Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry

Background  

Stable isotope tracing with ultra-high resolution Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) can provide simultaneous determination of hundreds to thousands of metabolite isotopologue species without the need for chromatographic separation. Therefore, this experimental metabolomics methodology may allow the tracing of metabolic pathways starting from stable-isotope-enriched precursors, which can improve our mechanistic understanding of cellular metabolism. However, contributions to the observed intensities arising from the stable isotope's natural abundance must be subtracted (deisotoped) from the raw isotopologue peaks before interpretation. Previously posed deisotoping problems are sidestepped due to the isotopic resolution and identification of individual isotopologue peaks. This peak resolution and identification come from the very high mass resolution and accuracy of FT-ICR-MS and present an analytically solvable deisotoping problem, even in the context of stable-isotope enrichment.     

Data filtration for more robust alignment of chromatograms of complex peptide mixtures

In carrying out proteomic researches using mass-spectrometry there often arises a need to compare experimental data with each other (e.g. control of pathology, the labeled to unlabelled samples). If for peptide identification in different experiments one uses only their exact mass measurements and the retention time in the chromatographic column, difficulties with the identification of chromatographic peaks belonging to the same substances in different chromatograms come up (retention time normalization). Due to inevitable discrepancies in chromatographic conditions of experiments (replacement of chromatographic columns, small changes in mobile phase flow rate or solvent concentration) retention times of the same peptides will diverge from experiment to experiment. In this paper we offer a reliable method for selecting peaks from mass-chromatograms corresponding to the same peptides, which can later be used for retention time normalization (either linear or any other monotone function).     

Estimation of descriptive parameters of overlapping chromatographic peaks: a simulation study

The characteristics of a non-linear optimization technique for resolution of overlapping chromatographic peaks are examined. A modified Meiron-Marquardt method was used. The estimates of the parameters of overlapping peaks in simulated chromatograms were investigated to indicate the limitations of present mathematical methods and, hopefully, to improve their ultimate utility. Gaussian shapes as well as exponential-Gaussian convolutes were used to simulate the chromatographic peaks. Effects on the overall performance of varying heights, widths, and separation of two peaks were determined. Random additive noise and base line drift were also simulated. For illustrative purposes, the performance of the parameter estimation techniques was expressed in terms of relative errors in estimating the second (or smaller) peak's area, height and location. The results presented indicate the relative importance of noise, skewness, height and width ratios and peak separation on the maximum resolution achievable by numerical methods in an automated chromatographic system.     

Dispersion of protein solutions in short, open capillary tubes as a method for rapid molecular weight determination

A method has been developed by which the molecular weight of proteins and other freely diffusing species can be estimated on the basis of chromatographic peak shapes developed by injection of a sample into an open capillary tube in a liquid chromatography system. In chromatographic peaks obtained from such a system, there are contributions from both convection and diffusion. Thus, peak shape is dependent upon the diffusion coefficient of the molecular species, the flow rate, and the length of the capillary tube. In the work reported here it has been found that for samples of different proteins ranging from 2000 to 14,000 molecular weight, each injected at the same mobile phase flow rate, the ratio (R) of h1, the height of the peak primarily due to convection, to h2, the height of the "makeup" peak, primarily due to diffusion from the capillary wall, is a direct measure of protein molecular weight. Linear plots of R vs molecular weight are obtained under certain conditions.     

Determination of tissue folate composition by affinity chromatography followed by high-pressure ion pair liquid chromatography

