Development of a novel nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based method for quantification of angiotensin peptides in HUVEC cultures

Background This study aimed to develop an analytical method using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the determination of angiotensin (Ang) I, Ang (1-9), Ang II, Ang (1-7), Ang (1-5), Ang III, Ang IV in human umbilical vein endothelial cell (HUVEC) culture supernatant. Methods HUVEC culture supernatant was added with gradient concentrations (0.05–1,000 ng/ml) of standard solutions of the Ang peptides. These samples underwent C18 solid-phase extraction and separation using a preconcentration nano-liquid chromatography mass spectrometry system. The target peptides were detected by a Q Exactive quadrupole orbitrap high-resolution mass spectrometer in the parallel reaction monitoring mode. Ang converting enzyme (ACE) in HUVECs was silenced to examine Ang I metabolism. Results The limit of detection was 0.1 pg for Ang II and Ang III, and 0.5 pg for Ang (1-9), Ang (1-7), and Ang (1-5). The linear detection range was 0.1–2,000 pg (0.05–1,000 ng/ml) for Ang II and Ang III, and 0.5–2,000 pg (0.25–1,000 ng/ml) for Ang (1-9) and Ang (1-5). Intra-day and inter-day precisions (relative standard deviation) were <10%. Ang II, Ang III, Ang IV, and Ang (1-5) were positively correlated with ACE expression by HUVECs, while Ang I, Ang (1-7), and Ang (1-9) were negatively correlated. Conclusion The nanoflow liquid chromatography-parallel reaction monitoring mass spectrometry-based methodology established in this study can evaluate the Ang peptides simultaneously in HUVEC culture supernatant.

2 was controlled by the Xcalibur software (v. 3.1) and the analysis software for mass spectrometry data was Skyline (v.4.1.0,MacCoss Lab,University of Washington,USA).

Construction of the HUVEC model cell lines with silenced ACE expression
Two shRNA sequences targeting ACE mRNA were designed based on the upstream and downstream sequences of the ACE gene in Genbank (number: NM_001178057.1→NP_001171528.1). A non-human homologous shRNA was used as the negative control. Homologous analysis was performed on the target sequence by BLAST to exclude the shRNA non-specific sequences. Ultimately, the shRNA sequences were determined. The 5' end of the coding strand of the above sequence was introduced into the AgeI cleavage site.The EcoRI cleavage site was introduced into the 5' end of the template strand. The sequence of the stem loop was 5'-CTCGAG-3'. The ACE targeted gene, shRNA sequences of the negative control gene, and the control sequences are shown in Supplementary Table S1. The above three ACE-shRNA sequences were ligated to the pLKO.1 vector plasmid to form a recombinant plasmid. The DH5α competence was used for the preparation of a large quantity of plasmids.
The 293FT cells were seeded in a 6-well plate and cultured at 37°C in 5% CO2 to a confluence of 70%-80%. The transfection mixture was prepared and divided into an interference group and a control group. The pLKO.1 recombinant plasmids containing the target sequence (1.6 μl), psPAX2 (0.8 μl), and pCMV-VSV-G (1.2 μl) were mixed with Lipofectamine 3000 at a ratio of 1:1. The mixed solution was added to the 6-well 3 plate and the virus supernatant was collected after 48 h.
The HUVECs were divided into three groups. One control group, named the pLKO.1 group, was transfected with the lentivirus packaged with the pLKO.1 plasmid of the negative control sequence. The other two groups were the interference groups, and the lentiviruses packaged with the ACE-shRNA-1 and ACE-shRNA-2 interference sequence plasmids were transfected, respectively. The cellular RNA was extracted after puromycin screening to detect the ACE mRNA levels.

Detection of the Ang peptides from HUVECs
For the detection of Ang I and its metabolites, three groups of cells were prepared in T25 flasks and passaged in 6-well plates. Three duplicate wells were set for each group. When the cells were grown to 70% confluence, the ECM-prf culture medium containing 1% ECGS was added to each well and cultured at 37°C, 5% CO2 for 24 h.
Then, Ang I at a final concentration of 1 μM was added. Culture was continued for 1 h and 100 μl of supernatant were extracted and added into an ice-pre-cooled low protein attachment LoBind centrifuge tube (1.5 ml, Eppendorf, Hamburg, Germany).
The samples were mixed and centrifuged at 4°C, 2000 ×g, for 4 min. The samples were processed LC-MS/MS detection.
For the detection of the ACE protein, the 6-well plates were washed with pre-cooled PBS once per well and 100 μl of RIPA buffer (containing 1% PMSF) was 4 added. After complete lysis, the samples were centrifuged at low temperature and the supernatant was extracted for BCA protein concentration assay. After mixing and boiling with the loading buffer, the protein sample was subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with TBST with 5% nonfat milk for 1 h. The ACE rabbit anti-human monoclonal antibody (1:1000, Abcam, Cambridge, United Kingdom) and β-actin rabbit anti-human monoclonal antibody (1:1000, Cell Signaling, Danvers, MA, USA) were added in the TBST with 5% nonfat milk and incubated at 4°C overnight.
The membranes were washed with TBST three times, 15 min each time. The horseradish peroxidase labeled mouse anti-rabbit secondary antibody (1:10,000, Jackson Immuno Research, West Grove, PA, USA) was incubated at room temperature for 2 h and washed with TBST three times, 15 min each time. The membranes were exposed in the darkroom. The Image J software was used to analyze the area and gray scale of the bands.

QRT-PCR detection of the ACE mRNA levels
The RNA extracted from each HUVEC model group was reversely transcribed to cDNA and detected using a real-time quantitative STRATAGENE Mx3000p PCR instrument. The primer sequences are shown in Supplementary Table S2. The 18s rRNA was used as the normalized internal standard and the 2 -ΔΔCT method was used to calculate the relative expression of ACE mRNA (Supplementary Table S3