Maureen T. Kachman

Proteomic Analysis of the Spore Coats of Bacillus subtilis and Bacillus anthracis

The outermost proteinaceous layer of bacterial spores, called the coat, is critical for spore survival, germination, and, for pathogenic spores, disease. To identify novel spore coat proteins, we have carried out a preliminary proteomic analysis of Bacillus subtilis and Bacillus anthracis spores, using a combination of standard sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation and improved two-dimensional electrophoretic separations, followed by matrix-assisted laser desorption ionization-time of flight and/or dual mass spectrometry. We identified 38 B. subtilis spore proteins, 12 of which are known coat proteins. We propose that, of the novel proteins, YtaA, YvdP, and YnzH are bona fide coat proteins, and we have renamed them CotI, CotQ, and CotU, respectively. In addition, we initiated a study of coat proteins in B. anthracis and identified 11 spore proteins, 6 of which are candidate coat or exosporium proteins. We also queried the unfinished B. anthracis genome for potential coat proteins. Our analysis suggests that the B. subtilis and B. anthracis coats have roughly similar numbers of proteins and that a core group of coat protein species is shared between these organisms, including the major morphogenetic proteins. Nonetheless, a significant number of coat proteins are probably unique to each species. These results should accelerate efforts to develop B. anthracis detection methods and understand the ecological role of the coat.

Two-dimensional liquid separations-mass mapping of proteins from human cancer cell lysates.

A review of two-dimensional (2D) liquid separation methods used in our laboratory to map the protein content of human cancer cells is presented herein. The methods discussed include various means of fractionating proteins according to isoelectric point (pI) in the first dimension. The proteins in each pI fraction are subsequently separated using nonporous (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC). The liquid eluent of the RP-HPLC separation is directed on-line into an electrospray ionization time-of-flight (ESI-TOF) mass spectrometer where an accurate value of the protein intact M(r) can be obtained. The result is a 2D map of pI versus M(r) analogous to 2D gel electrophoresis; however the highly accurate and reproducible M(r) serves as the basis for interlysate comparisons. In addition, the use of liquid separations allows for the collection of hundreds of purified proteins in the liquid phase for further analysis via peptide mass mapping using matrix assisted laser desorption ionization TOF MS. A description of the methodology used and its applications to analysis of several types of human cancer cell lines is described. The potential of the method for differential proteomic analysis for the identification of biomarkers of disease is discussed.

A protein molecular weight map of ES2 clear cell ovarian carcinoma cells using a two-dimensional liquid separations/mass mapping technique

A molecular weight map of the protein content of ES2 human clear cell ovarian carcinoma cells has been produced using a two‐dimensional (2‐D) liquid separations/mass mapping technique. This method uses a 2‐D liquid separation of proteins from whole cell lysates coupled on‐line to an electrospray ionization‐time of flight (ESI‐TOF) mass spectrometer to map the accurate intact molecular weight (Mr) of the protein content of the cells. The two separation dimensions involve the use of liquid isoelectric focusing as the first phase and nonporous silica reversed‐phase high‐performance liquid chromatography (HPLC) as the second phase of separation. The detection by ESI‐TOF‐MS provides an image of pI versus Mr analogous to 2‐D gel electrophoresis. Each protein is then identified based upon matrix‐assisted laser desorption/ionization (MALDI)‐TOF‐MS peptide mapping and intact Mr so that a standard map is produced against which other ovarian carcinoma cell lines can be compared. The accurate intact Mr together with the pI fraction, and peptide map serve to tag the protein for future interlysate comparisons. An internal standard is also used to provide a means for quantitation for future interlysate studies. In the ES2 cell line under study it is shown that nearly 900 Mr bands are detected over 17 pI fractions from pH 4 to 12 and a Mr range up to 85 kDa and that around 290 of these bands can be identified using mass spectrometric based techniques. The protein Mr is detected within an accuracy of 150 ppm and it is shown that many of the proteins in this human cancer sample are modified compared to the database. The protein Mr map may serve as a highly reproducible standard Web‐based method for comparing proteins from related human cell lines.

