1. Quantifying Ubiquitin Signaling
Alban Ordureau, Christian Münch, J. Wade Harper 
Molecular Cell 
Volume 58, Issue 4, Pages (May 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Figure 1
Quantitative Proteomics as a Framework for Understanding Signaling Pathways
(A) Two major classes of UB- and phosphorylation-driven pathways are depicted, one involving substrate phosphorylation and engagement by a CRL-based E3 and the second involving phosphorylation-dependent activation of the E3 itself, leading to substrate recruitment. Major questions related to the stoichiometry of individual steps in the pathway, as well as how UB chain synthesis and topology impact flux through the pathway, are also depicted. SCF, SKP-Cullin F-box-containing complex.
(B) Standard MS workflows involve isolation and peptide analysis of proteins from a sample of interest. Generation of quantitative data, generally in the context of cells from different states or treated with distinct stimuli, involves the use of various labeling strategies, including metabolic labeling of cells or tissues, isobaric tagging of peptides, and the use of heavy reference proteins or peptides to monitor and quantify specific target molecules. These labeling techniques can be merged with enrichment strategies that allow examination of particular organelles, protein complexes, or PTMs. Experimental design may also include temporal components. Together, these approaches allow for quantification of individual molecules and PTMs in space and time. SILAC, stable isotope labeling with amino acids in cell culture; PSAQ, protein standard absolute quantification; QCAT, concatemer of Q peptides; AQUA, absolute quantification; SRM, selected reaction monitoring; MRM, multiple reaction monitoring; PRM, parallel reaction monitoring; iTRAQ, isobaric tags for relative and absolute quantitation; TMT, tandem mass tags; ReDi, reductive dimethylation; ICAT, isotope-coded affinity tag, TiO2, titanium dioxide.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 2
Overview of Possible Proteomic Approaches for Measuring Relative and Absolute Levels of Proteins and Modifications
(A) Schematic showing how 10-plex TMT can be used to provide quantitative information for thousands of peptides and proteins. Samples from time courses, drug treatments, etc. are trypsinized, labeled with one of ten TMT reporter reagents, and subjected to LC-MS3 analysis. Reporter ion intensities from MS3 are used to determine the relative abundance of peptides, which are then summed across each protein. TMT can be merged with conventional triple SILAC to create 30-plex experiments.
(B) Schematic representation of NeuCode SILAC involving the use of six isotopologs of lysine, which can be distinguished by FT-MS. In combination with triplex ReDi labeling, this approach can provide 18-plex. Tryptic peptides are subjected to LC-MS2, and relative peak intensities measured.
(C) Heavy reference peptides against peptides and modified peptides of interest are synthesized, and known quantities added to samples post-trypsinization. Samples are analyzed by LC-MS1 using a high-resolution Orbitrap, and peak intensities measured for experimental sample relative to reference (AQUA) peptides. ∗, heavy labeled amino acid.
(D) MRM uses a pre-selected precursor peptide list to target peptides for isolation in quadruple-1 (Q1). These ions are fragmented in Q2, and desired fragment ions detected in Q3. To provide accurate quantification, heavy reference peptides can be added and monitored in parallel.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 3
Workflow for Detection and Analysis of UB and Other Modification in Proteins
The scheme depicts various possible experimental approaches for identification of sites of UB modification in their primary targets integrated with quantitative analysis of UB chain linkage types in specific proteins or in the context of specific organelles. Proteins purified from different conditions can be isolated with or without pre-selection of organelles or cellular sub-fractions, and specific proteins or complexes identified by affinity purification or UB chains and their conjugated proteins purified using various types of UB binding domains (see text). After trypsinization, peptides can be directly analyzed using UB-AQUA to identify and quantify UB chain linkages or peptides subjected to diGLY enrichment in order to identify primary ubiquitylation sites. Data analysis allows for the elucidation of how modifications are altered in response to different perturbations. HUPLC, ultra-high-performance liquid chromatography.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 4
Chain Linkage Analysis and Integration of Phosphorylation and Ubiquitylation Crosstalk in the PINK1-PARKIN Pathway
(A) UB code. Types of ubiquitylation events on proteins can range from monoubiquitylation and multi-monoubiquitylation to polyubiquitylation involving multiple chain linkage types and possibly including branched or mixed chain structures. Unanchored UB chains may also be present in cells.
(B) UB-AQUA. Schematic depiction of major UB-AQUA peptides including the major diGLY peptides and previously detected sites of phosphorylation in UB. Several additional UB-AQUA peptides used for locus concentration determination are not shown. UB-AQUA peptides containing phosphorylation sites can be used to determine the stoichiometry of phosphorylation. The predominant pS65 peptide identified is a partial tryptic cleavage product (residues 55–72), rather than the full tryptic cleavage product (residues 64–72), due to the inhibitory effect of the phosphate near the trypsin-cutting site.
(C) DUB restriction analysis or UbiCRest. Schematic depicting how linkage-specific DUBs can be used in vitro to analyze and determine the nature of the UB chains attached to targets.
(D) Schematic of a model for the PINK1-PARKIN pathway. Mitochondrial damage leads to PARKIN phosphorylation and activation of its ubiquitin ligase activity, as well as recruitment to mitochondria. Currently, quantitative studies suggest that ubiquitin phosphorylation on mitochondria requires chain synthesis by activated phospho-PARKIN. As PARKIN promotes chain synthesis, this increases the density of UB on mitochondria and provides a substrate for phosphorylation by PINK1. Multiple feedforward models, that are not mutually exclusive, are shown wherein ubiquitin phosphorylation on mitochondria leads to amplification of mitochondrial ubiquitylation. We hypothesize that maximal UB amplification (Model 2) involves recruitment of phospho-PARKIN to phospho-UB chains through a direct interaction, and maximal chain synthesis reflects the fact that phospho-PARKIN has significantly higher specific activity than unphosphorylated PARKIN bound to phospho-UB. Phospho-UB may also recruit non-phosphorylated PARKIN (Model 3) to promote chain synthesis or ubiquitylation of mitochondrial outer membrane proteins, albeit with rates lower than those seen with phospho-PARKIN. Moreover, binding of unphosphorylated PARKIN to phospho-UB may increase its local concentration in the vicinity of PINK1 to facilitate PARKIN phosphorylation, leading to further activation (Model 1). A combination of these mechanisms may occur.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions