Determination of cytokinins by mass spectrometry based on stable isotope dilution.

A method for quantitative determination of individual cytokinin species has been developed, based on gas chromatography-mass spectrometry and selected ion monitoring. Deuterated internal standards were prepared for analysis of N⁶-isopentenyladenosine, N⁶-isopentenyl-2-methylthioadenosine, zeatin riboside, and 2-methylthiozeatin riboside and were tested over the range of 1 to 20 ng of endogenous cytokinin per injection, relative to 100 ng of labeled standard. An isolation procedure for extracts of cabbage hearts as a model plant source has been developed that gives maximum recovery and minimum interference for gas chromatographic-mass spectrometric measurements. The present method differs from the commonly used bioassay by its selectivity for individual cytokinin components and shortened analysis time, including extractions, of 3 days vs several weeks.     

Automated comparative proteomics based on multiplex tandem mass spectrometry and stable isotope labeling

Comparative proteomic approaches using isotopic labeling and MS have become increasingly popular. Conventionally quantification is based on MS or extracted ion chromatogram (XIC) signals of differentially labeled peptides. However, in these MS-based experiments, the accuracy and dynamic range of quantification are limited by the high noise levels of MS/XIC data. Here we report a quantitative strategy based on multiplex (derived from multiple precursor ions) MS/MS data. One set of proteins was metabolically labeled with [13C6]lysine and [15N4]arginine; the other set was unlabeled. For peptide analysis after tryptic digestion of the labeled proteins, a wide precursor window was used to include both the light and heavy versions of each peptide for fragmentation. The multiplex MS/MS data were used for both protein identification and quantification. The use of the wide precursor window increased sensitivity, and the y ion pairs in the multiplex MS/MS spectra from peptides containing labeled and unlabeled lysine or arginine offered more information for, and thus the potential for improving, protein identification. Protein ratios were obtained by comparing intensities of y ions derived from the light and heavy peptides. Our results indicated that this method offers several advantages over the conventional XIC-based approach, including increased sensitivity for protein identification and more accurate quantification with more than a 10-fold increase in dynamic range. In addition, the quantification calculation process was fast, fully automated, and independent of instrument and data type. This method was further validated by quantitative analysis of signaling proteins in the EphB2 pathway in NG108 cells.     

Quantitative proteome analysis by solid-phase isotope tagging and mass spectrometry

The adaptation of sequences of chemical reactions to a solid-phase format has been essential to the automation, reproducibility, and efficiency of a number of biotechnological processes including peptide and oligonucleotide synthesis and sequencing. Here we describe a method for the site-specific, stable isotopic labeling of cysteinyl peptides in complex peptide mixtures through a solid-phase capture and release process, and the concomitant isolation of the labeled peptides. The recovered peptides were analyzed by microcapillary liquid chromatography and tandem mass spectrometry (microLC-MS/MS) to determine their sequences and relative quantities. The method was used to detect galactose-induced changes in protein abundance in the yeast Saccharomyces cerevisiae. A side-by-side comparison with the isotope-coded affinity tag (ICAT) method demonstrated that the solid-phase method for stable isotope tagging of peptides is comparatively simpler, more efficient, and more sensitive.     

Development of an isotope dilution mass spectrometry assay for HbA1c based on enzyme-cleaved peptide analysis

The hydrophobic contributions of 17 individual peptides, fused to the N-terminal of Bacillus stearothermophilus lactate dehydrogenase (LDH) were studied by hydrophobic interaction chromatography (HIC) and aqueous two-phase system (ATPS). The constructs were sequenced from a protein library designed with a five-amino acid randomised region in the N-terminal of an LDH protein. The 17 LDH variants and an LDH control lacking the randomised region were expressed in Escherichia coli. HIC and ATPS behaviour of the proteins indicated significant differences in protein hydrophobicity, even though the modifications caused only 1% increase in protein molecular weight and 2% variation in isoelectric points. HIC and ATPS results correlated well (R(2) = 0.89). Protein expression was clearly affected by N-terminal modification, but there was no evidence that the modification affected protein activity. A GluAsnAlaAspVal modification resulted in increased protein expression. In most cases, HIC and ATPS results compared favourably with those predicted on the basis of 34 amino acid residue hydrophobicity scales; assuming exposure of tag residues to solution. Exceptions included LeuAlaGlyValIle and LeuTyrGlyCysIle modifications, which were predicted, assuming full solution exposure, to be more hydrophobic than observed.     

