br The identity of RP MAP was confirmed by
The identity of RP8-MAP4 was confirmed by its molecular ion peak in its mass spectrum (Fig. S1). The [Mþ6H]6þ, [Mþ7H]7þ, [Mþ8H]8þ and [Mþ9H]9þ ions were indicative of its presence. The HPLC chromatogram indicated its purity was ~95.5% (Fig. S2). The result demonstrated that the retention time of tryptic peptide product was identical to that of reporter peptide (Fig. S2). The
Fig. 2. Schematic representation of (A) the structure of the peptide dendrimer RP8-MAP4 and (B) Michael addition reaction used to prepare the DPD probe.
Fig. 3. (A) Product ion spectrum of the reporter peptide AVLGDPFR. Identified ions are labeled. Immo refers to immonium ion that is produced in the secondary fragmentation by a combination of a type and y type cleavages of peptide backbone. (B) LC-MS/MS chromatogram of the reporter peptide AVLGDPFR. The MRM transitions were m/z 437.8 / m/z 72.0, m/z 437.8 / m/z 171.1 and m/z 437.8 / m/z 419.2.
extent of trypsin AngiotensinI was examined by LC-MS/MS. Its diges-tion efficiency was worked out from the response ratios of the tryptic peptide after digestion and 8 fold equimolar synthetic standard reporter peptide (Fig. S3). The estimated value was 99.1%, supporting the feasibility of embedding two reporter peptides in one substrate. Additionally, the other digestion product (ADTG)4K2KK-Male was also observed (Fig. S4). Notably, enzyme:-protein ratio of 1:100 to 1:20 (w/w) is normally recommended for trypsin digestion based on the frequency of lysine and arginine residues in proteins . At present, there is no recommendation available for peptide dendrimers. Thus, different ratios of tryp-sin:RP8-MAP4 ranging from 1:20 to 2:1 were evaluated here to optimize the trypsin concentration. As show in Fig. S5, the amount of the peptide product reached the plateau at the ratio of 1:5.
On the other end of RP8-MAP4, the complementary DNA sequence was conjugated to the dendrimer via Michael addition reaction (Fig. 2B). Specifically, the SH functional group of DNA reacted with the maleimide group of the “first” lysine. The final product RP8-MAP4-DNA was separated and detected by size exclusion liquid chromatography . As shown in Fig. 4A, the peaks of DNA and RP8-MAP4-DNA were partially overlapped. Simple change of mobile phase or optimization of HPLC gradient could not easily achieve the completeness of separation. Fortu-nately, these peaks were well resolved from that of RP8-MAP4. Thus, we used 40 times molar excess of RP8-MAP4 over DNA to shift the reaction equilibrium to the right side . To further confirm the depletion of DNA, HPLC analysis was performed and the result showed that almost no DNA was retained after the conjugation (Fig. S6). The product was further confirmed by agarose gel electrophoresis (Fig. S7). Finally, RP8-MAP4-DNA was collected
and condensed with high purity for the following miRNA quanti-fication as a DPD probe (Fig. 4B).
3.2. Characterization of DNA-Peptide dendrimer probe
The obtained RP8-MAP4-DNA was analyzed using a LCQ Deca XP Plus ion trap MS to provide definitive evidence for its existence. The signal at 18982.2 Da was consistent with the theoretical mass value of RP8-MAP4-DNA (Fig. 4C). In addition, the conjugated DNA did not have impact on the digestion efficiency. By adding trypsin to equal amounts of RP8-MAP4-DNA and RP8-MAP4, there was no significant difference in the released amount of reporter peptide (Data not shown).
For miRNA detection probe, its hybridization efficiency and specificity with target miRNA are critical factors contributing to its performance. In this study, the hybridization efficiency of RP8-MAP4-DNA was calculated by comparing the amount of hybrid-ized RP8-MAP4-DNA to the amount of immobilized target miR-21. In addition, factors including ionic strength, temperature and time were optimized to maximize the hybridization efficiency. As a result, a buffer containing 10 mM Tris, 100 mM KCl, 1 mM MgCl2, pH 7.4 was selected. Hybridization temperature of 45 C and hybridi-zation time of 4 h provided the highest efficiency (Fig. S8). Under these conditions, hybridization efficiency can achieve ~92.1%.
Probe specificity was also evaluated, due to the high degree of similarity between miRNA sequences . Some miRNA family members vary by a single nucleotide. Thus, single or double mis-matched miRNAs were employed in parallel with miR-21. Relative hybridization efficiency was worked out by comparing the reporter peptide amounts of perfectly matched and mismatched targets. The
Fig. 4. Size exclusion chromatograms of (A) RP8-MAP4, DNA and their products after conjugation and (B) purified RP8-MAP4-DNA at l ¼ 220 nm. The mobile phase consisted of 150 mM phosphate buffer containing 2 M sodium chloride (pH ¼ 7.0). The conditions of size exclusion chromatography were described in supplementary material. (C) Full scan spectrum of the DPD probe RP8-MAP4-DNA. Mass spectrometry detection was performed on a Thermo LCQ Deca XP Plus ion trap MS, which was operated with electrospray ionization in the positive mode.