Uncovering Effects of Ex Vivo Protease Activity during Proteomics and Peptidomics Sample Extraction in Rat Brain Tissue by Oxygen-18 Labeling

In
biological samples, proteins and peptides are altered by proteolytic
activity. The actual ex vivo form of the peptidome or proteome analyzed,
therefore, does not always reflect the natural in vivo state. Sample
stabilization and sample treatment are thereby decisive for how far
these two states diverge. To assess ex vivo formation of peptides,
we used enzymatic incorporation of oxygen-18 water during proteolysis
(PALeO approach) to label ex-vivo-formed peptides in rodent brain
tissue. Rates of ex-vivo-formed peptides were determined in 25 samples
that were stabilized and treated by six different protocols, whereby
samples were subjected to different conditions such as temperature,
urea concentration, and duration of treatment. Samples were measured
by nano LC-Orbitrap-MS, and incorporation of oxygen-18 was determined
by MS/MS database search and analysis of the precursor isotope pattern.
Extent of ex vivo degradations was affected relevantly by the sample
treatment protocol applied and stopped almost completely by heat stabilization.
Determination of the formation state by oxygen-18 incorporation by
MS/MS database search correlated well to more elaborate analysis of
the MS isotope pattern. Overall, oxygen-18 labeling in combination
with shotgun data-acquisition and MS/MS database search offers an
adjuvant and easily applicable tool to monitor sample quality and
fidelity in peptide and neuropeptide sample preparations.