A recent report from this laboratory described the use of affinity chromatography for the isolation of pure folates from tissue extracts (J. Selhub, B. Darcy-Vrillon, and D. Fell (1988) Anal. Biochem. 168, 247-251). The present study was undertaken to develop chromatographic procedures for quantitative analysis of the individual folates in the affinity-purified mixture. Methods were devised whereby mixtures containing pteroylglutamates (PteGlu1-7) were batch reduced to the dihydro, H2PteGlu1-7, and tetrahydro, H4Pte-Glu1-7, forms. The 5-methylH4PteGlu1-7 and the 10-formylH4PteGlu1-7 series were prepared from H4Pte-Glu1-7. These compounds were used to calibrate a liquid chromatographic system for the resolution of folate mixtures. This system included reverse-phase ion pair chromatography and a diode array detector. A mixture containing oxidized and reduced PteGlu1-7, a total of 35 derivatives, was separated into seven clusters arranged in an order of increasing number of glutamate residues. Each cluster was represented by two or more peaks which were due to folates that differed in the pteridine ring structure but had the same number of glutamate residues. In clusters containing mono and diglutamyl derivatives the 10-formyltetrahydro-, the tetrahydro-, and the dihydrofolate forms appeared as separate peaks while those representing folic acid and 5-methyl-tetrahydrofolate derivatives eluted in coinciding peaks. This hierarchy was maintained in the following clusters except for increasing tendency of the former three forms of folates to elute in the same peak. The number of glutamate residues of any eluting folate can be determined on the basis of retention time in relation to those of the clusters. The pteridine ring structure of that same folate can be determined on the basis of its elution position within that cluster and spectral characteristics determined by the diode array detection system. If that position is common for more than one derivative then identification is based on differential spectral properties. Using uv absorption signals at 280 nm to determine indiscriminate folate activity, absorption signals at 350 nm are used to identify folic acid and dihydrofolate derivatives and signals at 258 nm are used to identify 10-formyltetrahydrofolate derivatives. These principles were incorporated into mathematic expressions which were used for quantitative resolution of simulated mixtures containing oxidized and reduced PteGlu5 and for the analysis of folate composition in rat liver, human milk, and cows milk.     

Fractionation of Rat Liver tRNA by Reversed-Phase High Performance Liquid Chromatography: Isolation of ISO-tRNAsPro

Abstract

This paper illustrates the fractionation of cytoplasmic transfer ribonucleic acid from rat liver by reversed-phase high performance liquid chromatography using a gradient of acetonitrile/ammonium acetate. The procedure is fast, highly reproducible, and gives an excellent resolution of the numerous tRNA population: about 50 peaks with area peak percentages ranging from 0.001 to 5 can be monitored. Uncharged tRNA preparations exhibited a chromatographic profile different from aminoacylated tRNA, thus suggesting a possible strategy to distinguish between aminoacylated and nonacylated tRNA species. Moreover, a first approach to map the HPLC peaks was attempted by chromatographing preparations of tRNA which had been aminoacylated with individual ³H-labeled aminoacids. Here is reported the case of tRNA^Pro, which gave three well separated radioactive peaks, most likely corresponding to tRNA^Pro isoacceptor species.     

Study of the factors influencing peak asymmetry on chromatography using a molecularly imprinted polymer prepared by the epitope approach

Investigations of the effect of sample load on peak asymmetry during chromatography on molecularly imprinted polymer prepared by the epitope approach showed that the shape of the peaks for the template Tyr-Pro-Leu-Gly-NH₂ and for acetyl-L-tyrosine ethyl ester changed considerably until a split was observed. In contrast, the asymmetry of the peaks corresponding to oxytocin, which possesses the same C-terminus tripeptide as the template and interacts with the imprinted polymer, remained essentially unaltered. The circular dichroism (CD) spectra of these peptides showed significant dependence on peptide concentration, and the dependence was nearly the same for all the tested peptides. The addition of acetic acid influenced the CD spectra of YPLG and oxytocin but had no influence on the spectrum of acetyl-L-tyrosine ethyl ester. The shape differences in the chromatographic peaks seem to be associated with a solvation mechanism rather than with solute-solute complexation in solution. However, the observed differences in peak asymmetry cannot be completely explained by the mechanisms that have been postulated previously. Our results suggest the formation of triple complexes between a solute molecule (or molecules), an already adsorbed solute molecule, and an adjacent region of the polymeric stationary phase. These triple complexes may influence the retention of analytes and contribute to peak asymmetry.     

