Analysis of the topology of ubiquitin chains

Abstract

The small protein ubiquitin and its multiple polymers are encountered free in cells and as posttranslational modifications on all proteins. Different polyubiquitin three dimensional structures are shown to correlate uniquely with different cellular functions as part of the diverse ubiquitin signaling. At the same time, this multiplicity of structures provides serious challenges to the analytical biochemist. Globally applicable strategies are presented here for the analyses of polyubiquitins and of ubiquitinated proteins, which take advantage of the speed, specificity and sensitivity of top-down tandem mass spectrometry. Particular attention is given to the supervised interpretation of fragmentation as revealed in the MS/MS spectra of these branched proteins. The strategy is compatible with any MS activation technology, is applicable to all polyubiquitin linkage and chain types, can be extended to ubiquitin-like proteins, and will be compatible with and enhanced by continuing advances in LC-MS/MS instrumentation and interpretation software.

Top-Down Proteomic Characterization of Truncated Proteoforms

“…[17][18][19][20][21] An alternative or co-existing explanation is that proteins are hydrolyzed in situ by proteases active in the extracellular vesicles. This latter hypothesis is consistent with the previous identification of 49 proteases and the 37 components of the proteasome and immunoproteasome in MDSC-derived extracellular vesicles 11,22 and of 34 proteases in urinary extracellular vesicles. 23 Protease, proteasome and other enzymatic activities have recently been demonstrated to occur in situ in several kinds of extracellular vesicles.…”

“…Proteins in these categories have previously been reported to be abundant in extracellular vesicles shed by parental MDSC. 11,14,22 Analysis of the locations of the precursor proteins referenced to parental MDSC (Figure S-2) provides a similar distribution to that reported in a 2014 gene ontology analysis of exosomal proteins 14 with the major portion assigned to cytoplasm. Removal of initial methionine was detected in 171 of the 1020 proteoforms and was accompanied by N-terminal acetylation in another 135 proteoforms.…”

“…Additive modifications of intact proteins have been reported in previous, untargeted proteomic studies of MDSC extracellular vesicles. 10,11,14,15,22,30 In one of these studies 20 out of 75 intact proteoforms identified carried multiple modifications including acetylation, phosphorylation, cysteinylation, cysteine sulfonic acid, citrullination, mono-, diand tri-methylation and oxidation. 10 Thus, the list of truncated proteoforms that also carry additive modifications is likely to be expanded when improved search software becomes available.…”

“…Seven of these proteases have been found to be more abundant in extracellular vesicles than in parental MDSC, with enrichments ranging between 2.3-and 39-fold. 22 Thirty-four proteoforms (3.3 %) were recognized for whose production no proteases could be matched . The majority of cleavages with unassigned proteases occur after tyrosine or phenylalanine, Y/F-Xaa, which could be explained by a chymotrypsin-like protease such as found in the immunoproteasome.…”

Catherine Fenselau: A distinguished career dedicated to biomedical mass spectrometry

“…Because extracellular vesicles are a rich source of proteins modified by truncation, Prof. Fenselau advanced a strategy in which curated interpretation was coupled with top‐down MS/MS analysis to characterize, for the first time on a proteomic scale, both internal and terminal truncation products . A curated interpretive approach has also been used by her group to develop road maps for assigning branch sites and topology to polyubiquitins and other branched proteins . In combining curated interpretation with bioinformatic tools to address successfully these two challenging protein classes, Prof. Fenselau came full circle by returning to the subject of her early thesis research, the comprehension and exploitation of patterns of ion fragmentation.…”