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  • br Another concern is the difficulties involved


    Another concern is the difficulties involved in the preparation of peptide dendrimers, and high generation dendrimers in particular, while higher generation dendrimers are supposed to have greater signal amplification [25]. There is evidence indicating that the dendrimer yield normally dropped off with the increase of gener-ation, probably due to the large number of reactive sites and the challenge of purification [31]. As the compromise between probe size and amplification effect, MAPs with 4 amino terminals were proposed and four substrate peptides (AVLGDPFRAVLGDPFRADTG) containing two reporter peptides each were ligated to the MAPs to form (AVLGDPFRAVLGDPFRADTG)4K2KK-Male (RP8-MAP4). The C-terminal of RP8-MAP4 was maleimidohexanoic acid-modified for ligation of DNA via Michael addition reaction between maleimide and thiol groups (Fig. 2B). Notably, the substrate peptides in RP8-MAP4 might be partially resistant to enzymatic hydrolysis by blocking access of trypsin to possible cleavage sites [32]. Thus, a four-amino-acid peptide (ADTG) at the carboxyl terminus of the substrate peptide was employed as a spacer to circumvent potential steric hindrance from dendrimer scaffold in trypsin cleavage.
    The identity of RP8-MAP4 was confirmed by its molecular Poly-L-lysine 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 digestion 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 [33]. 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 [34]. 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 [35]. 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