Comparison of Internal Standard Approaches for SRM Analysis of Alpha-Synuclein in Cerebrospinal Fluid

Abstract

Absolute protein quantification by selected reaction monitoring (SRM, also MRM) is an alternative to immunoassays, and the gold standard here is the addition of stable-isotope labeled (SIL) proteins (PSAQ). Cerebrospinal fluid (CSF) is the preferred source of biomarkers for neurological diseases, and recent improvements in mass spectrometry enable the quantification of disease-relevant proteins in CSF. We used alpha-synuclein SRM to investigate alternatives to the PSAQ approach in human CSF regarding precision and accuracy, including SIL peptides, winged SIL (WiSIL) peptides, and quantitative protein epitope signature tags (QPrESTs). All approaches yielded precise results in CSF with CV values <15% in several runs for all four measured peptides. PSAQ and QPrEST also showed good accuracy (deviation ≤15%), whereas SIL and WiSIL peptides yielded deviations up to 54% that greatly depended on the measured peptide. Total protein concentration in CSF did not affect precision and accuracy. Thus, our study indicates that all four approaches are suitable for relative quantification of alpha-synuclein in CSF. QPrESTs are a valuable alternative to PSAQ in terms of precision and accuracy, although SIL and WiSIL peptides can yield accurate results as well when peptides are selected consciously.

Instant on-paper protein digestion during blood spot sampling

“…Both wSIL and PSAQ could correct for the entire sample preparation including enzymatic treatment. However, in a recent study by Oeckl et al 75 , no significant differences between the different IS approaches were found under controlled conditions. Others have demonstrated that PSAQ to have advantages 76,77 .…”

Diagnostic and Prognostic Performance of Neurofilaments in ALS

“…For implementation in the routine clinical practice, assay standardization, and characterization, and independent validation of the cut-offs are required. Indeed, the development of reference methods for NF measurements, e.g., by means of mass spectrometry (56, 57), and of certified reference materials for traceability of the calibrators and to demonstrate commutability among the different assays should be encouraged (58). Independent evaluation of the performance characteristics of the NF assays enables the public availability of data on the analytical quality of the different commercially available assays.…”

Proteomics and Metabolomics Approaches towards a Functional Insight onto AUTISM Spectrum Disorders: Phenotype Stratification and Biomarker Discovery

“…Given the ascertained performance of MS in the absolute quantification of proteins, as a strong alternative to immunoassays, the principal needs currently appear to be focused on the calibration methods, not only to improve the measurement precision but also to make data interchangeable by laboratories globally. Synthetic stable isotope labelled (SIL) proteins and peptides, used as internal standards (IS), present both advantages and drawbacks, due to availability, cost, reproducibility of biological features, and accuracy [ 98 ]. An open question is how recombinant SIL proteins, synthetic labelled tryptic peptides (tSIL), or even the so-called “flanked” or “winged” peptides, could be used with reasonable confidence to provide the best accuracy as an internal calibration (IC) in bottom-up experiments [ 99 ].…”