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

Figures

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  Figure 1. Optimization of the LCMS/MS parameters. Chromatogram of the seven target peptides and internal standard peptide by LC-MS/MS. The seven target peptides and the internal standard peptide could complete the column elution with acetonitrile concentrations of 10%–16%. In order to separate the elution peaks and not affect the peak shape, the separation stages were set using a gradient from 4% B at 2 min to 22% B at 47 min. It can be seen that the peaks of each target peptide and internal standard peptide were smooth and effectively separated.

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  Figure 2. Establishment of the PRM detection model. The ion dwell time for Ang I, Ang (1-9), Ang II, and Ang (1-7) was adjusted according to the number of precursor ions. Using the peptide map library pre-built using Skyline, the fragment ions in the PRM scan data were extracted. The fracture law and abundance of fragment ions were matched with the spectrum library. The mass spectral response was up to 106 orders of magnitude. The signal-to-noise ratio was high, and the mass spectral peak shape was smooth.

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  Figure 3. Determination of the sample stability over time. Stability (% of changes in concentration) of the seven angiotensin peptides kept in the injector at 7 °C over 8 h.

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  Figure 4. Determination of the number of sample loading on Ang detection. Evaluation of the number of sample passages through the reverse C18 solid-phase extraction column packing on the recovery rate of angiotensin peptides. We used three loadings on the cartridge as control and evaluated the other numbers of loading times on the recovery rate of Ang by comparing the peak area ratio of the same target peptide quantitative fragment ion. The control group used three passages.

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  Figure 5. Determination of the impact of ultrafiltration on sample recovery. Recovery rate of angiotensin peptides after ultrafiltration and solid-phase extraction compared with solid-phase extraction alone. **P < 0.01. ***P < 0.001.

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  Figure 6. ACE protein expression was successfully silenced in HUVECs. Western blotting showing that the expression of the ACE protein in the ACE-shRNA-1 and ACE-shRNA-2 groups was significantly reduced (95.6% and 78.2%, respectively) compared with the PLKO.1 group. **P < 0.01. ***P < 0.001.

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  Figure 7. ACE silencing induces differences in the Ang metabolic profile that can be detected by LC-MS/MS. To verify the practicability of the Ang assay established in this study, Ang I was added at a final concentration of 1 µM to three groups of HUVECs displaying different levels of ACE expression. After incubation for 1 h, Ang I and its major metabolites (Ang II, Ang (1-7) and Ang (1-5), Ang (1-9), Ang III and Ang IV) were detected by the nano-LC-MS/MS method established in this study. *P < 0.05. **P < 0.01. *** P < 0.001.