Total protein extraction for metaproteomics analysis of methane producing biofilm: the effects of detergents.

Protein recovery is crucial for shotgun metaproteomics to study the in situ functionality of microbial populations from complex biofilms but still poorly addressed by far. To fill this knowledge gap, we systematically evaluated the sample preparation with extraction buffers comprising four detergents for the metaproteomics analysis of a terephthalate-degrading methanogenic biofilm using an on-line two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS) system. Totally, 1018 non-repeated proteins were identified with the four treatments. On the whole, each treatment could recover the biofilm proteins with specific distributions of molecular weight, hydrophobicity, and isoelectric point. The extraction buffers containing zwitterionic and anionic detergents were found to harvest the proteins with better efficiency and quality, allowing identification up to 76.2% of total identified proteins with the LC-MS/MS analysis. According to the annotation with a relevant metagenomic database, we further observed different taxonomic profiles of bacterial and archaeal members and discriminable patterns of the functional expression among the extraction buffers used. Overall, the finding of the present study provides first insight to the effect of the detergents on the characteristics of extractable proteins from biofilm and the developed protocol combined with nano 2D-LC/MS/MS analysis can improve the metaproteomics studies on microbial functionality of biofilms in the wastewater treatment systems.

Community and proteomic analysis of methanogenic consortia degrading terephthalate.

Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122-130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H(2)/CO(2), and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.

Magnetic bead-based hydrophilic interaction liquid chromatography for glycopeptide enrichments.

Purification of glycopeptides prior to the analysis by mass spectrometry (MS) is demanded due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among various purification methods, hydrophilic interaction liquid chromatography (HILIC) has become a popular method in recent years. In this work, we reported a novel magnetic bead-based zwitterionic HILIC (ZIC-HILIC) material which was fabricated by coating a zwitterionic polymer synthesized by spontaneous acid-catalyzed polymerization of 4-vinyl-pyridinium ethanesulfonate monomer on iron oxide magnetic nanoparticles. The resulting magnetic ZIC-HILIC nanoparticles were shown to provide high specificity and high recovery yield (95-100%) for the enrichment of glycopeptides from a standard glycoprotein, fetuin, using a simple magnetic bar. In addition, we proposed a two-step HILIC enrichment strategy using magnetic ZIC-HILIC nanoparticles for a large scale analysis of glycoproteins in complex biological samples. Using this approach, we identified 85 N-glycosylation sites in 53 glycoproteins from urine samples. Two novel glycosylation sites on N513 of uromodulin and N470 of lysosomal alpha-glucosidase which have not yet been reported were identified by two-step HILIC approach. Furthermore, all these identified sites were confirmed by studies conducted using PNGase F deglycosylation and 18O enzymatic labeling.

Quantitative analysis of estrogens and estrogen metabolites in endogenous MCF-7 breast cancer cells by liquid chromatography-tandem mass spectrometry.

To study the roles of estrogens and estrogen metabolites (EMs) in breast carcinogenesis, we reported a quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) method utilizing selective reaction mode (SRM) to analyze estrogens and EMs in the extracellular and intracellular compartments of endogenous MCF-7 breast cancer cells through simple ethyl acetate (EA) extraction and dansyl chloride derivatization. Under a 35-min LC gradient elution on a reversed phase C18 column, the method was shown to simultaneously quantify 12 estrogens and EMs: estrone (E1) and its 2-, 4-, 16α-hydroxy derivatives (2-OHE1, 4-OHE1, 16α-OHE1), and 2-, 4-methoxy derivatives (2-MeOE1, 4-MeOE1); 17ß-estradiol (E2) and its 2-, 4-hydroxy derivative (2-OHE2, 4-OHE2) and 2- and 4-methoxy derivatives (2-MeOE2 and 4-MeOE2); and estriol (E3), using ethinylestradiol (EE2) as the internal standard (IS). Using a calibration curve-standard addition hybrid method, we were able to determine the amount of estrogens and EMs in not only the treated cells but also the non-treated cells. The limits of quantification (LOQs) were determined to range from 0.05-80 pg on column with an inter-batch accuracy around 72-123% and precision around 1-10%. Results indicated that trace amounts (<0.9 fg/cell) of E1 and E2 were present in both the extra- and intra-cellular compartments under non-treated condition but DMSO could induce E1 and E2 as well as trace amounts (<2.25 fg/cell) of EMs in the cell. E2 treatment substantially increased not only E1 and E2 in the intra-cellular (60 fg/cell) and extra-cellular (3000 fg/cell) compartment but also substantially induced EMs primarily in the extracellular compartment (0.6-25 fg/cell). These data implied that EMs could be quickly generated and distributed to the extracellular compartment by E2 within 24h of treatment and DMSO solvent could potentially induce slight estrogen effects.

Quantitative shot-gun proteomics and MS-based activity assay for revealing gender differences in enzyme contents for rat liver microsome.

