Thermospray liquid chromatography-mass spectrometry of nucleosides and of enzymatic hydrolysates of nucleic acids.

Nucleosides dissolved in aqueous buffered solutions undergo ionization during direct introduction of the solution into a mass spectrometer using a thermospray interface. The principal ions formed represent the protonated molecule, the corresponding protonated free base, and sugar. In addition to potential utility for characterization of new nucleosides, the technique can be used to monitor nucleosides separated from enzymatic hydrolysates by liquid chromatography. The selectivity of chromatographic detection is significantly greater than with UV absorbance alone so that independent detection of components of unresolved chromatographic peaks is usually possible. Detection limits, with signal/noise greater than 10 for most nucleosides, are approximately 0.1-1 ng per component for selected ion monitoring and 10-50 ng for full-scan mass spectra. Examples are given from the detection of modified nucleosides in enzymatic hydrolysates of 0.05 A260 units (2.5 micrograms) of rabbit liver tRNAVal and of unfractionated H. volcanii tRNA.     

MSight: an image analysis software for liquid chromatography-mass spectrometry

Images obtained from high-throughput mass spectrometry (MS) contain information that remains hidden when looking at a single spectrum at a time. Image processing of liquid chromatography-MS datasets can be extremely useful for quality control, experimental monitoring and knowledge extraction. The importance of imaging in differential analysis of proteomic experiments has already been established through two-dimensional gels and can now be foreseen with MS images. We present MSight, a new software designed to construct and manipulate MS images, as well as to facilitate their analysis and comparison.     

Characterization of glutathione conjugates of pyrrolylated amino acids and peptides by liquid chromatography-mass spectrometry and tandem mass spectrometry with electrospray ionization

High-performance liquid chromatography (HPLC) coupled with electrospray mass spectrometry (ES-MS) and tandem mass spectrometry (MS-MS) was used to identify the products formed upon reaction of lysine-containing peptides with the neurotoxicant 2,5-hexanedione (2,5-HD). In addition, secondary autoxidative reaction products of the resultant alkylpyrroles with the biological thiol, glutathione, were characterized. ES mass spectra of the HPLC-separated conjugates showed intense [M+H]⁺ ions as well as several ions formed by amide and C-S bond cleavage. The glutathione conjugates of pyrrolylated amino acids and peptides were analyzed by ES ionization and MS-MS, and product-ion spectra showed fragmentation pathways typical of glutathione conjugates. ES-MS-MS analysis of a synthetic nonapeptide modeling a sequence found in neurofilament proteins showed pyrrole formation after incubation with 2,5-HD, and sequence ions were used to assign the position of the pyrrole adduct. Subsequent reaction of the pyrrolylated peptide with reduced glutathione was evidenced by a shift in m/z of the sequence ions of the reaction products with or without prior methylation. The results demonstrate the utility of ES-MS and ES-MS-MS in the characterization of xenobiotic-modified peptides and confirm that stable pyrrole-thiol conjugates are formed by the reaction of biological thils with pyrrolylated peptides.     

Generation of hydroxyeicosatetraenoic acids by human inflammatory cells: analysis by thermospray liquid chromatography-mass spectrometry

Arachidonic acid was converted to a series of hydroxyeicosatetraenoic acids (HETEs) by mixed human inflammatory cells following stimulation with the calcium ionophore A23187. HETEs were purified by a simple one-step extraction procedure followed by HPLC. The HPLC was coupled to a Finnigan quadrupole mass spectrometer using the now commercially available thermospray liquid chromatography-mass spectrometry interface. The HPLC eluant was monitored 'on line' by the mass spectrometer. Soft ionisation occurs, generating intense molecular ion species in the negative ion mode (M - H-:m/z 319) for each of the isomeric HETEs. The (M + H+ - H2O) ion at m/z 303 is the major species in the positive ion spectra of HETEs. Mass spectra were obtained on-line post-HPLC for HETEs formed by the human cells, and the HPLC-MS profile compared with that obtained from standards; species corresponding to the 11-, 9- and 5-HETEs were observed.     

