Evaluation of the Sensitivity and Reproducibility of Targeted Proteomic Analysis Using Data-Independent Acquisition for Serum and Cerebrospinal Fluid Proteins.

There is a need for targeted analysis of biological fluids for diagnosis, prognosis, or monitoring the progression of diseases. Cerebrospinal fluid (CSF) and serum have been widely used for the development of protein analysis for neurodegenerative diseases and other diseases, respectively. Recently, data-independent acquisition (DIA) mass spectrometry (MS) has been developed to increase the throughput over data-dependent acquisition (DDA) on screening of a large number of samples and discovery of candidate targets. When it comes to target validation, the analytical performance of targeted analysis is critical. However, the inter- and intralaboratory analytical performances of the DIA-MS for targeted proteomic analysis of CSF and serum samples have not yet been investigated. In this study, we showed that the DIA-MS approach allowed us to identify and quantify 1732 CSF and 424 serum proteins, with 90% of proteins identified and quantified in at least 50% of DIA-MS runs. To evaluate the sensitivity, linearity, and dynamic range of the DIA approach, we included the stable isotope-labeled (SI) peptides into CSF and serum samples with serial dilutions. The lower limit of quantification (LLOQ) of peptides was 0.1-0.5 fmol, and the dynamic range was over 3.53 orders of magnitude, with excellent linearity (r2 < 0.978) in CSF and serum samples. Finally, the reproducibility of the DIA-MS approach was evaluated using entire proteins identified in CSF and serum samples. The intralaboratory three replicate results showed reliable reproducibility with 12.5 and 17.3% of the median coefficient of variation (CV) in both CSF and serum matrices, whereas the median CVs of interlaboratory three replicates were 23.8 and 32.0% in CSF and serum samples, respectively. The comparison of the quantitative result between replicates showed close similarity at intra- and interlaboratories with a median Pearson correlation value of >0.98 in CSF and serum, respectively. In conclusion, we demonstrate the capability of the DIA approach as a targeted proteomic analysis for candidate proteins from CSF and serum samples.