Highly Multiplexed Quantitative Mass Spectrometry Analysis of Ubiquitylomes

Abstract

SUMMARY System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000–9,000 ubiquitylation forms across ten samples simultaneously. Using this method, we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN- and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN- and PINK1-dependent ubiquitylation targets to date in a single experiment, and it also revealed a subset of proteins recruited to the mitochondria during mitophagy.

Global proteomics ofUbqln2-based murine models of ALS

“…The simplest hypothesis for the accumulation of PEG10 and TRIM32 in Ubqln2 -/tissues and cells is an impairment of their proteasomal degradation. In support of this hypothesis, ubiquitin-conjugated forms of TRIM32, PEG10, and CXX1B/RTL8 have all previously been observed to accumulate in HTC116 cells treated with proteasome inhibitors (Supplemental Figure 10A) (Rose et al, 2016). To examine their turnover in the context of Ubqln deficiency, WT and TKO HEK 293 cells were treated with the proteasome inhibitors or autophagy inhibitors.…”

Parkin‐mediated ubiquitylation redistributes MITOL/March5 from mitochondria to peroxisomes

“…Through the interaction with phosphorylated ubiquitin, Parkin also becomes phosphorylated, which triggers auto-activation and subsequent conformational changes [16][17][18]. Ubiquitylation of various mitochondrial proteins by the activated Parkin then leads to selective elimination of damaged mitochondria [19][20][21][22][23][24][25][26][27][28][29][30][31].…”

UbiFast, a rapid and deep-scale ubiquitylation profiling approach for biology and translational research

“…We next compared in-solution TMT labeling of K-ɛ-GG peptides to on-antibody TMT labeling of K-ɛ-GG peptides with respect to the total numbers of K-ɛ-GG peptides detected, the relative yield of K-ɛ-GG peptides (relative yield is the percentage of K-ɛ-GG peptides relative to the total peptides identified in the sample) and the efficiency of TMT labeling (Supplemental Figure 3 A-D, respectively). Jurkat peptide samples (1 mg, each) were enriched with the anti-Kɛ-GG antibody and peptides were labeled with a single TMT reagent, either while the peptides were bound to the antibody or using the in-solution labeling method previously described 19 (Supplemental Figure 3A). On-antibody TMT labeled samples resulted in 6087 K-ɛ-GG PSMs with a relative yield of 85.7%, while samples labeled in-solution resulted in 1255 K-ɛ-GG PSMs with a relative yield of 44.2% (Supplemental Figure 3B, C, Supplemental Table 4).…”

“…introduced a method where samples are enriched at the peptide level using the anti-K-ɛ-GG antibody prior to labeling with TMT10 reagents 19 . After elution from the antibody and labeling, the enriched peptides are subjected to high pH reversed-phase chromatography using spin columns, fractionated into six fractions, and analyzed by LC-SPS-MS3.…”