Identification of proteins in cell-free extracts using liquid chromatography-electrospray ionization mass spectrometry.

A rapid method for the identification and characterization of proteins in bacterial cell-free extracts has been developed using directly combined liquid chromatography-electrospray mass spectrometry. The usefulness of this technique is demonstrated for monitoring the expression and chemical modification of phosphoenolpyruvate-sugar phosphotransferase system (PTS) proteins from E. coli with molecular masses ranging from 9-65 kDa. The technique is characterized by minimal sample preparation, remarkable mass accuracy and resolution, reproducibility and the ability, unlike gel electrophoresis, to directly identify posttranslational modifications. The advantages of this technique over analogous matrix-assisted laser desorption ionization mass spectrometry approaches and its potential as a standard tool in the biomolecular research laboratory are discussed.

Characterization of prenylated xanthones and flavanones by liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

Reversed-phase liquid chromatography with atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) in the positive-ion mode was utilized to analyze crude ether extracts from the root bark of Maclura pomifera, a tree known to have a high content of prenylated xanthones and flavanones. Identification of three xanthones and two flavanones was based on their unique mass spectra. Under optimum conditions peaks corresponding to the [MH](+) ion and characteristic fragments for each compound were observed. (1)H NMR data were used to confirm the identities of two xanthones that had the same molecular mass and similar fragmentation patterns. Fragmentation of the analytes was achieved by application of an electrostatic potential at the entrance of the single quadrupole mass spectrometer. The optimum voltage for fragmentation was found to be related to the class of compounds analyzed and, within each class, to be dependent on the structure of the prenyl moiety. Collision-induced pathways consistent with precedent literature describing the MS characterization of similar compounds and with the observed fragmentation patterns are tentatively proposed.

Determination of tea catechins.

An overview of analytical methods for the measurement of biologically important tea catechins is presented. Liquid chromatography and capillary electrophoresis are the most cited techniques for catechin separation, identification and quantitation. Liquid chromatography with ultraviolet detection is frequently used; however, mass spectrometry, electrochemical, fluorescence and chemiluminescence detection are also utilized in cases where more sensitive or selective detection is needed. Two modes of capillary electrophoresis, capillary zone electrophoresis and micellar electrokinetic capillary chromatography, have been employed for the determination of catechins. Both modes of capillary electrophoresis are based on ultraviolet detection. Additional analytical techniques, such as gas chromatography, thin-layer chromatography, paper chromatography, spectrophotometry, biosensing, chemiluminescence and nuclear magnetic resonance spectroscopy have also been utilized for the determination of catechins and are reviewed herein.

Selection of column and gradient elution system for the separation of catechins in green tea using high-performance liquid chromatography.

A study of a variety of stationary phases and elution conditions for the liquid chromatographic (LC) determination of six biologically active green tea catechins has resulted in the development of two well-defined, reproducible systems for such analyses which overcome limitations of previously described methods. Comparison of six reversed-phase columns indicates that deactivated stationary phases, which utilize ultrapure silica and maximize coverage of the silica support, provide significantly improved separation and chromatographic efficiencies for catechin analyses using LC, compared to conventional monomeric or polymeric C18 columns. Evaluation of elution conditions used for the separations reveals that the presence of acid in the mobile phase (0.05% trifluoroacetic acid) is essential for both the complete resolution of the catechins present in tea and the efficient chromatography of these compounds. The efficacy of one of the developed systems was demonstrated by the quantitative measurement of the six biologically active catechins in aqueous infusions of green tea (Camellia sinensis). Overall precision values for the analyses were within the range 0.3-1% (relative standard deviation).

Mass spectrometry for direct determination of proteins in cells: applications in biotechnology and microbiology.

A number of different procedures have been developed in recent years that utilize mass spectrometry for the direct determination of proteins in complex mixtures of biological origin. Specific examples of these include the use of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or directly combined liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) for rapid profiling of protein expression in bacterial and eucaryotic cells and cell-free extracts. Approaches to sample cleanup, contaminant removal, and initial separation of analytes on-line for the direct determination of proteins in cells using MALDI- and ESI-MS are discussed. Advantages of these techniques over traditional biochemical methods are highlighted, and a critical review of their utility and potential as standard tools in the biomolecular and microbiological research laboratory is presented.

