NeuCode Proteomics Reveals Bap1 Regulation of Metabolism

Abstract

We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.

Figure 1. NeuCode SILAM allows protein quantification from partially labeled mouse tissues

A) Schematic comparing partial labeling in SILAM with NeuCode SILAM. In SILAM, a 50% labeled peptide (red) from a ‘heavy’ mouse will have an unlabeled counterpart (dark gray) that overlaps in mass with unlabeled (light gray) peptide from the ‘light’ mouse. NeuCode uses heavy to heavy ratios (blue:red) and ignores unlabeled peptides. B) In 4-plex NeuCode, mice are labeled with K₆₀₂, K_521, K₄₄₀, or K₀₈₀, tissues harvested, and protein lysates equally mixed for MS analysis. During MS, a low resolution MS1 scan at 30,000 (dotted line) is performed and used to trigger data-dependent MS2. Both light and heavy features can be observed due to partial labeling. During MS2 acquisition, a high resolution MS1 scan (480,000) resolves closely spaced NeuCode isotopologues (colored peaks). Isotopologue area under the curve signal is used to quantify peptide abundance.

Figure 2. Labeling Efficiency and Accuracy of NeuCode SILAM

A) Mice were labeled with either K₆₀₂ or K₀₈₀ for 3–30 days. Proteins extracted from nine tissues were LysC digested and analyzed by MS. Light and heavy peptides are quantified to assess percent labeling. K₆₀₂ and K₀₈₀ peptide samples from some tissues were also mixed at defined ratios to assess NeuCode accuracy. B) Heavy lysine % incorporation is plotted by tissue and labeling time. Box plots represent the average of n=3 animals per protein (K₆₀₂–blue; K₀₈₀-red). Box edges are the 25^th and 75^th percentiles, with the median protein shown. Whiskers extend to 1.5X interquartile range and outliers plotted as circles. C) Correlation plots of K₆₀₂:K₀₈₀ labeling for a representative biological replicate from each time point and tissue. D) Proteins from mice fed K₆₀₂ or K₀₈₀ were mixed in defined ratios (1:1, 5:1, and 10:1) for intestine, liver, and brain. Expected ratios are signified by dotted lines and measured ratios displayed as box plots as in 2B.

Figure 3. NeuCode SILAM Analysis Seven Days Following Bap1 Deletion

A) Bap1ko NeuCode experimental design. Mice were labeled 21 days with four NeuCode diets (K₆₀₂, K₅₂₁, K₄₄₀, K₀₈₀). During labeling, mice were treated with tamoxifen (tam) for 5 days to delete Bap1. Day 0 is the fifth and final tam injection (day 14 of labeling). Liver, pancreas, and spleen were harvested and protein lysates mixed as 4-plex and 2-plex. High resolution (480,000) MS1 scans reveal NeuCode-labeled peptides which are quantified by ‘area under the curve’. B) Glucose measurements during (day < 0) and after (day > 0) 5 daily tam injections. * p < .05; ** p < .01 in two-tailed unpaired T-test. n=6 per group. C) Bap1 mRNA measured by qPCR with primers spanning the deleted exons. n=6 per group. D) Number of proteins changing more than 1.5-fold with a nominal p < .05 following Bap1 deletion. E,F,G) Volcano plots of all proteins using log2 ratios and nominal p-values. Significantly enriched protein sets and individual proteins referred to in the text are colored and labeled.

Figure 4. Proteomic and pathological characterization of BMC-Bap1ko mice

A) Schematic of bone marrow rescue experiments and NeuCode labeling. B) Platelet measurements. N=3 mice per group, error bars=SEM. C) Time course of glucose measurements following Bap1 deletion in BMC mice. n = 11 Wt female, 10 Ko female, 9 Wt male, 9 Ko male mice. *p < .001, two-tailed unpaired T-test. D) Proteins changing >2-fold with nominal p < .05 in NeuCode SILAM data from each tissue following 3 months of Bap1 knockout with the final 3 weeks being fed NeuCode diets. N=3 Wt and Ko. E) Comparison between all proteins identified in NeuCode SILAM experiments in the liver at day 7 from Figure 3, and at day 90 post-Bap1 deletion. Select proteins are labeled. F) Log2 Ko:Wt ratios of enriched K-GG (ubiquitinated) peptides from Day 7 Bap1ko NeuCode livers are plotted against liver total protein ratios from the same samples analyzed in Figure 3F. H2A histone isoforms and outer membrane mitochondrial proteins Vdac1/2/3 and Maob are highlighted. Residue numbers derive from UniProt and include the start methionine.

Figure 5. Serum chemistry and pathology of BMC-Bap1ko mice

A,B) Serum chemistry for Bap1-BMC mice 2 or 12 wks post-deletion. Fed denotes ad-libitum feeding, and fasted denotes a 16 hr overnight fast. At 12 wks: Wt-fed (n=12), Wt fast (n=12), Ko-fed (n=14), Ko-fast (n=14). At 2 wks: Wt-fed (n=5), Wt-fast (n=12), Ko-fed (n=5), Ko-fast (n=14). * p <.001, unpaired two-tailed T-test. C,D) H&E staining of pancreas and liver of BMC-Bap1-Wt and Ko mice 2 wks and 12 wks post-deletion.

Figure 6. Metabolic characterization of BMC-Bap1Ko mice

A) Insulin staining of BMC-Bap1wt and BMC-Bap1ko pancreas 2wks post-deletion. Representative image from n=3 mice. B) Insulin release assay. Mice were fasted 12hr and serum insulin concentrations measured at times after glucose injection. Error bars: SEM. *p<.01. Replicated in 2 independent cohorts. C) Glucose tolerance assay. Mice were fasted for ~6hr and serum glucose measured at the times after glucose challenge. Error bars: SEM. *p<.001. Replicated in 3 independent cohorts. D) Pyruvate tolerance assay. Mice were fasted overnight (~16hr) and serum glucose measured at the times after pyruvate injection. Error bars: SEM. * p<.001. Replicated in 3 independent cohorts. E) Oil Red O staining of BMC-Bap1 Wt and Ko ad-libitum fed or fasted 12hr. Representative of n=3 mice per condition. F) BMC-Bap1-Wt and Ko mice were fed high fat chow for the indicated times starting 2 wks post-deletion. *p<1e-10.

Figure 7. Liver-specific Bap1 deletion causes neonatal mortality with metabolic defects

A–C) Label-free proteomics of gluconeogenenic, lipid chaperone, and cholesterol biosynthesis enzyme expression in the neonatal liver (12–24hr postnatal). n=3 mice. Error bars: SEM. *p<0.05. D) Blood glucose recorded 1–4hr post-partum for each indicated genotype. *p<.01. **p<1e-8. E) H&E staining of frozen neonatal liver section ~6hr post-partum. F) Cleaved caspase3 staining of liver frozen sections ~12hr post-partum. G) Frozen sections of liver stained with Oil Red O (Red) demonstrating intra-hepatic lipid of E18.5 embryos and P0 neonates (~12hr post-partum) with the designated genotypes.