Robust protein quantitation in chromatographic fractions using MALDI-MS of tryptic peptides

A method is introduced to evaluate protein concentrations using the height sum of all MALDI-MS peaks that unambiguously match theoretic tryptic peptide masses of the protein sought after. The method uses native chromatographic protein fractionation prior to digestion but does not require any depletion, labeling, derivatization, or preparation of a compound similar to the analyte. All peak heights of tryptic peptides are normalized with the peak height of a unique standard peptide added to the MALDI-MS samples. The sum of normalized peak heights, S(n), or the normalized mean peak height, M(n), reflects the concentration of the respective protein. For fractions containing various proteins, S(n) and M(n) can be used to compare concentrations of a protein between different fractions. For fractions with one predominating protein, they can be used to estimate concentration ratios between fractions, or to quantify the fractional protein concentration after calibration with pure protein solutions. Initial native fractionation retains the possibility to apply all conventional analytic procedures. Moreover, it renders the method relatively robust to MS mass accuracy. The method was validated with albumin, transferrin, alpha1-antitrypsin, and immunoglobulin G within highly complex chromatographic fractions of pathological and normal sera, which contained the respective intact native protein in dominating as well as minor concentrations. The correlation found between S(n) and the protein concentration as determined with ELISA showed that the method can be applied to select markers for distinguishing between normal and pathological serum samples.     

二维液相色谱在中药分析的应用

中药的物质体系复杂,对其组分的分离、鉴定和制备需要更高的分离度,二维液相色谱能将分离机理不同而又相互独立的两支 色谱柱串联构成分离系统,应用于复杂基质的中药材及中药复方制剂的分析,可显著提高峰容量和色谱峰鉴定的可靠性,降低色谱峰重叠, 使分离效率与分析通量大为提高。综述二维液相色谱基本原理及其在中药分析中的应用研究新进展。     

Characterization of quillaja bark extracts and evaluation of their purity using liquid chromatography–high resolution mass spectrometry

In the course of development of semi-preparative liquid chromatographic methods for the isolation of individual quillaja saponins from Quillaja saponaria (L.), some commercially available quillaja bark extracts revealed a distinctive and characteristic pattern of additional peaks in the chromatogram that could not be attributed to saponins commonly present in quillaja. To identify these peaks, analytical procedures based on HPLC coupled with high resolution MS detection were optimized which allowed the identification of the additional saponins Mi saponin A, Mi saponin B, Mi saponin C, madhucoside A and madhucoside B. These compounds are known to be the main saponins of the Indian plant Madhuca longifolia (L.). Tandem MS experiments were performed for the unambiguous assignment of the sapogenin. Madhuca saponins yielded a characteristic fragment of protobassic acid, whereas quillaja saponins showed a fragment of quillaic acid as expected. In addition, samples from madhuca seed kernels were analysed to verify the origin of the characteristic chromatographic peak pattern observed frequently in commercially available quillaja bark extracts.     

HPLC resolution of diacylglycerol moieties of natural triacylglycerols on a chiral phase consisting of bonded (R)-(+)-1-(1-naphthyl)ethylamine

Chiral phase high performance liquid chromatographic resolution of sn-1,2(2,3)- and X-1,3-diacylglycerols generated by partial Grignard degradation from natural triacylglycerols was carried out using a chiral column (25 cm x 4.6 mm i.d.) containing (R)-(+)-1-(1-napthyl)ethylamine polymer chemically bonded to 300A wide pore spherical silica (5 microns particles). The diacylglycerols were chromatographed as 3,5-dinitrophenyl-urethanes and detected at 226 or 254 nm UV. By an isocratic elution with n-hexane- 1,2-dichloroethane-ethanol 40:10:1 (v/v/v) as the mobile phase, the sn-1,2(2,3)-diacylglycerols from corn, linseed, and menhaden oils were resolved into two clearly distinguishable enantiomer groups, although some peak overlappings between the enantiomers were observed in the linseed and menhaden oil diacylglycerols. In addition to the excellent enantiomer resolution, each enantiomer and the X-1,3-isomers were partially resolved into several peaks, which could be tentatively identified on the basis of equivalent carbon number. It is concluded that chiral phase high performance liquid chromatography can be utilized for effective resolution, identification, and quantitation of enantiomeric diacylglycerols from complex natural mixtures.     

