Blood serum is a complex body fluid that contains various proteins ranging in concentration over at least 9 orders of magnitude. Using a combination of mass spectrometry technologies with improvements in sample preparation, we have performed a proteomic analysis with submilliliter quantities of serum and increased the measurable concentration range for proteins in blood serum beyond previous reports. We have detected 490 proteins in serum by on-line reversed-phase microcapillary liquid chromatography coupled with ion trap mass spectrometry. To perform this analysis, immunoglobulins were removed from serum using protein A/G, and the remaining proteins were digested with trypsin. Resulting peptides were separated by strong cation exchange chromatography into distinct fractions prior to analysis. This separation resulted in a 3-5-fold increase in the number of proteins detected in an individual serum sample. With this increase in the number of proteins identified we have detected some lower abundance serum proteins (ng/ml range) including human growth hormone, interleukin-12, and prostate-specific antigen. We also used SEQUEST to compare different protein databases with and without filtering. This comparison is plotted to allow for a quick visual assessment of different databases as a subjective measure of analytical quality. With this study, we have performed the most extensive analysis of serum proteins to date and laid the foundation for future refinements in the identification of novel protein biomarkers of disease.Molecular & Cellular Proteomics 1:947-955, 2002.

No abstract

No abstract

We have investigated the effects of DNA damage by (؎)-anti-benzo[a]pyrene diol epoxide (BPDE) and UV light on the formation of a positioned nucleosome in the Xenopus borealis 5S rRNA gene. Gel-shift analysis of the reconstituted products indicates that BPDE damage facilitates the formation of a nucleosome onto this sequence. Competitive reconstitution experiments show that average levels of 0.5, 0.9, and 2.1 BPDE adducts͞146 bp of 5S DNA (i.e., the size of DNA associated with a nucleosome core particle) yield changes of ؊220, ؊290, and ؊540 cal͞mol, respectively, in the free energy (⌬G) of nucleosome formation. These values yield increases of core histone binding to 5S DNA (K a ) of 1.4-, 1.6-, and 2.5-fold, compared with undamaged DNA. Conversely, irradiation with UV light decreases nucleosome formation. Irradiation at either 500 or 2500 J͞m 2 of UV light [0.6 and 0.8 cyclobutane pyrimidine dimer͞146 bp (on average), respectively] results in respective changes of ؉130 and ؉250 cal͞mol. This translates to decreases in core histone binding to irradiated 5S DNA (K a ) of 1.2-and 1.5-fold compared with undamaged DNA. These results indicate that nucleosome stability can be markedly affected by the formation of certain DNA lesions. Such changes could have major effects on the kinetics of DNA processing events.

An alternative sustainable fuel, biomass-derived fast pyrolysis oil or “bio-oil”, is coming into the market in Europe. Fast pyrolysis pilot and demonstration plants for fuel applications producing tonnes of bio-oil are in operation, and commercial plants are under design. There will be increasingly larger amounts of bio-oil transportation on water and by land, leading to a need for further specifications and supporting documentation. The properties of bio-oil are different from conventional liquid fuels and, therefore, may need to overcome both technical and marketing hurdles for its acceptability in the fuels market. Multiple material safety data sheets (MSDSs) are currently being used by different producers, but there is a desire to update these as more information becomes available. In order to standardize bio-oil, quality specifications are being adopted. The first bio-oil burner fuel standard in ASTM D7544 was approved in 2010. CEN standardization has been initiated in Europe. In the EU, a new chemical regulation system REACH (Registration, Evaluation and Authorisation of Chemicals) exists. Registration under REACH has to be perfomed if bio-oil is produced or imported into the EU. In the USA and Canada, bio-oil has to be filed under the TSCA (US Toxic Substances Control Act) and DSL (Domestic Substance List), respectively. In this paper, the state of the art on standardization is discussed, and new data for the transportation guidelines is presented. The focus is on flammability and toxicity.

The Xenopus borealis somatic 5S ribosomal RNA gene was used as a model system to determine the mutual effects of nucleosome folding and formation of ultraviolet (UV) photoproducts (primarily cis-syn cyclobutane pyrimidine dimers, or CPDs) in chromatin. We analyzed the preferred rotational and translational settings of 5S rDNA on the histone octamer surface after induction of up to 0.8 CPD/nucleosome core (2.5 kJ/m(2) UV dose). DNase I and hydroxyl radical footprints indicate that UV damage at these levels does not affect the average rotational setting of the 5S rDNA molecules. Moreover, a combination of nuclease trimming and restriction enzyme digestion indicates the preferred translational positions of the histone octamer are not affected by this level of UV damage. We also did not observe differences in the UV damage patterns of irradiated 5S rDNA before or after nucleosome formation, indicating there is little difference in the inhibition of nucleosome folding by specific CPD sites in the 5S rRNA gene. Conversely, nucleosome folding significantly restricts CPD formation at all sites in the three helical turns of the nontranscribed strand located in the dyad axis region of the nucleosome, where DNA is bound exclusively by the histone H3-H4 tetramer. Finally, modulation of the CPD distribution in a 14 nt long pyrimidine tract correlates with its rotational setting on the histone surface, when the strong sequence bias for CPD formation in this tract is minimized by normalization. These results help establish the mutual roles of histone binding and UV photoproducts on their formation in chromatin.