Liver microsomes are subcellular fractions that contain many metabolizing enzymes for drugs and endogeneous compounds. Some of these enzymes are regulated by sex hormonal control and exhibit sex-dependent expression pattern and metabolizing speed. Studying these enzymes, however, are complicated by the presence of isoforms such as cytochrome P450 (CYP450), which families share more than 50% amino acid identities. In this study, we applied quantitative shot-gun proteomics approach coupled with stable-isotope dimethyl labeling, two-dimensional reversed-phase peptide separation and tandem mass spectrometry (MS) to explore the gender-dependent expression of rat liver microsomal proteins. A total of 391 proteins were identified and quantified by this approach, and 56% of quantified proteins were enzymes. Although shot-gun approach is rarely used for identifying protein isoforms, we identified 53 isoforms by at least one unique peptide including 21 isoforms of CYP450s. Moreover, by quantitative and statistics assessment, we were able to classify them into 28 male dominant enzymes including CYP2C12 CYP2C11, CYP2C13, CYP2B3, CYP2C11, CYP2C70 and CYP3A2 which are known to be male specific, 21 female dominant enzymes including CYP2A1, CYP2C7, CYP2C12, CYP2D26, alcohol dehydrogenase 1, carboxylesterase 3, glutathione S-transferase, liver carboxylesterase 4, UDP-glucuronosyltransferase 2B1, and glyceraldehyde-3-phosphate dehydrogenase which are known to be female specific; and 125 sex-independent enzymes. However, most of the sex specificities revealed from this study, such as the male specificity of CYP2D1, were novel and not yet reported. We then conducted a mass spectrometry-multiple reaction mode (MS-MRM) based enzyme activity method to determine the catalyzing rate of CYP2D1 in male and female liver microsomes using carteolol as its specific substrate. The reaction rate catalyzed by CYP2D1 in female rats was determined to differ significantly with the rate in male rats. Moreover, the ratio (female/male) of reaction rate (0.68) was found to correlate with their relative protein abundance (0.72). This study revealed novel sex dependences of many rat liver enzymes and also demonstrated a unique MS-based analytical platform that could identify novel iso-enzymes and further quantify their abundance and enzyme activity.

Urinary CD14 as a potential biomarker for benign prostatic hyperplasia - discovery by combining MALDI-TOF-based biostatistics and ESI-MS/MS-based stable-isotope labeling.

PURPOSE: Quest for specific urinary biomarkers for benign prostatic hyperplasia (BPH). EXPERIMENTAL DESIGN: Proteomics studies were conducted with urines of the training set to discovering marker candidates that could differentiate BPH from normal subjects by matching results deduced from MALDI-TOF of individual samples and results deduced from nanoLC-ESI-MS/MS-based stable isotope dimethyl labeling of two pooled samples (BPH and normal). Samples were digested before analysis and such an approach takes into account the subject-to-subject variation and differential amount, as well as protein identification. Selected markers were validated by ELISA conducted on the training set and the test set as well as another set of urines collected from prostate cancer patients. RESULTS: Nine marker candidates were identified from proteomics studies; CD14, prostate-specific antigen and pancreatic α-amylase precursor were further selected for ELISA validation. Urinary CD14 is among the best match with high specificity (>81%) for both training and test sets. In addition, from the study of prostate cancer patients, CD14 also allows the distinction of BPH from cancer with high specificity (84-100%) when combined with urinary prostate-specific antigen. CONCLUSIONS AND CLINICAL RELEVANCE: Urinary CD14 is suggested to have a high specificity in the diagnosis of BPH in distinction from normal as well as cancer subjects.

The water-soluble Roussin's red ester acting as a potential photochemical NO-delivery agent: photolysis reactions, DNA cleavage and anticancer activity.

The water-soluble Roussin's red ester [(NO)(2)Fe(mu-SCH(2)CH(2)P(O)(CH(2)OH)(2))(2)Fe(NO)(2)] (1), a potential photochemical prodrug of an NO precursor, was synthesized from the reaction of HSCH(2)CH(2)P(O)(CH(2)OH)(2) (F) and [Fe(CO)(2)(NO)(2)]. The IR v(NO) stretching frequencies of complex 1 appear at 1759 (s), 1784 (s) and 1816 (w) cm(-1) in buffer (pH = 7.4). NO was released with a stoichiometry ratio Delta[NO]/Delta[1] = 3.6 +/- 0.2 when complex 1 was exposed to UV in deaerated aqueous phosphate buffer solution. Here light acts as an On/Off switch for NO release. Incubation of pBR322 supercoiled DNA with complex 1, followed by irradiation, produced DNA strand breakage. In contrast to the addition of carboxy-PTIO (NO radical scavenger), DNA strand breakage was not inhibited when the scavengers of hydroxyl radical and singlet oxygen were added. Complex 1 irradiated under a N(2) atmosphere exhibited the same cleavage efficiency as complex 1 irradiated under air. The results show that DNA strand cleavage efficiency depends on the concentration of complex 1, the pH value of the buffer, and the duration of the photolysis of complex 1. The conversion rate from supercoiled (SC form) to nicked circular (NC form) of complex 1 was 2.96 x 10(-2) s(-1). The results of a T4 ligase enzymatic assay reveals the nonhydrolytic DNA breakage mechanism. The NO-release ability of complexes 1, 2, and 3 follows the order 1 > 2 > 3. Upon UV-irradiation, complex 1 exhibits cytotoxicity against B16-F10 mouse melanoma cells.

Secondary structure, phospholipid membrane interactions, and fusion activity of two glutamate-rich analogs of influenza hemagglutinin fusion peptide.

Two synthetic mutants of influenza HA2 fusion peptide (residues 1-25), containing Glu on the polar (residues 4,8-E5(4,8)) or the hydrophobic (residues 3,7-E5(3,7)) face of the amphipathic helix, were synthesized and labeled with NBD at the N-terminus. Introduction of Glu residues into the fusion peptide leads to increased sensitivity of various biochemical properties to pH compared to the wild type. The E5 peptides showed a decrease of alpha-helix content and increase of beta-sheet structure. Lipid binding was diminished, but not abolished even at high pH. The E5 analogs penetrate the lipid bilayer less deeply than the wild type, especially at high pH. The N-terminal half of the peptide showed significant variation of the depth of the penetration into the lipid bilayer. Both E5 peptides were fusion active. The properties of E5(3,7) were more affected by the Glu substitution and showed greater variation with pH than E5(4,8).