 C for 2 h). The digest was Sep-PakCpurified, lyophilized, and resuspended in 2 ml of PNA-binding buffer (10 mm HEPES (pH 7.4), 150 mm NaCl, 1 mm CaCl2, 0.1 mm MgCl2, MnCl2, and ZnCl2) and injected to a pre-equilibrated 2.6-m-long PNA lectin-agarose Rhod-2 AM column (Vector Laboratories). The flow was set to 100 l min?1, and 1-ml fractions were collected. The sample was eluted with 3 1 column volume of 0.5 m galactose, 1 m galactose, and 1 m galactose, pH 3, respectively. All samples were desalted on homemade StageTips (Empore disk-C18, 3 m) (63, 64). The acidified samples were loaded around the activated StageTips; washed with 0.1% formic acid; eluted with 50% methanol, 0.1% Rabbit Polyclonal to PML formic acid; lyophilized; and finally dissolved in 0.1% formic acid. Samples were analyzed on an EASY-nLC 1000.