(M1230-01-01) Determination of One Significant Protein in Human Cerebrospinal Fluid (CSF) by LC-MS/MS

Purpose: One significant protein (MW~300KD) is a unique biomarker recognized as a cause of inherited Parkinson’s disease. Accurately and precisely quantitating this protein is crucial for developing targeted therapies to improve patient outcomes.

Methods: To analyze this significant protein in human cerebrospinal fluid (CSF), trypsin was used as proteolytic digestion enzyme. Recombinant commercial protein (ThermoFisher) was used to prepare standards and QC samples. Due to high endogenous levels of this significant protein in normal human CSF, artificial CSF fortified with BSA was used as a surrogate matrix. Digested specific peptides and the internal standard were extracted using Stable Isotope Standards and Capture by Anti-Peptide Antibodies method (SISCAPA) and analyzed by an Orbitrap Exploris 240 mass spectrometer, equipped with an Easy-Spray PepMap™ Neo UHPLC C18 columns. To separate these challenging peptides, these Neo UHPLC columns were chosen to feature easy connectivity, high reproducibility and excellent separation with 1500 bar pressure capability. Biotinylated antibodies mounting to Dynabeads™ M-280 Streptavidin showed highest sensitivity.

Results: Maximum separation efficiency was successfully achieved with outstanding MS sensitivity (~2 pg/mL) and sharp peaks. This innovative analytical approach was qualified and confirmed to be accurate and precise. The range of quantitation (2.00 pg/mL to 160 pg/mL) was successfully established for this significant protein based on the analysis of 1.00 mL of human cerebrospinal fluid sample.

Conclusion: The combination of SISICAPA-MS effectively bridges the gap between conventional immunoassays and mass spectrometry-based proteomics by leveraging the strengths of both technologies. It is an important trend in quantitative proteomics, particularly for isolating and measuring specific proteins/peptides with high sensitivity and specificity in biological fluids.

Acknowledgements: acknowledgement to Fan, Leimin



Examples of Chromatogram

Accuracy and Precision in two Batches