Bovine Liver Extract Research Articles

Mobile Phase Contaminants Affect Neutral Lipid Analysis in LC-MS-Based Lipidomics Studies.

Lipidomics is a well-established field, enabled by modern liquid chromatography mass spectrometry (LC-MS) technology, rapidly generating large amounts of data. Lipid extracts derived from biological samples are complex, and most spectral features in LC-MS lipidomics data sets remain unidentified. In-depth analyses of commercial triacylglycerol, diacylglycerol, and cholesterol ester standards revealed the expected ammoniated and sodiated ions as well as five additional unidentified higher mass peaks with relatively high intensities. The identities and origin of these unknown peaks were investigated by modifying the chromatographic mobile-phase components and LC-MS source parameters. Tandem MS (MS/MS) of each unknown adduct peak yielded no lipid structural information, producing only an intense ion of the adducted species. The unknown adducts were identified as low-mass contaminants originating from methanol and isopropanol in the mobile phase. Each contaminant was determined to be an alkylated amine species using their monoisotopic masses to calculate molecular formulas. Analysis of bovine liver extract identified 33 neutral lipids with an additional 73 alkyl amine adducts. Analysis of LC-MS-grade methanol and isopropanol from different vendors revealed substantial alkylated amine contamination in one out of three different brands that were tested. Substituting solvents for ones with lower levels of alkyl amine contamination increased lipid annotations by 36.5% or 27.4%, depending on the vendor, and resulted in >2.5-fold increases in peak area for neutral lipid species without affecting polar lipid analysis. These findings demonstrate the importance of solvent selection and disclosure for lipidomics protocols and highlight some of the major challenges when comparing data between experiments.

Enhancing the Signal-to-Noise of Diagnostic Fragment Ions of Unsaturated Glycerophospholipids via Precursor Exclusion Ultraviolet Photodissociation Mass Spectrometry (PEx-UVPD-MS).

Understanding and elucidating the diverse structures and functions of lipids has motivated the development of many innovative tandem mass spectrometry (MS/MS) strategies. Higher-energy activation methods, such as ultraviolet photodissociation (UVPD), generate unique fragment ions from glycerophospholipids that can be used to perform in-depth structural analysis and facilitate the deconvolution of isomeric lipid structures in complex samples. Although detailed characterization is central to the correlation of lipid structure to biological function, it is often impeded by the lack of sufficient instrument sensitivity for highly bioactive but low-abundance phospholipids. Here, we present precursor exclusion (PEx) UVPD, a simple yet powerful technique to enhance the signal-to-noise (S/N) of informative low-abundance fragment ions produced from UVPD of glycerophospholipids. Through the exclusion of the large population of undissociated precursor ions with an MS3 strategy, the S/N of diagnostic fragment ions from PC 18:0/18:2(9Z, 12Z) increased up to an average of 13x for PEx-UVPD compared to UVPD alone. These enhancements were extended to complex mixtures of lipids from bovine liver extract to confidently identify 35 unique structures using liquid chromatography PEx-UVPD. This methodology has the potential to advance lipidomics research by offering deeper structure elucidation and confident identification of biologically active lipids.

High Throughput Semiquantitative UHPSFC-MS/MS Lipid Profiling and Lipid Class Determination.

High throughput and high-resolution lipid analyses are important for many biological model systems and research questions. This comprises both monitoring at the individual lipid species level and broad lipid classes. Here, we present a nontarget semiquantitative lipidomics workflow based on ultrahigh performance supercritical fluid chromatography (UHPSFC)-mass spectrometry (MS). The optimized chromatographic conditions enable the base-line separation of both nonpolar and polar classes in a single 7-minute run. Ionization efficiencies of lipid classes vary 10folds in magnitude and great care must be taken in a direct interpretation of raw data. Therefore, the inclusion of internal standards or experimentally determined Response factors (RF) are highly recommended for the conversion of raw abundances into (semi) quantitative data. We have deliberately developed an algorithm for automatic semiquantification of lipid classes by RF. The workflow was tested and validated using a bovine liver extract with satisfactory results. The RF corrected data provide a more representative relative lipid class determination, but also the interpretation of individual lipid species should be performed on RF corrected data. In addition, semiquantification can be improved by using internal or also external standards when more accurate quantitative data are of interest but this requires validation for all new sample types. The workflow established greatly extends the potential of nontarget UHPSFC–MS/MS based analysis.