Covariance NMR in higher dimensions: application to 4D NOESY spectroscopy of proteins

Elucidation of high-resolution protein structures by NMR spectroscopy requires a large number of distance constraints that are derived from nuclear Overhauser effects between protons (NOEs). Due to the high level of spectral overlap encountered in 2D NMR spectra of proteins, the measurement of high quality distance constraints requires higher dimensional NMR experiments. Although four-dimensional Fourier transform (FT) NMR experiments can provide the necessary kind of spectral information, the associated measurement times are often prohibitively long. Covariance NMR spectroscopy yields 2D spectra that exhibit along the indirect frequency dimension the same high resolution as along the direct dimension using minimal measurement time. The generalization of covariance NMR to 4D NMR spectroscopy presented here exploits the inherent symmetry of certain 4D NMR experiments and utilizes the trace metric between donor planes for the construction of a high-resolution spectral covariance matrix. The approach is demonstrated for a 4D (13)C-edited NOESY experiment of ubiquitin. The 4D covariance spectrum narrows the line-widths of peaks strongly broadened in the FT spectrum due to the necessarily short number of increments collected, and it resolves otherwise overlapped cross peaks allowing for an increase in the number of NOE assignments to be made from a given dataset. At the same time there is no significant decrease in the positive predictive value of observing a peak as compared to the corresponding 4D Fourier transform spectrum. These properties make the 4D covariance method a potentially valuable tool for the structure determination of larger proteins and for high-throughput applications in structural biology.     

Isolation and characterization of different molecular and chromatographic forms of heparin-binding growth factor 1 from bovine brain

Bovine brain heparin-binding growth factor 1 (HBGF-1), a single polypeptide (Mr 17,400) with an amino-terminal acetylalanine and three cysteines within the sequence, isolates in multiple truncated and chromatographic forms. The relative yields of the various forms of HBGF-1 depend upon the methods used for purification. Extraction of brain tissue at neutral pH in the presence of protease inhibitors yielded intact acetylala-HBGF-1 and Asn21-HBGF-1 in a ratio of 2.3 to 1. Omission of the protease inhibitors during extraction markedly reduced the yield of acetylala-HBGF-1 and generated predominantly a mixture of Asn21-HBGF-1 and Phe15-HBGF-1. Acetylala-HBGF-1 and Asn21-HBGF-1 can be separated by cation-exchange chromatography prior to further purification. Isolated acetylala-HBGF-1 and Asn21-HBGF-1 distributed into three chromatographic peaks each on reverse-phase high-performance chromatography. Reduction of samples with dithiothreitol prior to reverse-phase chromatography reduced the three peaks of each molecular species into a single peak. Exposure of a single chromatographic peak of HBGF-1 to pH 8 in the absence of a reducing agent generated two or more additional chromatographic peaks upon subsequent chromatography. Although each chromatographic form of different molecular species of HBGF-1 exhibited potent mitogenic activity, reduction of HBGF-1 forms prior to reverse-phase chromatography appeared to increase the specific mitogenic activity of both purified molecular forms.     

Gas chromatography — mass spectrometry of catechol estrogens

The gas chromatographic and mass spectrometric properties of 19 catechol estrogens and catechol estrogen methyl ethers are reported. The gas chromatographic behaviour of the TMS-derivatives on the stationary phases OV-1, OV-3, OV-7, and OV-17 is examined and correlated with their molecular weight, shape, and polarity. The characteristic mass spectrometric features of the compounds result from the aromatic ring A, which is able to stabilize positive charge within the molecular ions. Consequently the molecular ions form the base peaks of the spectra. Fragmentation patterns highly specific for the catechols as well as for their monomethyl and dimethyl ethers are discussed and substantiated by determination of metastable ions and high resolution mass measurements.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