Quantitative Proteomics Analysis of Cell Cycle-regulated Golgi Disassembly and Reassembly

During mitosis, the stacked structure of the Golgi undergoes a continuous fragmentation process. The generated mitotic fragments are evenly distributed into the daughter cells and reassembled into new Golgi stacks. This disassembly and reassembly process is critical for Golgi biogenesis during cell division, but the underlying molecular mechanism is poorly understood. In this study, we have recapitulated this process using an in vitro assay and analyzed the proteins associated with interphase and mitotic Golgi membranes using a proteomic approach. Incubation of purified rat liver Golgi membranes with mitotic HeLa cell cytosol led to fragmentation of the membranes; subsequent treatment of these membranes with interphase cytosol allowed the reassembly of the Golgi fragments into new Golgi stacks. These membranes were then used for quantitative proteomics analyses by combining the isobaric tags for relative and absolute quantification approach with OFFGEL isoelectric focusing separation and liquid chromatography-matrix assisted laser desorption ionization-tandem mass spectrometry. In three independent experiments, a total of 1,193 Golgi-associated proteins were identified and quantified. These included broad functional categories, such as Golgi structural proteins, Golgi resident enzymes, SNAREs, Rab GTPases, cargo, and cytoskeletal proteins. More importantly, the combination of the quantitative approach with Western blotting allowed us to unveil 84 proteins with significant changes in abundance under the mitotic condition compared with the interphase condition. Among these proteins, several COPI coatomer subunits (α, β, γ, and δ) are of particular interest. Altogether, this systematic quantitative proteomic study revealed candidate proteins of the molecular machinery that control the Golgi disassembly and reassembly processes in the cell cycle.

Genotyping of Apolipoprotein E by Matrix- assisted Laser Desorption/Ionization Time-of- flight Mass Spectrometry

The genotyping of the various isoforms of Apolipoprotein E (apo E) has been performed using matrix-assisted laser desorption/ionization (MALDI-MS). The polymerase chain reaction was used to amplify the specific apo E gene sequence followed by digestion with Cfo I (Clostridium formicoaceticum), for generating restriction fragments for rapid and accurate mass analysis. An exonuclease I digestion step was introduced to remove the unused primers after PCR, which can otherwise interfere in the mass spectral analysis. By replacing the gel electrophoresis detection step with MALDI-MS, restriction isotyping of the apo E gene was achieved. Genotyping of an unknown sample and obtained from an independent diagnostic laboratory demonstrated the validity of the MALDI-MS method for the routine analysis of apo E.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950–Metabolites in Frozen Human Plasma

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950–Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Microparticle surface protein are associated with experimental venous thrombosis: a preliminary study.

Microparticles are small membrane vesicles released from activated cells and are associated with thrombosis and inflammation. Microparticle contain a unique subset of surface protein derived form the parent cell and may be responsible for their diverse biological functions. To identify these proteins, juvenile baboons (Papio anubis, n = 4) underwent iliac vein thrombosis with 6-hour balloon occlusion. Plasma samples were taken at baselines and at 2 days postthrombosis for microparticle analysis. Microparticles were extracted from platelet-poor plasma, digest separately with trypsin and tagged using isobaric tagging for relative and absolute quantitation reagents. The digests were subjected to 2-dimensional liquid chromatographic separation followed by matrix-assisted laser desorption/ionization tandem mass spectrometry. Peak lists were generated and searched against all primate sequences. For protein identity, a minimum of 2 peptides at 95% confidence interval was required. Later, isobaric tagging for relative and absolute quantitation ratios were generated comparing relative protein level of day 2 to baseline. The proteomic analysis was performed twice for each blood sample, totaling 8 experiments. Proteins were considered elevated of depressed if the isobaris tagging for relative and absolute quantitation ratio deviated by 20% changes from normal and a P value less than .05. Significantly, 7 proteins were differentially expressed on day 2 compared to baseline, and appeared in at least 3 animals and regulated in at least 4 experiment. Among these 7 proteins, upregulated proteins include various forms of fibrinogen and alpha-1-antichymotrypsin and downregulated proteins include immunoglobulins. These proteins influence thrombosis and inflammation through hemostatic plug formation (fibrinogen), inhibiting neutrophil adhesion (alpha-1-antichymoptrypsin), and immunoregulation (immunoglobulins). Further studies are needed to confirm the mechanistic role of these proteins in the pathogenesis of venous thrombosis.

