Ubiquitinomics: History, methods, and applications in basic research and drug discovery

Abstract

The ubiquitin‐proteasome system (UPS) was discovered about 40 years ago and is known to regulate a multitude of cellular processes including protein homeostasis. Ubiquitylated proteins are recognized by downstream effectors, resulting in alterations of protein abundance, activity, or localization. Not surprisingly, the ubiquitylation machinery is dysregulated in numerous diseases, including cancers and neurodegeneration. Mass spectrometry (MS)‐based proteomics has emerged as a transformative technology for characterizing protein ubiquitylation in an unbiased fashion. Here, we provide an overview of the different MS‐based approaches for studying protein ubiquitylation. We review various methods for enriching and quantifying ubiquitin modifications at the peptide or protein level, outline MS acquisition, and data processing approaches and discuss key challenges. Finally, we examine how MS‐based ubiquitinomics can aid both basic biology and drug discovery research.

Non-lysine ubiquitylation: Doing things differently

“…While some direct evidence of N-terminal ubiquitylation was obtained in these early studies by mass spectrometry ( Ben-Saadon et al, 2004 ; Coulombe et al, 2004 ), until recently no quantitative proteomics data was available on this modification. The global profiling of isopeptide-linked ubiquitylation sites was revolutionized a decade ago by the development of an antibody recognizing the diglycine remnant present on ubiquitylated lysines after tryptic digestion ( Xu et al, 2010 ), fast becoming the method of choice for enrichment and identification of ubiquitylated sites ( Kim et al, 2011 ; Udeshi et al, 2013 ; Steger et al, 2022 ). A major drawback of this technique however is that, due to the nature of the antibody epitope, it does not enrich for peptides originating from N-terminally ubiquitylated proteins.…”

“…Mass spectrometry is usually considered the gold standard to identify protein ubiquitylation sites ( Steger et al, 2022 ) but unless machines are specifically programmed to search for non-lysine ubiquitylation this information is simply lost amongst the thousands of unassigned spectra ( Pathan et al, 2017 ). Ubiquitin proteomics is typically accompanied by an enrichment step, often involving purification using a linkage-specific ubiquitin binder ( Hjerpe et al, 2009 ; Emmerich et al, 2013 ; Michel et al, 2017 ; Antico et al, 2021 ) or peptide enrichment using an antibody recognizing the diGly remnant attached to ubiquitylated lysines following tryptic digest ( Xu et al, 2010 ).…”

Does the Ubiquitination Degradation Pathway Really Reach inside of the Chloroplast? A Re-Evaluation of Mass Spectrometry-Based Assignments of Ubiquitination

“…One could also consider omitting sample alkylation since this will avoid di-carbamidomethylation of lysines (and other amino acid residues) while still obtaining relatively high PSM match rates Include appropriate negative affinity-enrichment controls to determine the true false discovery rate of the K-ε-GG footprint (or K + 114) and that affinity enrichment for-K-ε-GG containing peptides is highly selective. Use parallel alternative methods to enrich for peptides with ubiquitination; there are several independent methods now available. ,,, Perform an open search on the acquired MS data to ensure that the mass modifications in the data are consistent with the intended sample handling. For the analysis of MS data, include only the most relevant (variable) modifications (ideally based on open searches) and site scoring algorithms to reduce false discovery rate of ubiquitination (in particular +57.022 and +114.0429 Da on lysine, cysteine, and peptide N terminus). The sequence database used for MS data analysis should include all proteins of the target organism (e.g., Arabidopsis predicted protein sequences in Araport11 or TAIR10) as well as additional proteins that may be in the sample (including contaminants such as keratin, trypsin, and E. coli proteins) not just proteins of interest. …”

“…60,61 The determination of the nature of the substrate ubiquitination is technically challenging. 36,37,61 Figure 2D of Sun et al 1 showed a linkage analysis indicating that 32% was from ubiquitin K48 (no further information was provided on this analysis). It was stated that "because K48 was the most abundant linkage type, much of the chloroplast ubiquitinome can be assumed to be primed for proteasomal degradation."…”

“…Based on our own analysis presented here, the most recent reviews on "ubiquitinomics", 22,23,25,36,37,61,62 and the primary literature, we recommend the following for the MS-based determination of possible in vivo ubiquitination of intrachloroplast proteins of viable and intact chloroplasts: i. Ensure that the plants are not under significant chloroplast stress (e.g., due to high light treatment) as this will result in (a) rapid release of chloroplast proteins into the cytosol as demonstrated by confocal microscopy of plants expressing chloroplast-targeted green fluorescent protein (GFP) in the f lu mutant 63,64 and (b) ATGdependent and independent autophagy of swollen or otherwise damaged chloroplasts or selected chloroplast content and delivery and degradation in the vacuole.…”