Targeted peptide quantitation by mass spectrometry is a rapidly emerging field. Traditionally it relied on the development and validation of multiple reaction monitoring assays that could comply with a high level of sensitivity, specificity, accuracy and reproducibility in complex biological samples. However, with the introduction of high‐resolution mass spectrometers, other acquisition modes could provide more comprehensive datasets for identification and quantification but also for in‐depth data mining. The objective of this study was to evaluate two analytical approaches, parallel‐reaction monitoring (PRM) and data‐independent analysis (DIA) using a hybrid Quadrupole–Orbitrap mass spectrometer for the quantification of neuropeptides in animal spinal cord tissues. Mouse spinal cord tissues were harvested, homogenized and neuropeptides extracted using a C18 solid‐phase extraction protocol. Chromatography was achieved using a Thermo Biobasic C8 100 × 1 mm (5 μm) column. The initial mobile phase conditions consisted of acetonitrile and water (both containing 0.1% of formic acid) at a ratio of 5:95. An 11 min linear gradient was applied up to a ratio of 50:50 and maintained for 3 min. The flow rate was fixed at 75 μL/min and 2 μL of sample was injected. Mass spectrometry analyses were performed using a Thermo Q Exactive Plus MS using PRM and DIA approaches. Quantitative data using an isotopic dilution and a label‐free strategy were obtained for both methods and statistically compared. Using both approaches, we were able to clearly detect endogenous neuropeptides. However, with DIA, mass spectra alone could not distinguish Leu‐Enk and Met‐Enk. We used a Bland–Altman plot (Difference plot) to analyze the agreement between both approaches and no systematic bias was observed. Further statistical analyses, including variance analysis, showed more variability in DIA compared with PRM mode. Further analyses were performed using a label‐free approach and confirmed an increase of the variance using a DIA approach.

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