Purification of bovine liver transglutaminase by gel filtration

Transglutaminases (TGases) are enzymes that catalyze transfer of acyl group and covalent crosslinks formation between peptide-bound glutaminyl residues and amino groups. TGases have many industrial applications and have been purified from various sources. TGase was purified from the bovine liver extract by gel filtration on Sephacryl S-200 HR column. TGase activity was measured using CBZ-l-glutaminylglycine & hydroxylamine and the enzyme was characterized with respect to its response to different temperatures, pHs and salt concentrations. TGase was purified by yield 36.7%, had a weight 74 kDa, a high pH (pH = 8) and temperature (45 °C) optimum. The enzyme was observed to be stable at temperatures below 55 °C and was stable within a narrow pH range of 6.5–8.0. Purified TGase showed Ca2+ dependent characteristics and tended to retain activity at a high NaCl concentration. These results revealed that purified TGase can be used as a potential alternative to other sources.

Specific PCR method for detection of species origin in biochemical drugs via primers for the ATPase 8 gene by electrophoresis

A PCR method is described to identify the species origin of various animal and human tissue-derived biochemical drugs. Four commercialized drugs, including spermary tablets, compound embryonic bovine liver extract tablets, spleen aminopeptide solution, and placenta polypeptide injection, were used as a proof-of-principle in this study. Primers were designed to amplify conservative regions of mitochondrial cytochrome b and ATPase 8 genes from beef, pork, lamb and human DNA, respectively. The specificity of primers for ATPase 8 gene is found to be higher than those for cytochrome b under the given experimental conditions. The amplicon sizes of ATPase 8 were 212, 271, 293 and 405bp for pork, beef, lamb and human tissue, respectively. The minimum detectable concentration of DNA sample for species identification is 0.05-0.5pg·μL-1. The species origin can be distinguished by this method in extremely low concentrations of template DNAs extracted. Conceivably, this PCR method for meat authentication may be extended to quality control of other biochemical drugs and raw materials. Graphical abstract A specific PCR method was developedfor thedetection of species origin in biochemical drugs via species-specific primers targeting mitochondrial ATPase 8 genes. The PCR products were separated by gel electrophoresis and species origins were indicated by comparison to references.

Locating Carbon-Carbon Double Bonds in Unsaturated Phospholipids by Epoxidation Reaction and Tandem Mass Spectrometry.

The presence of carbon-carbon double bonds (C═Cs) in unsaturated phospholipids is closely related to lipid conformations and physiochemical activities. Previously, we have demonstrated that epoxidation reaction facilitated by low-temperature plasma (LTP) enabled the structural analysis of unsaturated fatty acids (FAs). Epoxidation of the C═C leads to the production of an epoxide, which can be easily cleaved via collision-induced dissociation (CID) to produce diagnostic ions indicative of the C═C bond locations in FAs. In this work, we further developed this method for analysis of phospholipids. Tandem mass spectrometry analysis with epoxidation reaction was performed in both positive and negative ion mode to analyze phosphatidylcholines (PCs), phosphatidic acids (PAs), phosphatidylethanolamines (PEs), phosphatidylglycerols (PGs), and phosphatidylinositols (PIs). The developed method was applied in a shotgun lipidomics approach to characterize phospholipids in a bovine liver extract.

Using In Silico Fragmentation to Improve Routine Residue Screening in Complex Matrices.

Targeted residue screening requires the use of reference substances in order to identify potential residues. This becomes a difficult issue when using multi-residue methods capable of analyzing several hundreds of analytes. Therefore, the capability of in silico fragmentation based on a structure database ("suspect screening") instead of physical reference substances for routine targeted residue screening was investigated. The detection of fragment ions that can be predicted or explained by in silico software was utilized to reduce the number of false positives. These "proof of principle" experiments were done with a tool that is integrated into a commercial MS vendor instrument operating software (UNIFI) as well as with a platform-independent MS tool (Mass Frontier). A total of 97 analytes belonging to different chemical families were separated by reversed phase liquid chromatography and detected in a data-independent acquisition (DIA) mode using ion mobility hyphenated with quadrupole time of flight mass spectrometry. The instrument was operated in the MSE mode with alternating low and high energy traces. The fragments observed from product ion spectra were investigated using a "chopping" bond disconnection algorithm and a rule-based algorithm. The bond disconnection algorithm clearly explained more analyte product ions and a greater percentage of the spectral abundance than the rule-based software (92 out of the 97 compounds produced ≥1 explainable fragment ions). On the other hand, tests with a complex blank matrix (bovine liver extract) indicated that the chopping algorithm reports significantly more false positive fragments than the rule based software. Graphical Abstract.

Practical application of in silico fragmentation based residue screening with ion mobility high-resolution mass spectrometry.

