In-capillary enzymatic digestion beyond trypsin for the sensitive targeted bottom-up analysis of protein biomarkers by capillary electrophoresis-mass spectrometry.

BACKGROUND

Targeted bottom-up proteomics is of great interest for the straightforward, accurate, and sensitive measurement of specific protein biomarkers from surrogate peptide fragments. However, this approach typically relies on off-line enzymatic digestion with trypsin, a time-consuming step that may be inadequate for covering certain sequence regions containing important post-translational modifications (PTMs).

RESULTS

In this study, we present an in-line enzymatic digestion strategy for the targeted bottom-up analysis of α-synuclein (α-syn), which is a protein biomarker of Parkinson's disease (PD). The method is based on in-capillary enzymatic digestion capillary electrophoresis-mass spectrometry (ICD-CE-MS) with endoproteinase GluC as an alternative to trypsin, which fails to map Ser-129, a phosphorylated site in PD. The method was optimized and validated using a recombinant α-syn standard, focusing on the detection of potentially phosphorylated peptides. Under optimized conditions, the method was repeatable at 100 μg mL-1 of digested protein, yielding percent relative standard deviation (%RSD) values of 3.4 % for peak area and 7.2 % for migration time (n = 5) for the (124-131) α-syn peptide. Linearity between 25 and 200 μg mL-1 (R2 > 0.995) was satisfactory, with a limit of detection of 15 μg mL-1 and a protein sequence coverage of 77.1 %. The method was successfully applied to analyze endogenous α-syn in human blood.

SIGNIFICANCE: ICD-CE-MS with GluC extends the scope of targeted bottom-up proteomics beyond trypsin-based workflows, offering a straightforward and fully integrated platform for the sensitive targeted bottom-up analysis of challenging protein biomarkers in biological fluids. This strategy can also be applied to the analysis of protein biopharmaceuticals.