Targeted degradomics in protein terminomics and protease substrate discovery

Abstract

Targeted degradomics integrates positional information into mass spectrometry (MS)-based targeted proteomics workflows and thereby enables analysis of proteolytic cleavage events with unprecedented specificity and sensitivity. Rapid progress in the establishment of protease-substrate relations provides extensive degradomics target lists that now can be tested with help of selected and parallel reaction monitoring (S/PRM) in complex biological systems, where proteases act in physiological environments. In this minireview, we describe the general principles of targeted degradomics, outline the generic experimental workflow of the methodology and highlight recent and future applications in protease research.

Targeted Proteomics Comes to the Benchside and the Bedside: Is it Ready for Us?

“…The origin of these peptides can be broadly categorized into two types: first as a by‐product of the maturation and degradation cycle of a protein, secondly as a result of translation of short open reading frames (sORFs) present within both coding and noncoding regions. Above 10 lakh sORFs have been identified in humans, which emphasizes on the role of sORFs in cells …”

“…Above 10 lakh sORFs have been identified in humans, which emphasizes on the role of sORFs in cells. [64] These two classes of peptides are now acknowledged to be present in the cell but with rare functional annotation. [65][66][67] The likely reason for this disparity is the lack of tools to measure the presence and activity of these peptides.…”

Degradomics in Biomarker Discovery

“…Besides actual cleavage site peptides (N‐ or C‐termini), a peptide sequence spanning over the investigated cleavage site can also be selected as a target sequence. Such sequences enable the evaluation of the abundance of cleaved versus uncleaved peptide as a relative estimate of the extent of processing . The selected target peptide must be unique to a specific protein and compatible with MS analysis (ionization potential, hydrophobicity, length, etc.).…”

“…In comparison to immunological assays, proteomic analysis still does not regularly achieve lower than 10% coefficient of variation (CV) levels, which is a common precision acceptance criteria requested by regulatory agencies . Targeted quantitative proteomics approaches such as SRM/MRM represent a significant improvement over classical shotgun approaches due to their improved specificity, sensitivity, and reproducibility . Despite that, the reproducibility of SRM is currently still not completely comparable to reproducibilities of standard immunological biomarker assays, since SRM is capable of achieving CV levels in the range of 10% while automated ELISA assays can regularly achieve CV levels below 4% 75b.…”

“…[75] Targeted quantitative proteomics approaches such as SRM/MRM represent a significant improvement over classical shotgun approaches due to their improved specificity, sensitivity, and reproducibility. [76] Despite that, the reproducibility of SRM is currently still not completely comparable to reproducibilities of standard immunological biomarker assays, since SRM is capable of achieving CV levels in the range of 10% while automated ELISA assays can regularly achieve CV levels below 4%. [75b] Sample preparation steps were found to be the main source of CV variability in LC-MS methodologies.…”