A screening concept for residues in complex matrices based on liquid chromatography coupled to ion mobility high-resolution mass spectrometry LC/IMS-HRMS is presented. The comprehensive four-dimensional data (chromatographic retention time, drift time, mass-to-charge and ion abundance) obtained in data-independent acquisition (DIA) mode was used for data mining. An in silico fragmenter utilizing a molecular structure database was used for suspect screening, instead of targeted screening with reference substances. The utilized data-independent acquisition mode relies on the MSE concept; where two constantly alternating HRMS scans (low and high fragmentation energy) are acquired. Peak deconvolution and drift time alignment of ions from the low (precursor ion) and high (product ion) energy scan result in relatively clean product ion spectra. A bond dissociation in silico fragmenter (MassFragment) supplied with mol files of compounds of interest was used to explain the observed product ions of each extracted candidate component (chromatographic peak). Two complex matrices (fish and bovine liver extract) were fortified with 98 veterinary drugs. Out of 98 screened compounds 94 could be detected with the in silico based screening approach. The high correlation among drift time and m/z value of equally charged ions was utilized for an orthogonal filtration (ranking). Such an orthogonal ion mobility based filter removes multiply charged ions (e.g. peptides and proteins from the matrix) as well as noise and artefacts. Most significantly, this filtration dramatically reduces false positive findings but hardly increases false negative findings. The proposed screening approach may offer new possibilities for applications where reference compounds are hardly or not at all commercially available. Such areas may be the analysis of metabolites of drugs, pyrrolizidine alkaloids, marine toxins, derivatives of sildenafil or novel designer drugs (new psychoactive substances). Copyright © 2017 John Wiley & Sons, Ltd.

Structural Characterization of Phosphatidylcholines Using 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Advances in mass spectrometry have made it a preferred tool for structural characterization of glycerophospholipids. Collisional activation methods commonly implemented on commercial instruments do not provide fragmentation patterns that allow elucidation of certain structural features, including acyl chain positions on the glycerol backbone and double bond positions within acyl chains. In the present work, 193 nm ultraviolet photodissociation (UVPD) implemented on an Orbitrap mass spectrometer is used to localize double bond positions within phosphatidylcholine (PC) acyl chains. Cleavage of the carbon-carbon bonds adjacent to the double bond provides a diagnostic mass difference of 24 Da and enables differentiation of double-bond positional isomers. The UVPD method was extended to the characterization of PCs in a bovine liver extract via a shotgun strategy. Positive mode higher energy collisional dissociation (HCD) and UVPD, and negative mode HCD were undertaken in a complementary manner to identify species as PCs and to localize double bonds, respectively.

A Role for the Mitochondrial Protein Mrpl44 in Maintaining OXPHOS Capacity.

We identified Mrpl44 in a search for mammalian proteins that contain RNase III domains. This protein was previously found in association with the mitochondrial ribosome of bovine liver extracts. However, the precise Mrpl44 localization had been unclear. Here, we show by immunofluorescence microscopy and subcellular fractionation that Mrpl44 is localized to the matrix of the mitochondria. We found that it can form multimers, and confirm that it is part of the large subunit of the mitochondrial ribosome. By manipulating its expression, we show that Mrpl44 may be important for regulating the expression of mtDNA-encoded genes. This was at the level of RNA expression and protein translation. This ultimately impacted ATP synthesis capability and respiratory capacity of cells. These findings indicate that Mrpl44 plays an important role in the regulation of the mitochondrial OXPHOS capacity.

Reliability of veterinary drug residue confirmation: High resolution mass spectrometry versus tandem mass spectrometry

Confirmation of suspected residues has been a long time domain of tandem triple quadrupole mass spectrometry (QqQ). The currently most widely used confirmation strategy relies on the use of two selected reaction monitoring signals (SRM). The details of this confirmation procedure are described in detail in the Commission Decision 93/256/EC (CD). On the other hand, high resolution mass spectrometry (HRMS) is nowadays increasingly used for trace analysis. Yet its utility for confirmatory purposes has not been well explored and utilized, since established confirmation strategies like the CD do not yet include rules for modern HRMS technologies.It is the focus of this paper to evaluate the likelihood of false positive and false negative confirmation results, when using a variety of HRMS based measurement modes as compared to conventional QqQ mass spectrometry. The experimental strategy relies on the chromatographic separation of a complex blank sample (bovine liver extract) and the subsequent monitoring of a number of dummy transitions respectively dummy accurate masses. The term “dummy” refers to precursor and derived product ions (based on a realistic neutral loss) whose elemental compositions (CxHyNzOdCle) were produced by a random number generator. Monitoring a large number of such hypothetical SRM’s, or accurate masses inevitably produces a number of mass traces containing chromatographic peaks (false detects) which are caused by eluting matrix compounds. The number and intensity of these peaks were recorded and standardized to permit a comparison among the two employed MS technologies. QqQ performance (compounds which happen to produce a response in two SRM traces at identical retention time) was compared with a number of different HRMS1 and HRMS2 detection based modes. A HRMS confirmation criterion based on two full scans (an unfragmented and an all ion fragmented) was proposed. Compared to the CD criteria, a significantly lower probability of false positive and false negative findings is obtained by utilizing this criterion.

