Mass Spectrometry-Based Quantification of Tau in Human Cerebrospinal Fluid Using a Complementary Tryptic Peptide Standard

Here, we report a method for the
generation of complementary tryptic
(CompTryp) isotope-labeled peptide standards for the relative and
absolute quantification of proteins by mass spectrometry (MS). These
standards can be digested in parallel with either trypsin (Tryp-C)
or trypsin-N (Tryp-N), to generate peptides that significantly overlap
in primary sequence having C- and N-terminal arginine and lysine residues,
respectively. As a proof of concept, an isotope-labeled CompTryp standard
was synthesized for Tau, a well-established biomarker in Alzheimer’s
disease (AD), which included both N- and C-terminal heavy isotope-labeled
(15N and 13C) arginine residues and flanking
amino acid sequences to monitor proteolytic digestion. Despite having
the exact same mass, the N- and C-terminal heavy Tau peptides are
distinguishable by retention time and MS/MS fragmentation profiles.
The isotope-labeled Tau CompTryp standard was added to human cerebrospinal
fluid (CSF) followed by parallel digestion with Tryp-N and Tryp-C.
The native and isotope-labeled peptide pairs were quantified by parallel
reaction monitoring (PRM) in a single assay. Notably, both tryptic
peptides were effective at quantifying Tau in human CSF, and both
showed a significant difference in CSF Tau levels between AD and controls.
Treating these CompTryp Tau peptide measurements as independent replicates
also improved the coefficient of variation and correlation with Tau
immunoassays. More broadly, we propose that CompTryp standards can
be generated for any protein of interest, providing an efficient method
to improve the robustness and reproducibility for MS analysis of clinical
and research samples.