Quantitative analysis of a novel glucosylated phospholipid by liquid chromatography-mass spectrometry

Building upon the demonstrated presence of a new glyceroglycolipid, phosphatidylglucoside (PtdGlc), in rat embryonic brain tissues, we have developed a method to identify minute amounts of PtdGlc in cultured cells by using nano-flow high-performance liquid chromatography and negative-ion-mode electrospray linear-ion trap time-of-flight mass spectrometry (LC-MS). A normal-phase silica gel-based column enabled us to separate PtdGlc from other lipid classes. PtdGlc was identified from its tandem mass spectrometry spectrum and from its retention time in the column. Using an internal standard collection and LC-MS, we obtained the linearity of PtdGlc at a range of 6.3-800 fmol per injection. We applied this method to analyze quantitative changes in PtdGlc in C6 glioma cells after cellular differentiation into GFAP-positive glial cells. PtdGlc in C6 glioma cells consisted exclusively of C18:0/C20:0 fatty acyl chains. Differentiation induced by the addition of anti-PtdGlc antibody plus cAMP in culture medium significantly increased the glycolipid content.     

A new hydrolase specific for taurine-conjugates of bile acids.

Through the investigation of the bile acid-deconjugation activities of human intestinal anaerobes, a new enzyme was discovered in Peptostreptococcus intermedius which hydrolyzed specifically the taurine-conjugates, but not the glycine-conjugates of bile acids. However, the enzymes in Streptococcus faecalis and Lactobacillus brevis hydrolyzed chiefly the glycine-conjugates.     

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.     

A sensitive and specific liquid chromatography-mass spectrometry method for determination of metacavir in rat plasma

A sensitive and specific liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method has been developed and validated for the quantification of metacavir in rat plasma using tinidazole as an internal standard (I.S.). Following ethyl acetate extraction, the analytes were separated on a Shim-pack ODS (4.6 microm, 150 mm x 2.0 mm I.D.) column and analyzed in selected ion monitoring (SIM) mode with a positive ESI interface using the respective [M+H](+) ions, 266 for metacavir and 248 for tinidazole. The method was validated over the concentration range of 1-600 ng/mL for metacavir. Between and within-batch precisions (R.S.D.%) were all within 15% and accuracy (%) ranged from 92.2 to 105.8%. The lower limit of quantification (LLOQ) was 1 ng/mL. The extraction recovery was on average 89.8%. The validated method was used for the pharmacokinetic study of metacavir in rats.     

Mining phosphopeptide signals in liquid chromatography-mass spectrometry data for protein phosphorylation analysis

Protein phosphorylation is a key post-translational modification that governs biological processes. Despite the fact that a number of analytical strategies have been exploited for the characterization of protein phosphorylation, the identification of protein phosphorylation sites is still challenging. We proposed here an alternative approach to mine phosphopeptide signals generated from a mixture of proteins when liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis is involved. The approach combined dephosphorylation reaction, accurate mass measurements from a quadrupole/time-of-flight mass spectrometer, and a computing algorithm to differentiate possible phosphopeptide signals obtained from the LC-MS analyses by taking advantage of the mass shift generated by alkaline phosphatase treatment. The retention times and m/z values of these selected LC-MS signals were used to facilitate subsequent LC-MS/MS experiments for phosphorylation site determination. Unlike commonly used neutral loss scan experiments for phosphopeptide detection, this strategy may not bias against tyrosine-phosphorylated peptides. We have demonstrated the applicability of this strategy to sequence more, in comparison with conventional data-dependent LC-MS/MS experiments, phosphopeptides in a mixture of alpha- and beta-caseins. The analytical scheme was applied to characterize the nasopharyngeal carcinoma (NPC) cellular phosphoproteome and yielded 221 distinct phosphorylation sites. Our data presented in this paper demonstrated the merits of computation in mining phosphopeptide signals from a complex mass spectrometric data set.     

Gas-liquid chromatography of bile acids: a new liquid phase for both acetate and trimethylsilyl derivatives.

Polymetaphenoxylene (PPE-20) has been found to be more useful than cyclohexane dimethanol succinate (HI-EFF-8-BP) for trimethylsilyl derivatives of bile acids and to be preferable to trifluoropropyl substituted silicone (OV-210, QF-1) for analysis of their acetate derivatives.     