Precolumn affinity capillary electrophoresis for the identification of clinically relevant proteins in human serum: application to human cardiac troponin I.

An approach has been developed to the on-line extraction and identification of clinical disease-state marker proteins in human serum. Fabrication of capillaries with integral packed beds for the online determination of human cardiac troponin I (cTnI), a diagnostic marker for myocardial infarction, at clinically relevant levels (2 nmol/L) in serum is demonstrated. The technique, termed precolumn affinity capillary electrophoresis (PA-CE), utilizes a short (approximately 5 mm) packed bed of porous silica containing covalently immobilized monoclonal anti-cTnI antibodies directly integrated within a separation capillary for the selective retention of cTnI from a complex matrix. Following a rinsing step to eliminate nonspecifically bound serum proteins and other impurities from the column, desorption of the antigen into the separation region of the PA-CE capillary for subsequent measurement of femto-molar amounts of cTnI by CE is effected by the injection of an appropriate elution buffer. Advantages of this approach over previously reported affinity preconcentration techniques, related applications for PA-CE technology, and its potential for use in the development of a certified reference material for cTnI in serum are discussed.

Heterogeneity in human cardiac troponin I standards.

The LC-MS analysis of recombinant cardiac troponin I (cTnI) and cTnI extracted from human hearts showed a high degree of structural heterogeneity among all samples. The examined recombinant cTnI samples indicated posttranslational modifications, presumably due to their purification (i.e., 2-mercaptoethanol adducts and carbamylation) and related to their expression (i.e., an N-terminal expression tag). The extracted cTnI samples, while having a higher degree of structural heterogeneity, showed less structural variance between samples than the recombinant proteins. The LC-MS analysis of the extracted cTnI samples provided evidence of posttranslational modification by phosphorylation, acetylation, proteolytic cleavage, and intrachain disulfide bond formation.

Quantitative measurement of dihydrouridine in RNA using isotope dilution liquid chromatography-mass spectrometry (LC/MS).

A method has been developed for the microscale determination of 5,6-dihydrouridine, the most common post-transcriptional modification in bacterial and eukaryotic tRNA. The method is based on stable isotope dilution liquid chromatography-mass spectrometry (LC/MS) using [1,3-15N2]dihydrouridine and [1,3-15N2]uridine as internal standards. RNA samples were enzymatically digested to nucleosides before addition of the internal standards and subsequently analyzed by LC/MS with selected ion monitoring of protonated molecular ions of the labeled and unlabeled nucleosides. Sample quantities of approximately 1 pmol tRNA and 5 pmol 23S rRNA were analyzed for mole% dihydrouridine. Dihydrouridine content of Escherichia coli tRNASer(VGA) and tRNAThr(GGU) as controls were measured as 2.03 and 2.84 residues/tRNA molecule, representing accuracies of 98 and 95%. Overall precision values for the analyses of E. coli tRNASer(VGA) and E. coli tRNAThr(GGU), unfractionated tRNA from E. coli and 23S rRNA from E. coli were within the range 0.43-2.4%. The mole% dihydrouridine in unfractionated tRNA and 23S rRNA from E. coli were determined as 1.79 and 0.0396%, corresponding to 1.4 and 1.1 residues/RNA molecule respectively.

Preliminary application of liquid chromatography-electrospray-ionization mass spectrometry to the detection of 5-methyltetrahydrofolic acid monoglutamate in human plasma.

Liquid chromatography (LC) in direct combination with mass spectrometry (MS) has been shown to be a good analytical technique for the selective separation and detection of labile folate monoglutamates. Reversed-phase LC and electrospray-ionization MS conditions were developed and optimized for the separation and detection of 5-methyltetrahydrofolic acid, 5-formyl tetrahydrofolic acid, tetrahydrofolic acid, dihydrofolic acid and folic acid in aqueous samples. Representative and reproducible positive ion mass spectra were generated for each folate under mild MS conditions. The selective MS detection and identification of endogenous 5-methyltetrahydrofolic acid in human plasma was accomplished through the development of a straightforward C18-based solid-phase extraction procedure. This procedure allows for the qualitative assessment of 5-methyltetrahydrofolic acid in plasma. Based upon an isotope-dilution internal standard calibration study with standards, the LC-MS limit of quantitation for 5M-THF was estimated to be 0.39 ng/mnl.