Co-production of two new peptide antibiotics by a bacterial isolate Paenibacillus alvei NP75.

Two new peptide antibiotics were secreted by a Gram-positive bacterial strain isolated from fermented tomato fruit. Based on its 99% 16S rDNA sequence similarity with Paenibacillus alvei, the isolate was designated as P. alvei NP75. Among these two peptides, one is active against Gram-positive pathogens while the other against Gram-negative pathogens; thus these peptides were named as paenibacillin P and paenibacillin N, respectively. After the purification of those peptide antibiotics from the cell free culture supernatant by RP-HPLC, they were analyzed for their temperature sensitivity and susceptibility to proteases. Higher-temperature tolerant paenibacillin N was easily degraded by proteinase K, while the temperature sensitive paenibacillin P was not affected by any of the proteases used in this study other than a specific protease that was secreted by the same NP75 strain. Mass-spectrometry analysis of the above peptide antibiotics further confirmed their distinction among the known peptide antibiotics. We are reporting first of its kind the co-production of two different new peptide antibiotics from a single bacterial isolate of P. alvei strain.

Comparative analysis of the extracellular proteomes of two Clostridium sordellii strains exhibiting contrasting virulence

Clostridium sordellii is a toxin-producing anaerobic bacillus that causes severe infections in humans and livestock. C. sordellii infections can be accompanied by a highly lethal toxic shock syndrome (TSS). Lethal toxin (TcsL) is an important mediator of TSS. We recently obtained a clinical strain of C. sordellii (DA-108) lacking the TcsL-encoding tcsL gene, which was not fatal in rodent models of infection, in contrast to a tcsL(+) reference strain (ATCC9714). Protein preparations derived from cell-free, stationary phase cultures obtained from ATCC9714 were lethal when injected into mice, while those obtained from DA-108 were not, a difference that was attributed to the unique presence of TcsL in the ATCC9714-derived proteins. We questioned whether there were other major differences between the extracellular proteomes of these two strains, apart from TcsL. Two-dimensional gel electrophoresis was conducted using crude cell-free supernatants from these strains and 14 differentially expressed proteins were subjected to mass spectrometric analysis. Nine of these 14 proteins were more highly expressed by DA-108 and 5 by ATCC9714. Twelve of the 14 proteins isolated from the 2-D gels were putatively identified by mass spectrometry. Several of these proteins were identical, possibly reflecting enzymatic cleavage, degradation, and/or post-translational modifications. Excluding identical sequences, only 5 unique proteins were identified. Four proteins (ferredoxin-nitrite reductase; formate acetyltransferase; Translation Elongation Factor G; and purine nucleoside phosphorylase) were over-expressed by DA-108 and 1 (N-acetylmuramoyl-l-alanine amidase) by ATCC9714. These results support the concept that TcsL is the major determinant of C. sordellii TSS during infection.

Glioblastoma Therapy Can Be Augmented by Targeting IDH1-Mediated NADPH Biosynthesis

NADPH is a critical reductant needed in cancer cells to fuel the biosynthesis of deoxynucleotides and antioxidants and to sustain stress-survival responses after radiation-induced DNA damage. Thus, one rational strategy to attack cancer cells is to target their heavy reliance on NADPH. Here, we report that the isocitrate dehydrogenase IDH1 is the most strongly upregulated NADPH-producing enzyme in glioblastoma (GBM). IDH1 silencing in GBM cells reduced levels of NADPH, deoxynucleotides, and glutathione and increased their sensitivity to radiation-induced senescence. Rescuing these metabolic restrictions was sufficient to reverse IDH1-mediated radiosensitization. In a murine xenograft model of human GBM, we found that IDH1 silencing significantly improved therapeutic responses to fractionated radiotherapy, when compared with either treatment alone. In summary, our work offers a mechanistic rationale for IDH1 inhibition as a metabolic strategy to improve the response of GBM to radiotherapy. Cancer Res; 77(4); 960-70. ©2016 AACR.