A liquid chromatography-mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites

The ability to measure estrogen metabolites (EMs) quantitatively is important for investigating their individual roles in cancer screening, treatment and prevention, as well as in a host of other hormone-related disorders. In this protocol we describe a method that is capable of quantitating 15 distinct EMs in urine. Endogenous EMs are quantitatively measured using a liquid chromatography-tandem mass spectrometry method in which the spectrometer operates in a selected reaction monitoring mode. This method is capable of quantifying estrone and its 2-, and 4- and 16alpha-hydroxy and its 2-, 4-methoxy derivatives, and 2-hydroxyestrone-3-methyl ether; 17beta-estradiol and its 2-hydroxy, and 2- and 4-methoxy derivates, and estriol, 16-epiestriol, 17-epiestriol, and 16-ketoestradiol. The method requires only 0.5 ml of urine and approximately 60 urine samples can be quantitatively analyzed per week.     

Quantifying reproducibility for differential proteomics: noise analysis for protein liquid chromatography-mass spectrometry of human serum

SUMMARY: Using replicated human serum samples, we applied an error model for proteomic differential expression profiling for a high-resolution liquid chromatography-mass spectrometry (LC-MS) platform. The detailed noise analysis presented here uses an experimental design that separates variance caused by sample preparation from variance due to analytical equipment. An analytic approach based on a two-component error model was applied, and in combination with an existing data driven technique that utilizes local sample averaging, we characterized and quantified the noise variance as a function of mean peak intensity. The results indicate that for processed LC-MS data a constant coefficient of variation is dominant for high intensities, whereas a model for low intensities explains Poisson-like variations. This result leads to a quadratic variance model which is used for the estimation of sample preparation noise present in LC-MS data.     

A highly sensitive high-performance liquid chromatography-mass spectrometry method for quantification of fludarabine triphosphate in leukemic cells

A high-performance liquid chromatography (HPLC)-mass spectrometry (MS) method has been developed for the analysis of 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate (F-ara-ATP) from biological samples. Quantification is carried out by selected ion monitoring of the parent ion. Baseline separation of the monophosphate (F-ara-AMP) and diphosphate (F-ara-ADP) is achieved using the volatile ion-pairing reagent dimethylhexylamine. This method is selective and sensitive with an on-column detection limit of approximately 50 fmol. It also permits simultaneous monitoring of endogenous adenosine phosphates. The utility of the assay has been demonstrated by the analysis of F-ara-ATP in human leukemic cells after incubation with 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A) at clinically relevant concentrations.     

Improvement of recovery and repeatability in liquid chromatography-mass spectrometry analysis of peptides

Poor repeatability of peak areas is a problem frequently encountered in peptide analysis with nanoLiquid Chromatography coupled on-line with Mass Spectrometry (nanoLC-MS). As a result, quantitative analysis will be seriously hampered unless the observed variability can be corrected in some way. Currently, labeling techniques or addition of internal standards are often applied for this purpose. However, these procedures are elaborate and error-prone and may render complex samples even more complex. Moreover, whenever poor repeatability results from variable recovery, not just quantification, but also sensitivity is affected. We have studied the parameters influencing the repeatability of chromatographic peak areas for a model set of proteolytic peptides (i.e., a cytochrome c tryptic digest) in nanoLC-MS analysis. It is demonstrated that repeatability issues are mainly due to poor recovery of peptides from the sample vial. Problems are largely resolved by addition of an organic modifier to the sample vial to improve solubility of the peptides, but care needs to be taken not to lose peptides due to reduced affinity for reversed-phase materials. Good results are obtained when applying dimethylsulfoxide (DMSO) for this purpose. When applying DMSO, repeatability increases, and the limit of detection (LOD) decreases. For the most hydrophobic peptides, a gain in LOD of at least an order of magnitude is obtained. In an aqueous sample containing 0.1% formic acid (FA), it is possible to detect 100-200 fmol of peptide, whereas +/-10 fmol can be detected in a sample containing 5% FA and 25% DMSO (10 microL injections).     