Assessment of differential protein abundance from the observed properties of detected peptides is an essential part of protein profiling based on shotgun proteomics. However, the abundance observed for shared peptides may be due to contributions from multiple proteins that are affected differently by a given treatment. Excluding shared peptides eliminates this ambiguity but may significantly decrease the number of proteins for which abundance estimates can be obtained. Peptide sharing within a family of biologically related proteins does not cause ambiguity if family members have a common response to treatment. On the basis of this concept, we have developed an approach for including shared peptides in the analysis of differential protein abundance in protein profiling. Data from a recent proteomics study of lung tissue from mice exposed to lipopolysaccharide, cigarette smoke, and a combination of these agents are used to illustrate our method. Starting from data where about half of the implicated database protein involved shared peptides, 82% of the affected proteins were grouped into families, based on FASTA annotation, with closure on peptide sharing. In many cases, a common abundance relative to control was sufficient to explain ion-current peak areas for peptides, both unique and shared, that identified biologically related proteins in a peptide-sharing closure group. On the basis of these results, we propose that peptide-sharing closure groups provide a way to include abundance data for shared peptides in quantitative protein profiling by high-throughput mass spectrometry.

Most naturally occurring mammalian cancers and immortalized tissue culture cell lines share a common characteristic, the overexpression of full-length HMGA1 (high mobility group A1) proteins. The HMGA1 protooncogene codes for two closely related isoform proteins, HMGA1a and HMGA1b, and causes cancerous cellular transformation when overexpressed in either transgenic mice or "normal" cultured cell lines. Previous work has suggested that the in vivo types and patterns of the HMGA1 post-translational modifications (PTMs) differ between normal and malignant cells. The present study focuses on the important question of whether HMGA1a and HMGA1b proteins isolated from the same cell type have identical or different PTM patterns and also whether these isoform patterns differ between nonmalignant and malignant cells. Two independent mass spectrometry methods were used to identify the types of PTMs found on specific amino acid residues on the endogenous HMGA1a and HMGA1b proteins isolated from a non-metastatic human mammary epithelial cell line, MCF-7, and a malignant metastatic cell line derived from MCF-7 cells that overexpressed the transgenic HMGA1a protein. Although some of the PTMs were the same on both the HMGA1a and HMGA1b proteins isolated from a given cell type, many other modifications were present on one but not the other isoform. Furthermore, we demonstrate that both HMGA1 isoforms are di-methylated on arginine and lysine residues. Most importantly, however, the PTM patterns on the endogenous HMGA1a and HMGA1b proteins isolated from nonmetastatic and metastatic cells were consistently different, suggesting that the isoforms likely exhibit differences in their biological functions/activities in these cell types.Overexpression of the HMGA1 gene (formerly known as HMGIY (1)) is such a consistent feature of tumors that it has been suggested to be a "diagnostic" biochemical marker of both neoplastic transformation and cancer progression (2-8). Elevated levels of HMGA1 gene products and HMGA1 proteins have been observed in almost every cancer type investigated and are correlated with increasing degrees of malignancy and metastatic potential of a number of cancer types (6, 9, 10).Additionally, it was demonstrated that overexpression of HMGA1 proteins in "normal" rat 1a cells and a human breast adenocarcinoma cell line caused neoplastic transformation and malignant metastatic progression of these cells, respectively (11,12). Together, these results demonstrate that overexpression of full-length HMGA1 proteins in tumor cells is extremely widespread and biologically important.The HMGA1a (formerly known as HMG-I) and HMGA1b (formerly known as HMG-Y) isoform proteins are architectural transcription factors that are derived from alternatively spliced mRNA transcripts coded for by the HMGA1 gene located on human chromosome 6 (locus 6p21) with HMGA1b (95 amino acids; ϳ10.6 kDa) containing an internal 11-amino acid deletion compared with HMGA1a (106 amino acids; ϳ11.5 kDa) (13). Both isoforms contain three independent DNA-bin...