Screening of Processed Foods for Transgenic Proteins from Genetically Engineered Plants Using Targeted Mass Spectrometry.

Screening of food products for the presence of material from genetically engineered (GE) plants is typically done using deoxyribonucleic acid (DNA)-based methods to detect the presence of transgenic DNA. In this study, we have demonstrated the feasibility of using targeted mass spectrometry (MS) to detect a protein expressed by transgenic DNA to confirm the presence of GE plant material in processed foods. Scheduled parallel reaction monitoring (sPRM) was used to detect the enzyme, 5-enolpyruvulshikimate-3-phosphate synthase, from Agrobacterium sp. strain CP4 (CP4 EPSPS), which confers glyphosate tolerance in transgenic crops. Five CP4 EPSPS surrogate peptides and their corresponding retention times identified via data-dependent LC/MS/MS analysis of a glyphosate-tolerant soybean certified reference material, GTS 40-3-2, were used to develop the sPRM assay. The assay was used to screen four soy-based infant formulas, four corn-based cereals, corn tortilla chips, and cornmeal for the presence of CP4 EPSPS. At least four of the five selected surrogate peptides were detected in nine of the products analyzed, suggesting that targeted MS can serve as a complementary analytical method to DNA-based methods for the detection of material from GE plants in processed foods.

Oncogenesis driven by the Ras/Raf pathway requires the SUMO E2 ligase Ubc9.

The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.

Automethylation activities within the mixed lineage leukemia-1 (MLL1) core complex reveal evidence supporting a "two-active site" model for multiple histone H3 lysine 4 methylation.

The mixed lineage leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. In this investigation, we report that the suppressor of variegation, enhancer of zeste, trithorax (SET) domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I subdomain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. We also report that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex with WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, we observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di-, or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, we demonstrate that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a "two-active site" model for multiple H3K4 methylation by the MLL1 core complex.

Biotinylation of lysine method identifies acetylated histone H3 lysine 79 in Saccharomyces cerevisiae as a substrate for Sir2.

Although the biological roles of many members of the sirtuin family of lysine deacetylases have been well characterized, a broader understanding of their role in biology is limited by the challenges in identifying new substrates. We present here an in vitro method that combines biotinylation and mass spectrometry (MS) to identify substrates deacetylated by sirtuins. The method permits labeling of deacetylated residues with amine-reactive biotin on the ε-nitrogen of lysine. The biotin can be utilized to purify the substrate and identify the deacetylated lysine by MS. The biotinyl-lysine method was used to compare deacetylation of chemically acetylated histones by the yeast sirtuins, Sir2 and Hst2. Intriguingly, Sir2 preferentially deacetylates histone H3 lysine 79 as compared to Hst2. Although acetylation of K79 was not previously reported in Saccharomyces cerevisiae, we demonstrate that a minor population of this residue is indeed acetylated in vivo and show that Sir2, and not Hst2, regulates the acetylation state of H3 lysine 79. The in vitro biotinyl-lysine method combined with chemical acetylation made it possible to identify this previously unknown, low-abundance histone acetyl modification in vivo. This method has further potential to identify novel sirtuin deacetylation substrates in whole cell extracts, enabling large-scale screens for new deacetylase substrates.

Evolutionarily conserved protein ERH controls CENP-E mRNA splicing and is required for the survival of KRAS mutant cancer cells.

Cancers with Ras mutations represent a major therapeutic problem. Recent RNAi screens have uncovered multiple nononcogene addiction pathways that are necessary for the survival of Ras mutant cells. Here, we identify the evolutionarily conserved gene enhancer of rudimentary homolog (ERH), in which depletion causes greater toxicity in cancer cells with mutations in the small GTPase KRAS compared with KRAS WT cells. ERH interacts with the spliceosome protein SNRPD3 and is required for the mRNA splicing of the mitotic motor protein CENP-E. Loss of ERH leads to loss of CENP-E and consequently, chromosome congression defects. Gene expression profiling indicates that ERH is required for the expression of multiple cell cycle genes, and the gene expression signature resulting from ERH down-regulation inversely correlates with KRAS signatures. Clinically, tumor ERH expression is inversely associated with survival of colorectal cancer patients whose tumors harbor KRAS mutations. Together, these findings identify a role of ERH in mRNA splicing and mitosis, and they provide evidence that KRAS mutant cancer cells are dependent on ERH for their survival.

Quantitative proteomics of parotid saliva in primary Sjögren's syndrome.