High Accuracy Mass Measurement Approach in the Identification of Phospholipids in Lipid Extracts: 7 T Fourier-transform Mass Spectrometry and MS/MS Validation

Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, KoreaReceived September 29, 2010, Accepted February 1, 2011In the present study, the approach of high accuracy mass measurements for phospholipid identifications wasevaluated using a 7 T ESI-FTMS/linear ion trap MS/MS. Experiments were carried out for porcine brain,bovine liver, and soybean total lipid extracts in both positive and negative ion modes. In total, 59, 55, and 18phospholipid species were characterized in the positive ion mode for porcine brain, bovine liver, and soybeanlipid extracts, respectively. Assigned lipid classes were PC, PE, PEt, PS, and SM. In the negative ion mode,PG, PS, PA, PE, and PI classes were observed. In the negative ion mode, for porcine brain, bovine liver, andsoybean lipid extracts, 28, 34, and 29 species were characterized, respectively. Comparison of our results withthose obtained by other groups using derivatization-LC-APCI MS and nano-RP-LC-MS/MS showed that ourapproach can characterize PC species as effectively as those methods could. In conclusion, we demonstratedthat high accuracy mass measurements of total lipid extracts using a high resolution FTMS, particularly, 7TFTMS, plus ion-trap MS/MS are very useful in profiling lipid compositions in biological samples. Abbreviation: PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PEt, phosphatid-ylethanol; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphtidylserine; SM, sphingomyelinKey Words : Lipids, Fourier transform ion cyclotron resonance mass spectrometer, Linear ion trap, Accuratemass measurements, Lipid databaseIntroductionFourier transform-ion cyclotron resonance mass spectro-metry (FT-ICR MS or FTMS) offers an ultrahigh massresolution and extraordinary mass accuracy, which has thusled to its wide applications in protein/peptide analysis.

Purification of alcohol dehydrogenase from bovine liver crude extract by dye–ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye–ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge ( x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000–7.0% dextran T500–0.1 M potassium phosphate buffers and 16% PEG 1000–12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000–potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6 h with minimum loss of its enzymatic activity.

The chaperone activity of protein disulfide isomerase is affected by cyclophilin B and cyclosporin A in vitro.

To elucidate the function of protein disulfide isomerase (PDI), we screened for PDI-binding proteins in a bovine liver extract using affinity column chromatography. One of the binding proteins was identified by SDS-PAGE and N-terminal amino acid sequence analysis to be cyclophilin B (Cyp B). Use of the BIACORE system revealed that purified bovine Cyp B bound specifically to bovine PDI with a K(D) value of 1.19 x 10(-5) M. Interestingly, the binding affinity between PDI and Cyp B was strengthened by preincubation of the Cyp B with cyclosporin A (CsA), yielding a K(D) value of 3.67 x 10(-6) M. Although the interaction between PDI and Cyp B affected neither the isomerase activity of PDI nor the peptidyl-prolyl cis-trans isomerase activity of Cyp B, Cyp B increased the chaperone activity of PDI. However, the complex of Cyp B and CsA completely inhibited the chaperone activity of PDI. Thus, PDI and Cyp B appear to cooperate with each other to regulate the functional expression of proteins in vivo.

Isolation, purification and identification of intracellular protein targets of ochratoxin A: mitochondrial aldehyde dehydrogenase class 2 is a high-affinity ochratoxin A-binding protein

