Abstract
Purpose :
Proteases are essential for many biological pathways and their activity must be controlled to prevent and aberrant proteolysis, which underscores the onset of several human disorders, including eye diseases. Thus, profiling the endogenous protein fragments of a biological sample can help uncovering pathological proteolytic events.
N-terminomics is a proteomics workflow that applies to the identification of endogenous N-terminal ends (N-tails) of peptides after their chemical modification before trypsin digestion and mass spectrometry (MS) analysis. The distribution of N-terminal residues then helps predicting proteases whose activity goes dysregulated.
Here, we have developed a workflow to explore the “N-terminome" of human vitreous humor (VH) for studying proteolytic events of retinal diseases.
Methods :
VH (n=2) was collected from subjects diagnosed with macular pucker during surgery. All procedures were approved by the local ethic committee and done respecting the Helsinki declaration. Samples were dried, resuspended in denaturing buffer (6M guanidine-HCl) and normalized after BCA assay. Indeed, 500 μg of total proteins/sample were either digested with trypsin or labeled with Tandem Mass Tag (TMT) pro-0 to chemically block the peptides N-termini before trypsin digestion. Both samples were run on a UHPLC-MS Orbitrap, analyzed by FragPipe (searching against a FASTA human database) and processed by R.
Results :
The TMT-labelled and tryptic sample performed similarly for global protein identification and quantification (>500 proteins/single injection). Labelling efficiency of lysines was optimal as well as missed cleavages in TMT-labelled samples.
Notably, >150 TMT-labelled N-tails peptides were identified and many of them originated from structural proteins and adhesion molecules (e.g., vitronectin, collagen, dystroglycan, etc.).
Aminoacids distribution indicated a net prevalence of hydrophobic (P, L) or polar (G, S) residues in P1 and P1’ position.
Conclusions :
N-terminomics can be applied to studying endogenous proteolytic events in VH samples and is currently being used to profiling the N-terminome of subjects diagnosed with retinal detachment (vs pucker). This technique is expected to uncover proteolytic events of pathological relevance in retinal diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.