Abstract
Posttranslational modifications of proteins increase the complexity of the cellular proteome and enable rapid regulation of protein functions in response to environmental changes. Protein ubiquitylation is a central regulatory posttranslational modification that controls numerous biological processes including proteasomal degradation of proteins, DNA damage repair and innate immune responses. Here we combine high-resolution mass spectrometry with single-step immunoenrichment of di-glycine modified peptides for mapping of endogenous putative ubiquitylation sites in murine tissues. We identify more than 20,000 unique ubiquitylation sites on proteins involved in diverse biological processes. Our data reveals that ubiquitylation regulates core signaling pathways common for each of the studied tissues. In addition, we discover that ubiquitylation regulates tissue-specific signaling networks. Many tissue-specific ubiquitylation sites were obtained from brain highlighting the complexity and unique physiology of this organ. We further demonstrate that different di-glycine-lysine-specific monoclonal antibodies exhibit sequence preferences, and that their complementary use increases the depth of ubiquitylation site analysis, thereby providing a more unbiased view of protein ubiquitylation.
Highlights
Ubiquitin is a small 76-amino-acid protein that is conjugated to the -amino group of lysines in a highly orchestrated enzymatic cascade involving ubiquitin activating (E1), ubiquitin conjugating (E2), and ubiquitin ligase (E3) enzymes [1]
It should be noted that the di-glycine remnant is not specific for proteins modified by ubiquitin and proteins modified by NEDD8 or ISG15 generate an identical di-glycine remnant on modified lysines making it impossible to distinguish between these modifications by mass spectrometry
We identified more than 20,000 ubiquitylation sites from five different murine tissues and report the largest ubiquitylation dataset obtained from mammalian tissues to date
Summary
Tissues—Mouse tissues were dissected from C57BL/6 mice, rinsed with PBS and frozen immediately in liquid nitrogen. The samples were split in two equal parts; one part was used to enrich modified peptides with 100 g of purified di-glycine-lysine-specific GX41 monoclonal antibody (Lucerna). Peptides were immunoprecipitated using 40 l of di-glycine-lysine antibody resin provided in the Ubiquitin Remnant Motif Kit (Cell Signaling Technology, Danvers, MA). The average enrichment of di-glycine-lysine in the peptide fractions was 35% (supplemental Fig. S1A). Di-glycine-lysine containing peptides have a higher molecular weight and a lower hydropathicity compared with the unmodified peptides in the enriched peptide fractions (supplemental Fig. S1B). MS Analysis—Peptide fractions were analyzed on a hybrid linear ion-trap Orbitrap (LTQ-Orbitrap Velos, Thermo Scientific) or quadrupole Orbitrap (Q-Exactive, Thermo Scientific) mass spectrometer equipped with a nanoflow HPLC system (Thermo Scientific) as described (36 –38). Di-glycine modified lysines were required to be located internally in the peptide sequence. Properties of the enriched peptides were determined using the ProtParam tool from the Biopython software package [47]
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