An affinity chromatography technique was utilised to isolate and purify the soluble intracellular protein targets of ochratoxin A (OA). OA was covalently immobilised on a beaded agarose matrix. Several proteins (approximately nine) in the soluble fraction of crude protein extracts of different bovine tissues were specifically bound to the immobilised OA. The binding of these proteins to the modified agarose could not be dissociated with mild buffer (buffer plus 0.5 M NaCl and 1% Tween 20), but the proteins were competitively eluted with free 25 mM OA in mild buffer. Only one major protein (55 kDa) remained tightly bound to the immobilised OA when the column was incubated with bovine liver extract and then washed extensively with higher concentrations of salt and detergent (2 M buffer plus NaCl and 5% Tween 20). The bound protein was eluted competitively with 25 mM OA and further purified using SDS-PAGE. The N-terminal sequence of 24 residues of the high-affinity OA-binding protein was shown to be AATQAVPTPNQQPEVLYNQIFINN. This sequence is identical to the 24 residues of the N-terminal sequence of bovine mitochondrial aldehyde dehydrogenase class 2 (ALDH2, EC 1.2.1.3). The molecular weight and tissue distribution of ALDH2 are the same as those of the high-affinity protein isolated in this study. Immobilised OA can serve as an excellent affinity matrix for the identification of target proteins of OA and for the purification of ALDH2 and possibly other proteins. © 1999 Society of Chemical Industry

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF)

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3`-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA Sec is not recognized by elongation factor EF-1α, but is recognized specifically by a Sec-tRNA Sec protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA Sec, providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.

Trace 5-methylaminomethyl-2-selenouridine in bovine tRNA and the selenouridine synthase activity in bovine liver.

We measured the amount of Se in bovine liver tRNA. tRNA was chromatographed on a BD-cellulose column and Se-rich tRNA was eluted from the column in front of a main tRNA peak. There was 0.3 mmol Se/mol of tRNA and this level is about one tenth that of Escherichia coli tRNA. This suggests the presence of an enzyme that modifies tRNA with Se in bovine liver. We isolated the activity of this enzyme (selenouridine synthase) by chromatography of bovine liver extracts on a DEAE-cellulose column. ATP and selenophosphate synthetase, as well as selenouridine synthase and tRNA, were necessary for the reaction. 75Se was used to label the reaction products, which were analyzed by TLC after digestion with ribonuclease T2. The position of the 75Se-nucleotide on a TLC plate was identical to that of the Se-nucleotide, 5-methylaminomethyl-2-seleno-Up, prepared from 75Se-tRNA in E. coli.

Haptotactic migration of pancreatic cancer cells induced by bioactive components in bovine liver extract.

Migration into subendothelial tissue in remote organ by cancer cells are crucial events for organ-specific metastasis, including liver metastasis. This study aims to investigate the chemoattractive ingredients in liver extract, which induce migration of liver metastatic cancer cells. Cell migrations of SU.86.86 cells, human pancreatic cancer cells raised from liver metastasis, toward the bovine liver extract were studied by chemotaxis or haptotaxis assay. Bovine liver extract was partially purified by chromatographic or gel elutriation method. The soluble fraction of the liver extract did not induce chemotactic migration of SU.86.86 cells. However, the insoluble fraction induced a remarkable haptotactic migratory response. C18 column unbound insoluble fractions eluted from SDS-PAGE induced chemotactic migration of SU.86.86 cells. Such bioactive components in liver extract may play an important role in the development of liver metastasis.

Haptotactic migration of pancreatic cancer cells induced by bioactive components in bovine liver extract

Haptotactic migration of pancreatic cancer cells induced by bioactive components in bovine liver extract

A new translational elongation factor for selenocysteyl-tRNA in eucaryotes

In eucaryotes, selenocysteine (SeCys) was found in some selenoproteins, but SeCys-tRNA was not recognized by EF-1α. A different translational elongation factor for SeCys-tRNA must therefore supply SeCys-tRNA to the machinery of selenoprotein translation. I found this factor in bovine liver extracts with a UGA-programmed ribosome binding assay. The activity of binding of [ 75Se]SeCys-tRNA to the UGA-programmed ribosomes was eluted in fractions 57–65 using a CM-Sephadex C-25 column, and separated from EF-1α (the activity of binding of [ 14C]Phe-tRNA to the UUU-programmed ribosomes) in fractions 25–37. EF-1α in fraction 25 could discriminate (UUU) 10 for [ 14C]Phe-tRNA. A factor in fraction 57 could discriminate (UGA) 10 for [ 75Se]SeCys-tRNA. The elution pattern of activity of binding of [ 75Se]SeCys-tRNA to the UGA-programmed ribosomes was almost identical to that of activity of protecting [ 75Se]SeCys-tRNA against alkaline hydrolysis (SePF activity) [FEBS Lett. 347 (1994) 137–1421]. These two activities might depend on the same factor. The activity of binding of [ 75Se]SeCys-tRNA to the UGA-programmed ribosomes directly indicates that a factor in fraction 57 is a new translational elongation factor for SeCys-tRNA in eucaryotes.