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1.  Proteomic Analyses Reveal Divergent Ubiquitylation Site Patterns in Murine Tissues*  
Molecular & Cellular Proteomics : MCP  2012;11(12):1578-1585.
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.
doi:10.1074/mcp.M112.017905
PMCID: PMC3518112  PMID: 22790023
2.  The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator 
Nucleic Acids Research  2012;40(9):3913-3928.
Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
doi:10.1093/nar/gkr1300
PMCID: PMC3351161  PMID: 22234878
3.  A Proteome-wide, Quantitative Survey of In Vivo Ubiquitylation Sites Reveals Widespread Regulatory Roles* 
Molecular & Cellular Proteomics : MCP  2011;10(10):M111.013284.
Post-translational modification of proteins by ubiquitin is a fundamentally important regulatory mechanism. However, proteome-wide analysis of endogenous ubiquitylation remains a challenging task, and almost always has relied on cells expressing affinity tagged ubiquitin. Here we combine single-step immunoenrichment of ubiquitylated peptides with peptide fractionation and high-resolution mass spectrometry to investigate endogenous ubiquitylation sites. We precisely map 11,054 endogenous putative ubiquitylation sites (diglycine-modified lysines) on 4,273 human proteins. The presented data set covers 67% of the known ubiquitylation sites and contains 10,254 novel sites on proteins with diverse cellular functions including cell signaling, receptor endocytosis, DNA replication, DNA damage repair, and cell cycle progression. Our method enables site-specific quantification of ubiquitylation in response to cellular perturbations and is applicable to any cell type or tissue. Global quantification of ubiquitylation in cells treated with the proteasome inhibitor MG-132 discovers sites that are involved in proteasomal degradation, and suggests a nonproteasomal function for almost half of all sites. Surprisingly, ubiquitylation of about 15% of sites decreased more than twofold within four hours of MG-132 treatment, showing that inhibition of proteasomal function can dramatically reduce ubiquitylation on many sites with non-proteasomal functions. Comparison of ubiquitylation sites with acetylation sites reveals an extensive overlap between the lysine residues targeted by these two modifications. However, the crosstalk between these two post-translational modifications is significantly less frequent on sites that show increased ubiquitylation upon proteasome inhibition. Taken together, we report the largest site-specific ubiquitylation dataset in human cells, and for the first time demonstrate proteome-wide, site-specific quantification of endogenous putative ubiquitylation sites.
doi:10.1074/mcp.M111.013284
PMCID: PMC3205876  PMID: 21890473
4.  RNA-DNA sequence differences spell genetic code ambiguities 
Artificial DNA, PNA & XNA  2011;2(3):69-70.
A recent paper in Science by Li et al. 20111 reports widespread sequence differences in the human transcriptome between RNAs and their encoding genes termed RNA-DNA differences (RDDs). The findings could add a new layer of complexity to gene expression but the study has been criticized. 
PMCID: PMC3324336  PMID: 22567189
gene expression; RNA editing; RNA-DNA differences; transcription; transcriptome
5.  Mass Spectrometric Analysis of Lysine Ubiquitylation Reveals Promiscuity at Site Level* 
Molecular & Cellular Proteomics : MCP  2010;10(3):M110.003590.
The covalent attachment of ubiquitin to proteins regulates numerous processes in eukaryotic cells. Here we report the identification of 753 unique lysine ubiquitylation sites on 471 proteins using higher-energy collisional dissociation on the LTQ Orbitrap Velos. In total 5756 putative ubiquitin substrates were identified. Lysine residues targeted by the ubiquitin-ligase system show no unique sequence feature. Surface accessible lysine residues located in ordered secondary regions, surrounded by smaller and positively charged amino acids are preferred sites of ubiquitylation. Lysine ubiquitylation shows promiscuity at the site level, as evidenced by low evolutionary conservation of ubiquitylation sites across eukaryotic species. Among lysine modifications a significant overlap (20%) between ubiquitylation and acetylation at site level highlights extensive competitive crosstalk among these modifications. This site-specific crosstalk is not prevalent among cell cycle ubiquitylations. Between SUMOylation and ubiquitylation the preferred interaction is through mixed-chain conjugation. Overall these data provide novel insights into the site-specific selection and regulatory function of lysine ubiquitylation.
doi:10.1074/mcp.M110.003590
PMCID: PMC3047152  PMID: 21139048
6.  A Dual Pressure Linear Ion Trap Orbitrap Instrument with Very High Sequencing Speed* 
Since its introduction a few years ago, the linear ion trap Orbitrap (LTQ Orbitrap) instrument has become a powerful tool in proteomics research. For high resolution mass spectrometry measurements ions are accumulated in the linear ion trap and passed on to the Orbitrap analyzer. Simultaneously with acquisition of this signal, the major peaks are isolated in turn, fragmented and recorded at high sensitivity in the linear ion trap, combining the strengths of both mass analyzer technologies. Here we describe a next generation LTQ Orbitrap system termed Velos, with significantly increased sensitivity and scan speed. This is achieved by a vacuum interface using a stacked ring radio frequency ion guide with 10-fold higher transfer efficiency in MS/MS mode and 3–5-fold in full scan spectra, by a dual pressure ion trap configuration, and by reduction of overhead times between scans. The first ion trap efficiently captures and fragments ions at relatively high pressure whereas the second ion trap realizes extremely fast scan speeds at reduced pressure. Ion injection times for MS/MS are predicted from full scans instead of performing automatic gain control scans. Together these improvements routinely enable acquisition of up to ten fragmentation spectra per second. Furthermore, an improved higher-energy collisional dissociation cell with increased ion extraction capabilities was implemented. Higher-collision energy dissociation with high mass accuracy Orbitrap readout is as sensitive as ion trap MS/MS scans in the previous generation of the instrument.
doi:10.1074/mcp.M900375-MCP200
PMCID: PMC2816009  PMID: 19828875
7.  Transient Marker System for Iterative Gene Targeting of a Prototrophic Fungus▿  
Applied and Environmental Microbiology  2007;73(22):7240-7245.
Auxotrophic microorganisms are often used for genetic engineering, because their biosynthetic deficiency can be complemented by the transforming DNA and allows selection for transformants that have become prototrophic. However, when complementation is obtained by ectopic expression this may lead to unpredictable side effects on the phenotype and, consequently, misinterpretation of experimental data. There are various ways to overcome the problem of auxotrophy, but the most reliable is to restore the function of the defective biosynthetic gene at the native genomic locus. This can be done by either sexual crossing or further genetic engineering. For fungal species lacking a perfect state or situations in which gene targeting is generally cumbersome we have developed a concept that allows transient disruption of pyrG. When the gene is in the disrupted state, multiple rounds of gene targeting can be performed with the strain. Once the desired genome engineering is completed, pyrG function can be rapidly returned to wild type by a simple selection scheme.
doi:10.1128/AEM.01839-07
PMCID: PMC2168213  PMID: 17921280
8.  The RBCC Gene RFP2 (Leu5) Encodes a Novel Transmembrane E3 Ubiquitin Ligase Involved in ERAD 
Molecular Biology of the Cell  2007;18(5):1670-1682.
RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-δ, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
doi:10.1091/mbc.E06-03-0248
PMCID: PMC1855009  PMID: 17314412

Results 1-8 (8)