The analysis of recombinant interleukin-2 by thermospray liquid chromatography-mass spectrometry

The complete sequence of recombinant human interleukin-2 expressed in Escherichia coli has been confirmed by thermospray liquid chromatography-mass spectrometry (TS-LC-MS) of a tryptic digest derived from 100 micrograms (7 nmol) of reduced carboxymethylated interleukin-2. The preparation was shown by this method to contain predominantly unprocessed N-terminal initiator Met, with some authentic N-terminal Ala; the rest of the protein was as predicted from the DNA sequence, though some deamidated material was noted. TS-LC-MS proved to be a rapid and efficient method for surveying the protein tryptic peptide products allowing all the data to be collected in one chromatographic run; all tryptic fragments were identified by their molecular ions including those for the larger peptides (Mr 1500-3500) which, due to the presence of doubly and triply charged molecular ions, were brought within the mass range of the instrument (1800 Da). It is proposed that TS-LC-MS is a good general method for analyzing recombinant protein digests with respect to sequence confirmation, processing, and post-translational modification, and since each chromatographic peak is identified allows for subsequent monitoring of the protein by LC using uv detection. The method suffers from the disadvantages that all the sample is consumed during the experiment and that no fragment (sequence) ions are generally observed.     

Bile acids LIII: Application of reverse-phase high-pressure liquid chromatography to the analysis of conjugated bile acids in bile samples

The common conjugated bile acids of deproteinated bile from the human or the rat can be separated by high-pressure liquid chromatography and quantitated within 30 min with a 4-mm × 30-cm “fatty-acid analysis” column (Waters Associates) in 2-propanol/8.8 mm potassium phosphate buffer (pH 2.5) 160:340, coupled to a uv flow detector set at 193-nm wavelength. Detection limits were at least 0.1 nmol for the tauro-conjugates and 0.2 nmol for the glyco-derivatives.     

Determination of haloperidol and its reduced metabolite in human plasma by liquid chromatography-mass spectrometry with electrospray ionization

A sensitive and accurate liquid chromatographic-electrospray mass spectrometric (LC-ES-MS) method for the determination of haloperidol (H) and reduced haloperidol (RH) in human plasma is presented, using chlorohaloperidol as the internal standard. A 2-ml volume of plasma subjected to basic (NaOH) extraction, acid (HCl) back-extraction, acid wash and basic (NaOH) re-extraction. The extraction solvent was hexane-isoamyl alcohol (99:1, v/v) for the whole procedure. A Nucleosil C₁₈ column (150×1 mm) was used for high-performacne liquid chromatography, together with 2 mM HCOONH₄-acetonitrile (55:45, v/v; pH 3.0) as the mobile phase. For each drug, four characteristic ions were monitored. Linearity was assessed in the ranges 0.1–50 and 0.25–50 ng/ml for H and RH, respectively. Recoveries were 58 and 70% and detection limits were 0.075 and 0.100 ng/ml for H and RH, respectively. Correlation coefficients were better than 0.999 for both compounds. R.S.D.s for repeatability and reproducibility at 0.25 ng/ml were 11.1 and 8.5% for H and 9.4 and 11.2% for RH, respectively. One of the main advantages of (LC-ES-MS) over other detection systems is the increase in selectivity obtained by monitoring three ions of confirmation for each of the drugs.     

A geometric approach for the alignment of liquid chromatography-mass spectrometry data

MOTIVATION: Liquid chromatography coupled to mass spectrometry (LC-MS) and combined with tandem mass spectrometry (LC-MS/MS) have become a prominent tool for the analysis of complex proteomic samples. An important step in a typical workflow is the combination of results from multiple LC-MS experiments to improve confidence in the obtained measurements or to compare results from different samples. To do so, a suitable mapping or alignment between the data sets needs to be estimated. The alignment has to correct for variations in mass and elution time which are present in all mass spectrometry experiments. RESULTS: We propose a novel algorithm to align LC-MS samples and to match corresponding ion species across samples. Our algorithm matches landmark signals between two data sets using a geometric technique based on pose clustering. Variations in mass and retention time are corrected by an affine dewarping function estimated from matched landmarks. We use the pairwise dewarping in an algorithm for aligning multiple samples. We show that our pose clustering approach is fast and reliable as compared to previous approaches. It is robust in the presence of noise and able to accurately align samples with only few common ion species. In addition, we can easily handle different kinds of LC-MS data and adopt our algorithm to new mass spectrometry technologies. AVAILABILITY: This algorithm is implemented as part of the OpenMS software library for shotgun proteomics and available under the Lesser GNU Public License (LGPL) at www.openms.de.