The diagnosis of primary Sjögren's syndrome (pSS) is difficult due to the lack of specific laboratory and clinical tests. As an initial step for the global discovery of changes in the abundance of parotid salivary proteins in pSS, a pooled sample was compared to that from healthy control subjects by multidimensional protein identification technology (MudPIT). A total of 1246 proteins were identified by MudPIT. The abundance of 477 of these proteins did not change, 529 were only detected in either the pSS or HC sample, while 206 of these proteins were significantly upregulated ≥ twofold and 34 were downregulated ≤ 0.5. Ingenuity Pathway Analyses of differentially expressed proteins identified by MudPIT resulted in the identification of 100 significant pathways. The same samples were quantified in parallel using RP MS. Fifty-eight of 71 proteins identified by RP overlapped with MudPIT results. Five proteins were further analyzed by targeted label-free quantification to confirm the similar relative differential expression observed by RP and MudPIT approaches. The present study supports the use of MS for global discovery and validation of marker proteins for improved and early diagnosis of pSS.

Microwave assisted acid cleavage for denaturation and proteolysis of intact human adenovirus.

Microwave assisted acid cleavage was applied directly to intact adenovirus type 5 to achieve denaturation and proteolysis in a single reaction. The speed of the digestion, coupled with the simplicity of MALDI analysis, allowed peptide products to be profiled in less than 5 min. Identification of peptides from a range of proteins by MALDI-TOF confirms that both denaturation and proteolysis were achieved using low concentrations of acetic acid (12.5%) and short incubations (1.5 to 2 min) at high temperatures (140° C). These conditions favor production of peptides that carry Asp on their C-termini. When this cleavage reaction is carried out in highly enriched H(2) (18)O, a single atom of (18)O is introduced site-specifically into the carboxyl terminal. The labeling reaction is applied to label reporter peptides from human adenovirus type 5 harvested from HeLa cells. Small peptide products of endogenous processing were also observed.

Identification of beta-lactamase in antibiotic-resistant Bacillus cereus spores.

Beta-lactamase type I is reported for the first time to occur in the sporulated form in a penicillin-resistant Bacillus species. The enzyme was readily characterized from the B. cereus 5/B line (ATCC 13061) by mass spectrometry and two-dimensional gel electrophoresis.

Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network.

Cell interactions with the extracellular matrix (ECM) can regulate multiple cellular activities and the matrix itself in dynamic, bidirectional processes. One such process is local proteolytic modification of the ECM. Invadopodia of tumor cells are actin-rich proteolytic protrusions that locally degrade matrix molecules and mediate invasion. We report that a novel high-density fibrillar collagen (HDFC) matrix is a potent inducer of invadopodia, both in carcinoma cell lines and in primary human fibroblasts. In carcinoma cells, HDFC matrix induced formation of invadopodia via a specific integrin signaling pathway that did not require growth factors or even altered gene and protein expression. In contrast, phosphoproteomics identified major changes in a complex phosphosignaling network with kindlin2 serine phosphorylation as a key regulatory element. This kindlin2-dependent signal transduction network was required for efficient induction of invadopodia on dense fibrillar collagen and for local degradation of collagen. This novel phosphosignaling mechanism regulates cell surface invadopodia via kindlin2 for local proteolytic remodeling of the ECM.

Polo-like kinase 4 autodestructs by generating its Slimb-binding phosphodegron.

Polo-like kinase 4 (Plk4) is a conserved master regulator of centriole assembly. Previously, we found that Drosophila Plk4 protein levels are actively suppressed during interphase. Degradation of interphase Plk4 prevents centriole overduplication and is mediated by the ubiquitin-ligase complex SCF(Slimb/ßTrCP). Since Plk4 stability depends on its activity, we studied the consequences of inactivating Plk4 or perturbing its phosphorylation state within its Slimb-recognition motif (SRM). Mass spectrometry of in-vitro-phosphorylated Plk4 and Plk4 purified from cells reveals that it is directly responsible for extensively autophosphorylating and generating its Slimb-binding phosphodegron. Phosphorylatable residues within this regulatory region were systematically mutated to determine their impact on Plk4 protein levels and centriole duplication when expressed in S2 cells. Notably, autophosphorylation of a single residue (Ser293) within the SRM is critical for Slimb binding and ubiquitination. Our data also demonstrate that autophosphorylation of numerous residues flanking S293 collectively contribute to establishing a high-affinity binding site for SCF(Slimb). Taken together, our findings suggest that Plk4 directly generates its own phosphodegron and can do so without the assistance of an additional kinase(s).

Identification of protein SUMOylation sites by mass spectrometry using combined microwave-assisted aspartic acid cleavage and tryptic digestion.