The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles.

The influence of posttranscriptional modification on structural stabilization of tRNA from hyperthermophilic archaea was studied, using Pyrococcus furiosus (growth optimum 100 degrees C) as a primary model. Optical melting temperatures (Tm) of unfractionated tRNA in 20 mM Mg2+ are 97 degrees C for P. furiosus and 101.5 degrees C for Pyrodictium occultum (growth optimum, 105 degrees C). These values are approximately 20 degrees C higher than predicted solely from G-C content and are attributed primarily to posttranscriptional modification. Twenty-three modified nucleosides were determined in total digests of P. furiosus tRNA by combined HPLC-mass spectrometry. From cells cultured at 70, 85, and 100 degrees C, progressively increased levels of modification were observed within three families of nucleosides, the most highly modified forms of which were N4-acetyl-2'-O-methylcytidine (ac4Cm), N2,N2,2'-O-trimethylguanosine (m2(2)Gm), and 5-methyl-2-thiouridine (m5s2U). Nucleosides ac4Cm and m2(2)Gm, which are unique to the archaeal hyperthermophiles, were shown in earlier NMR studies to exhibit unusually high conformational stabilities that favor the C3'-endo form [Kawai, G., et al. (1991) Nucleic Acids Symp. Ser. 21, 49-50; (1992) Nucleosides Nucleotides 11, 759-771]. The sequence location of m5s2U was determined by mass spectrometry to be primarily at tRNA position 54, a site of known thermal stabilization in the bacterial thermophile Thermus thermophilus [Horie, N., et al. (1985) Biochemistry 24, 5711-5715]. It is concluded that selected posttranscriptional modifications in archaeal thermophiles play major stabilizing roles beyond the effects of Mg2+ binding and G-C content, and are proportionally more important and have evolved with greater structural diversity at the nucleoside level in the bacterial thermophiles.

Solid phase synthesis of 5'-diphosphorylated oligoribonucleotides and their conversion to capped m7Gppp-oligoribonucleotides for use as primers for influenza A virus RNA polymerase in vitro.

We have synthesized four different 5'-diphosphorylated oligoribonucleotides, varying in length from 11 to 13 nucleotides by a new solid phase method. After deprotection and partial purification the 5'-diphosphorylated oligoribonucleotides could be converted to capped (m7Gppp) oligoribonucleotides using guanylyl transferase. Radiolabelled capped oligoribonucleotides acted as primers for the influenza A virus RNA polymerase in vitro. The solid phase method described here should also allow the addition of 5'-diphosphates to synthetic oligodeoxyribonucleotides and be capable automation.

Separation and identification of twelve catechins in tea using liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry.

A method has been developed for the direct microscale determination of 12 catechins in green and black tea infusions. The method is based on liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry (LC/APCI-MS). Standard catechin mixtures and tea infusions were analyzed by LC/APCI-MS with detection of protonated molecular ions and characteristic fragment ions for each compound. The identities of eight major catechins and caffeine in tea were established based on LC retention times and simultaneously recorded mass spectra. In addition, monitoring of the catechin-specific retro Diels-Alder fragment ion at m/z 139 throughout the chromatogram provided a unique fingerprint for catechin content in the samples that led to the identification of four minor chemically modified catechin derivatives in the infusions. This report is the first to describe the comprehensive determination of all 12 reported catechins in a single analysis. The utility of LC/APCI-MS for providing routine separation and identification of catechins at femtomole to low-picomole levels without extraction or sample pretreatment, and its potential as a standard analytical tool for the determination of polyphenols in natural products and biological fluids, are discussed.

Conformational flexibility in RNA: the role of dihydrouridine.

