br another injection The sample injection volume was set
another injection. The sample injection volume was set at 5 mL. Mass parameters for the analysis were the following: ESI des- olvation temperature: 350 C, desolvation gas flow: 10 L min 1,
capillary voltage: 4 kV, Q1 and Q3: unit resolution; nebulizer pressure: 35 psi. The Mass was operated in the positive MRM mode. An Agilent Mass Hunter Workstation Software (version B.06.00) was used for data collection and processing.
3. Results and discussion
3.1. Preparation of DNA-Peptide dendrimer probe
In this study, the DPD probe contains reporter peptide for signal detection, branched dendrimer scaffold for signal amplification and a piece of DNA sequence for target miRNA recognition. To obtain the probe product with good performance and high quality, several aspects deserve careful thought and consideration.
Classical dendrimers are normally formed by chemical polymers such as polyamidoamine (PAMAM), polypropyleneimine (PPI) and polyglycerol (PG) . Different from these dendrimers, peptide dendrimers contain peptide bonds. The most known peptide den-drimers are multiple antigen peptides (MAPs) . Their branches consist of amino acids, and peptides can be easily bound to the surface amino groups via peptide bond . In most cases, MAPs are based upon a polylysine skeleton constructed by layers of lysine residues with an increase in the number of Lys branching (i.e., one, two, four and etc., also termed as generations). The alkylamino side-chain of lysine is a good branching point for introduction of functional Geneticin . Another feature of MAPs is their ‘‘wedge-like shape’’ without a core and growth in one direction, which fa-cilitates exposure of the “first” lysine residue and its conjugation with other functional groups . In this study, we attached the substrate peptides at the branches of MAPs and conjugated the complementary DNA at the other end (Fig. 2A).
Peptide dendrimer containing substrate peptides was synthe-sized stepwise on solid phase . For selection of substrate/re-porter peptide, the criteria have been extensively described. Briefly, reporter peptide should offer a sufficient response, can liberate efficiently after digestion, and can generate high-quality multiple reaction monitoring (MRM) . Besides, the sequence of reporter peptide should not be identical with any proteins to exclude protein interference in the analysis. Following these criteria, several pairs of substrate and reporter peptides have been developed in our lab, such as GDKAVLGVDPFR/AVLGVDPFR [24,30]. However, these peptides may not be appropriate for this work due to the presence
of peptide dendrimer scaffold and the subsequent DNA conjugation reaction. There are two major concerns. First, the anchored pep-tides should avoid containing lysine because of the same amino acid residues residing on the dendrimer surface. This extra lysine may lead to byproduct formation in the process of peptide den-drimer decoration. Specifically, the peptide may ligate to each other via its lysine residue. Thus, an alternative reporter peptide AVLGDPFR containing arginine instead of lysine was selected as the reporter peptide. Its doubly-charged ion at m/z 437.8 gives the maximum mass response in the scan mode. As shown in Fig. 3A, the corresponding sequence-specific b ions and y ions were identified in its product ion spectrum. The stable isotope-labeled peptide replaced with that same residue was synthesized to serve as an internal standard. Specifically, the isotopically labeled [D8] Val (V*) was used to replace the nature Val at position 2 to form AV*LGDPFR, which added 8 Da molecular mass compared to the nature peptide and the final mass was 881.5 Da. Finally, the MRM transitions for reporter peptide 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 and were used for quantification in the following analysis (Fig. 3B).
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 . 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 . 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 . 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.