Small-ubiquitin-like modifier (SUMO) is a posttranslational modifier of protein substrates at lysine residues that conjugates to proteins in response to various changes in the cell. As a result of SUMO modification, marked changes in transcription regulation, DNA repair, subcellular localization and mitosis, among other cellular processes, are known to occur. However, although the identification of ubiquitylation sites by mass spectrometry is aided in part by the presence of a small di-amino acid GlyGly "tag" that remains on lysine residues following tryptic digestion, SUMOylation poses a particular challenge as the absence of a basic residue near to the SUMO C-terminus results in a significant 27 or 32-amino-acid sequence branch conjugated to the substrate peptide. MS/MS analyses of these branch peptides generally reveal abundant fragment ions resulting from cleavage of the SUMO tail, but which obscure those needed for characterizing the target peptide sequence. Other approaches for identifying SUMO substrates exist and include overexpression of the SUMO isoforms using an N-terminal histidine tag, as well as site-directed mutagenesis of the C-terminal end of the SUMO sequence. Here, we employ combined enzymatic/chemical approaches, which serve to shorten the SUMO tag and thus help to simplify SUMO spectra, making interpretation of mass spectra and location of the SUMOylation site easier. As described in this report, we demonstrate a method for identifying SUMOylation sites using three commercially available SUMO- modified isoforms and by employing acid-only and acid/trypsin cleavage strategies. These approaches were carried out using MALDI-time-of-flight (TOF) and LC/MS instrumentation, along with collision induced dissociation (CID) and electron transfer dissociation (ETD).

Side chain specificity of ADP-ribosylation by a sirtuin.

Endogenous mono-ADP-ribosylation in eukaryotes is involved in regulating protein synthesis, signal transduction, cytoskeletal integrity, and cell proliferation, although few cellular ADP-ribosyltransferases have been identified. The sirtuins constitute a highly conserved family of protein deacetylases, and several family members have also been reported to perform protein ADP-ribosylation. We characterized the ADP-ribosylation reaction of the nuclear sirtuin homolog Trypanosoma brucei SIR2-related protein 1 (TbSIR2RP1) on both acetylated and unacetylated substrates. We demonstrated that an acetylated substrate is not required for ADP-ribosylation to occur, indicating that the reaction performed by TbSIR2RP1 is a genuine enzymatic reaction and not a side reaction of deacetylation. Biochemical and MS data showed that arginine is the major ADP-ribose acceptor for unacetylated substrates, whereas arginine does not appear to be the major ADP-ribose acceptor in reactions with acetylated histone H1.1. We performed combined ab initio quantum mechanical/molecular mechanical molecular dynamics simulations, which indicated that sirtuin ADP-ribosylation at arginine is energetically feasible, and involves a concerted mechanism with a highly dissociative transition state. In comparison with the corresponding nicotinamide cleavage in the deacetylation reaction, the simulations suggest that sirtuin ADP-ribosylation would be several orders slower but less sensitive to nicotinamide inhibition, which is consistent with experimental results. These results suggest that TbSIR2RP1 can perform ADP-ribosylation using two distinct mechanisms, depending on whether or not the substrate is acetylated.

Evaluation of microwave-accelerated residue-specific acid cleavage for proteomic applications.

Microwave-accelerated proteolysis using acetic acid has been shown to occur specifically on either or both sides of aspartic acid residues. This chemical cleavage has been applied to ovalbumin and several model peptides to test the effect on some of the more common post-translational modifications. No oxidation of methionine or cysteine was observed; however, hydrolysis of phosphate groups proceeds at a detectable rate. Acid cleavage was also extended to the yeast ribosome model proteome, where it provided information on 74% of that proteome. Aspartic acid occurs across the proteome with approximately half the frequency of the combined occurrence of the trypsin residues lysine and arginine, and implications of this are considered.

Analysis of a model virus using residue-specific chemical cleavage and MALDI-TOF mass spectrometry.

A nonenzymatic proteomics strategy is applied to the rapid identification of viruses. The approach provides solubilization and subsequent digestion of viral coat proteins in under 30 s. Acid digestions were carried out using a laboratory-quality microwave system equipped with temperature, pressure, and power controls, which allowed for precise optimization of experimental parameters. Under optimal conditions, this method provides an efficient alternative to traditional enzymatic digestion-based methods for virus identification. Following rapid microwave heating of a suspension of a model virus, RNA bacteriophage MS2, 13 chemical digestion products were detected in parallel with the coat protein precursor using MALDI-TOF MS. Because of the high sequence coverage obtained, the bacteriophage MS2 coat protein was identified with high confidence and the specificity of the identification allowed for the discrimination between bacteriophage MS2 and other closely related RNA bacteriophages.

Rapid chemical digestion of small acid-soluble spore proteins for analysis of Bacillus spores.

A method for the rapid identification of Bacillus spores is proposed, based on the selective release and chemical digestion of small, acid-soluble spore proteins (SASPs). Microwave-assisted acid hydrolysis of SASPs from B. anthracis str. Sterne and B. subtilis str. 168 was accomplished in a single step requiring only 90 s of heating. The peptide products of the chemical digestion were identified by postsource decay sequencing with a MALDI-TOF-MS equipped with a curved-field reflectron. The specificity of the observed SASP peptides was evaluated using a cross-species sequence search. The incomplete nature of the acid digestion under these conditions allowed detection of the digest products along with the proteins from which they originated, which increased species identification confidence. The feasibility of this approach for the rapid identification of Bacillus species was further demonstrated by analyzing a mixture of B. subtilis str. 168 and B. anthracis str. Sterne spores.