In order to further understand the structural role of the modified nucleoside dihydrouridine in RNA the solution conformations of Dp and ApDpA were analyzed by one- and two-dimensional proton NRM spectroscopy and compared with those of the related uridine-containing compounds. The analyses indicate that dihydrouridine significantly destabilizes the C3'-endo sugar conformation associated with base stacked, ordered, A-type helical RNA. Equilibrium constants (Keq = [C2'-endo]/[C3'-endo]) for C2'-endo-C3'-endo interconversion at 25 degrees C for Dp, the 5'-terminal A of ApDpA and D in ApDpA are 2.08, 1.35 and 10.8 respectively. Stabilization of the C2'-endo form was shown to be enhanced at low temperature, indicating that C2'-endo is the thermodynamically favored conformation for dihydrouridine. DeltaH values show that for Dp the C2'-endo sugar conformation is stabilized by 1.5 kcal/mol compared with Up. This effect is amplified for D in the oligonucleotide ApDpA and propagated to the 5'-neighboring A, with stabilization of the C2'-endo form by 5.3 kcal/mol for D and 3.6 kcal/mol for the 5'-terminal A. Post-transcriptional formation of dihydrouridine therefore represents a biological strategy opposite in effect to ribose methylation, 2-thiolation or pseudouridylation, all of which enhance regional stability through stabilization of the C3'-endo conformer. Dihydrouridine effectively promotes the C2'-endo sugar conformation, allowing for greater conformational flexibility and dynamic motion in regions of RNA where tertiary interactions and loop formation must be simultaneously accommodated.

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.

Capillary liquid chromatography/electrospray mass spectrometry for the separation and detection of catechins in green tea and human plasma.

The separation and detection of biologically active green tea catechins has been accomplished using capillary liquid chromatography/electrospray mass spectrometry (cLC/ESI-MS). Microscale determination (approximately 20 ng) of all six catechins in a green tea infusion, and the most extensively studied catechin, (-)epigallocatechin gallate (EGCG), in human plasma is demonstrated by cLC/ESI-MS with selected ion monitoring of protonated molecular ions. The overall quality of the analysis is shown to be dependent on the use of a capillary column with a deactivated, monomeric C18 stationary phase. The high chromatographic separation efficiency of this packed-capillary column, combined with the high sensitivity and selectivity afforded by the mass spectrometer as detector, provide a reliable approach to the analysis of picomolar quantities of these interesting compounds in complex matrices.

Posttranscriptional modification of tRNA in psychrophilic bacteria.

Posttranscriptional modification in tRNA is known to play a multiplicity of functional roles, including maintenance of tertiary structure and cellular adaptation to environmental factors such as temperature. Nucleoside modification has been studied in unfractionated tRNA from three psychrophilic bacteria (ANT-300 and Vibrio sp. strains 5710 and 29-6) and one psychrotrophic bacterium (Lactobacillus bavaricus). Based on analysis of total enzymatic hydrolysates by liquid chromatography-mass spectrometry, unprecedented low amounts of modification were found in the psychrophiles, particularly from the standpoint of structural diversity of modifications observed. Thirteen to 15 different forms of posttranscriptional modification were found in the psychrophiles, and 10 were found in L. bavaricus, compared with approximately 29 known to occur in bacterial mesophiles and 24 to 31 known to occur in the archaeal hyperthermophiles. The four most abundant modified nucleosides in tRNA from each organism were dihydrouridine, pseudouridine, 7-methylguanosine, and 5-methyluridine. The molar abundances of the latter three nucleosides were comparable to those found in tRNA from Escherichia coli. By contrast, the high levels of dihydrouridine observed in all three psychrophiles are unprecedented for any organism in any of the three phylogenetic domains. tRNA from these organisms contains 40 to 70% more dihydrouridine, on average, than that of the mesophile E. coli or the psychrotroph L. bavaricus. This finding supports the concept that a functional role for dihydrouridine is in maintenance of conformational flexibility of RNA, especially important to organisms growing under conditions where the dynamics of thermal motion are severely compromised. This is in contrast to the role of modifications contained in RNA from thermophiles, which is to reduce regional RNA flexibility and provide structural stability to RNA for adaptation to high temperature.