Assessment of protein purity by chromatography and multiwavelength detection

Commercial protein preparations were analyzed for purity on a size-exclusion column coupled to a photodiode array detector. Two methods that are commonly supplied by the manufacturers of these detectors were used to establish the purity of the eluting peaks. These methods are based on (i) a visual inspection of the superposition of normalized spectra taken upslope, at the apex, and downslope and (ii) the variation of the ratio of the absorbance measured at two different wavelengths. The results obtained were compared with those of a newly developed method, based on a comparison of at least eight peak spectra by means of their correlation coefficients. It was found that the first two methods were not reliable in the case of protein peaks containing a spectrally similar impurity, while with the third method it was possible to detect the presence of only 2% (w/w) of a spectrally closely resembling protein contaminant (r = 0.9997), eluting with a chromatographic resolution of 0.37 sigma. Analysis of the samples with polyacrylamide gel electrophoresis indicated that the new method is at least equally suited to assess protein purity, provided a minimal resolution is achieved by the chromatographic system.     

Isocratic high-performance liquid chromatographic method for the separation of testosterone metabolites

An isocratic reversed-phase high-performance liquid chromatographic (HPLC) method using an Ultrasphere IP column has been developed for the determination of testosterone and its metabolites after incubation of 4-¹⁴C-labelled or unlabelled testosterone with rat liver microsomes. Compounds were eluted with methanol-water-tetrahydrofuran (35:55:10, v/v, pH 4.0) and detected by ultraviolet (UV) absorption at 245 nm. UV or on-line radioactivity detection can be used although, due to differences in detector cell volumes, peak resolution is slightly better with UV detection. Selectivity was validated by collecting HPLC peaks and verifying their identity by gas chromatography-mass spectrometry after derivatization by N,O-bis(trimethylsily)trifluoroacetamide-trimethylchlorosilane. A three-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method, using corticosterone as internal standard. The method is applicable to the measurement of cytochrome P-450 isoenzyme activities in rat liver.     

Membrane chromatography of DNA: conformation-induced capacity and selectivity

The chromatographic purification of biological macromolecules requires a novel approach to overcome some of the pore size limitations of commercially available resins. Membrane adsorbers offer the potential for better resolution as well as productivity. Sharp peaks are gained by the rapid exchange rate with the adsorbing membranes associated with the convective flow path, in contrast to the pore diffusion requirement for resin exchange. The resolution advantage is preserved even when the very short bed heights of membranes are exploited for the purpose of exceptionally high flow rates and productivity.Breakthrough experiments were used to assess the membrane dynamic loading capacities of flexible macromolecules using supercoiled (SC) DNA as a model system. In contrast to reports for smaller biomolecules such as proteins and antibodies, the dynamic capacity for DNA was found to be highly dependent on flow rates and concentrations. Increasing flow rates induced DNA elongation, which increased the surface coverage and, in turn, lowered the capacity. Increasing concentrations beyond C*, the overlap concentration, led to exclusion-volume interactions, which reduced the size of DNA and increased the membrane adsorber capacity. In the chromatographic mode, membranes with a strongly positive charge were able to resolve various isoforms of DNA, surpassing the capabilities of analogous chromatographic resins. In this study, we found that the convective-flow-induced-structural behavior of DNA is responsible for the resolution in separation.     

Large scale chiral chromatography for the separation of an enantiomer to accelerate drug development

There are several approaches to produce enantiomerically pure drug substances, such as recrystallization, catalytic process (ligand and enzyme), indirect chromatographic resolution, and direct chromatographic resolution. However, the use of preparative chromatography with chiral stationary phases seems to be most effective for early phase projects, where the time and resources on the developments need to be minimized to get the drug candidates into the clinical studies. We showed that by following a well-defined process, chiral chromatography can be easily scaled up from an analytical system to a pilot plant system. We also used the results from a multicolumn continuous chromatography (MCC) study to conclude that MCC can be a cost-effective production method for chiral manufacturing.     

High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

ABSTRACT

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.