A) Schematic overview of diGly peptide enrichment protocol.
B) Extracts from HCT116 cells treated as indicated were immunoblotted with α-ubiquitin, α-tubulin or α-NRF2.
C) The total number of diGly-modified lysines (sites) and proteins identified and quantified from four biological replicate experiments are shown with the determined false discovery rate. The correlation of the log2 ratios for all quantified peptides after 8hr Btz treatment between two biological replicates is plotted (inset: correlation coefficients).
D) Distribution of the sites per protein observed from diGly enrichment after 8hr Btz treatment.
E) H:L log2 ratios of all quantified diGly-containing peptides from untreated (blue bars) or 8hr Btz treated (red bars) cells.
F) Log2 ratio (top) and TSCs (bottom) of ubiquitin-derived diGly peptides after 8hr Btz treatment. Each bar represents the average log2 ratio or TSC from all quantified diGly peptides (Error bars: SEM, n=4).
G) Log2 ratios of H:L (treated:untreated) for sites quantified in both Btz and epoxomicin experiments.

A) Schematic of experiment.
B) Left, extracts from 8 hr Btz treated cells before and after mock or USP2cc treatment were immunoblotted with the indicated antibodies. Right, the total number of diGly sites (black bar) after Btz treatment and the number of sites in which their log2 ratio was decreased greater than 50% with USP2cc (grey bar).
C) Log2 ratios of all quantified diGly peptides from Btz treated cells either untreated (blue bars) or treated (red bars) with USP2cc.
D) Log2 ratios of all ubiquitin diGly sites (left) and representative known diGly modified proteins (right) from Btz treated heavy cells mixed with untreated light cells (blue bars) or light Btz treated, mock incubated and heavy Btz treated cells incubated with USP2cc post-lysis (red bars).
E) The fraction of the total quantified diGly sites with log2 ratios less than −1.0 (black bars) that were also unchanged after Btz treatment (grey bar) from cells treated as indicated.
F) Log2 ratios of representative peptides either known to be neddylated (CUL5) or putatively neddylated (NEDD8) from cells treated with Btz for 8 hours (blue bars), treated with Btz for 8 hours and subsequently treated with USP2cc post-lysis (red bars), untreated (green bars), or treated with MLN4924 for 2 hours (purple bars).
All error bars represent the SEM of multiple MS1 quantifications for the indicated site.

A) Cells were either untreated (K0) or treated with Btz for 0, 2, 4, or 8 hours (K8). Depicted is a heat map representing the hierarchical clustering of each peptide grouped using K-means clustering according to their log 2 H:L Btz response curves. The total number of peptides in each group is indicated.
B) Average log2 ratios of all peptides in each group with increasing time of Btz treatment (black line) and four representative peptides are depicted. Error bars: SEM of all peptide measurements in the group (black line) or multiple MS1 quantifications for the indicated peptide.

A) The distribution of diGly site (red, top) or protein (blue, bottom) log2 ratios (H:L) from heavy cells treated with Btz for 0, 2, 4, or 8 hours. The protein level data was obtained by quantifying SCX fractionated whole cell extracts for each time point. Numbers indicate total number of quantified peptides.
B) Percentage of diGly-containing peptides (red) or proteins (blue) that change two fold or more in each Btz treated time point.
C) Immunoblot of extracts from the Btz time course.
D) Site and protein level changes during the Btz time course as quantified by mass spectrometry. The diGly-modified peptide that was quantified is indicated. All measurements represent the average log2 ratio for all quantified peptides with error bars representing the SEM.
E) TSCs for diGly-modified peptides derived from the 50 proteins with the largest protein level TSCs.

A) Distribution of log2 ratios from K8 cells treated with Btz for 8 hours (green line) or with Btz and CHX (red line) for 8 hours mixed with untreated K0 cells. Log2 ratios from K0, Btz treated cells mixed with K8 labeled cells treated with Btz and CHX (blue line).
B) Schematic of SILAC switching experiment.
C) The distribution of the fractional incorporation of the K8 label from each experiment for diGly-containing peptides quantified after diGly enrichment (red) or from total protein (blue).

A) Extracts from cells treated as indicated were immunoblotted for HIF1α or CUL5.
B) Schematic of experimental setup for CRL substrate identification using the NEDD8 E1 inhibitor, MLN4924.
C) The log2 ratios for the indicated diGly peptides for Btz treated cells (red bars), or Btz and MLNL4924 treated K8 labeled cells mixed with Btz treated K0 cells (blue bars). Error bars represent the SEM of all diGly peptide ratios measured.
D) The log2 ratios for the indicated site across multiple experiments as indicated. The number inside of the red box indicates the total number of sites assigned to each diGly abundance profile.
E) The log2 ratios for the indicated diGly peptides from Btz and MLNL4924 treated K8 labeled cells mixed with Btz treated K0 cells between two replicate experiments.
F) Extracts from cells treated as indicated were immunoblotted with α-NEDD8 antibodies.
G) Log2 ratios corresponding to three diGly sites within NEDD8 from multiple experiments as indicated. All error bars represent the SEM of multiple MS1 quantifications for the indicated site.

A) Fraction of all and diGly-modified lysines occurring in regions with positive and negative local charges at physiological pH.
B) Heat map (log(10) of the p-value) depicting significance of enrichment (red) or depletion (blue) for each amino acid within +/− six residues of each diGly site, evaluated using a Binomial test. ‘x’ represents the absence of an amino acid and was used when lysines and diGly sites fell within 6 amino acids of the N- or C-terminus of the protein.
C) The log(10) p-value associated with enrichment or depletion of selected amino acids near sites of diGly modification.
D) Pfam domains that were found to be enriched (green), disfavored (red), or unchanged (grey) on diGly-modified proteins anywhere within proteins containing the domain (rectangles) or within the domain itself (ovals). p < 0.01; Hypergeometric test, with Benjamini-Hochberg correction for multiple hypothesis testing. Domain names in red were also found to exhibit enriched or depleted levels of modification in a previous proteomic survey of lysine acetylation.
E) (Left) The fraction of diGly-modified lysines (black bar) and all non-diGly-modified lysines (grey bar) observed here compared to all lysines within the human proteome (left) and to acetylated-lysines previously described (right). (Right) The fraction of diGly-modified lysines (black bar) and all non-diGly-modified lysines (grey bar) observed here compared to all lysines observed in proteins demonstrated to be acetylated (left) and to acetylated-lysines (right). p-value calculated by the Binomial test.