Profiling of urinary formestane and confirmation by isotope ratio mass spectrometry

Formestane (F, androst-4-en-4-ol-3,17-dione) is an irreversible aromatase inhibitor with the ability to suppress the estrogen production from anabolic steroids. Consequently, F is mentioned on the World Anti-Doping Agency (WADA) prohibited list and because studies have shown that F is produced endogenously in small amounts, a threshold for urinary excreted F of 150 ng/mL was introduced. Lower concentrations could be due to endogenous production and need further investigation to prove the exact origin through determination of the carbon isotope ratio.However, because the current screening methods are a lot more sensitive, F is detected in practically every urine sample. A strict implementation of this WADA rule would imply that almost every urine sample needs additional investigation to verify an exogenous or endogenous origin. The main aim of this study was to propose and introduce a lower concentration limit of 25 ng/mL beneath which the detected F is considered as being endogenous and no further investigation is needed. The data presented in this paper suggests that this threshold provides a good balance between a sufficiently large detection window and not having to perform isotope ratio mass spectrometry (IRMS) analyses on negative urine samples.     

Quantification of growth hormone in serum by isotope dilution mass spectrometry

Interassay variation of antibody-based routine tests hampers comparability of measurement results for growth hormone (GH) between different laboratories and decision making in clinical practice. Here it is demonstrated that quantification of GH by isotope dilution mass spectrometry (IDMS) constitutes a way to obtain precise and reliable results that can be referred to in evaluation of performance of commercial test kits. With the IDMS method developed, tryptic cleavage products YSFLQNPQTSLCFSESIPTPSNR (T6) and LEDGSPR (T12) of GH are quantified by liquid chromatography tandem mass spectrometry (LC-MS/MS) using isotopically labeled forms of the peptides as internal standards. The GH cleavage fragments are obtained by whole serum tryptic proteolysis and then extracted from the resulting mixture by semipreparative reversed-phase LC followed by strong cation exchange chromatography. Analysis of blank serum spiked with recombinant 22-kDa GH at different concentration levels would result in a mean recovery of 101.6%, a standard deviation (SD) of 2.5%, a combined uncertainty (u_c) of 3.0%, and a limit of quantification (LOQ) of 1.7 μg/L when quantifying T6 as a GH-derived fragment, whereas recovery = 100.7%, SD = 2.4%, u_c = 2.5%, and LOQ = 2.7 μg/L were found with T12. The potential to acquisition of reference values is exemplified by application to serum materials used in a recent quality assessment exercise for routine laboratories.     

Discovering mercury protein modifications in whole proteomes using natural isotope distributions observed in liquid chromatography-tandem mass spectrometry

The identification of peptides that result from post-translational modifications is critical for understanding normal pathways of cellular regulation as well as identifying damage from, or exposures to xenobiotics, i.e. the exposome. However, because of their low abundance in proteomes, effective detection of modified peptides by mass spectrometry (MS) typically requires enrichment to eliminate false identifications. We present a new method for confidently identifying peptides with mercury (Hg)-containing adducts that is based on the influence of mercury's seven stable isotopes on peptide isotope distributions detected by high-resolution MS. Using a pure protein and E. coli cultures exposed to phenyl mercuric acetate, we show the pattern of peak heights in isotope distributions from primary MS single scans efficiently identified Hg adducts in data from chromatographic separation coupled with tandem mass spectrometry with sensitivity and specificity greater than 90%. Isotope distributions are independent of peptide identifications based on peptide fragmentation (e.g. by SEQUEST), so both methods can be combined to eliminate false positives. Summing peptide isotope distributions across multiple scans improved specificity to 99.4% and sensitivity above 95%, affording identification of an unexpected Hg modification. We also illustrate the theoretical applicability of the method for detection of several less common elements including the essential element, selenium, as selenocysteine in peptides.     

Application of stable isotope dilution assays based on liquid chromatography-tandem mass spectrometry for the assessment of folate bioavailability

A pilot study was performed to prove the suitability of stable isotope dilution assays for assessing the bioavailability of endogenous folates in foods. By using [2H(4)]folic acid, [2H(4)]tetrahydrofolate, [2H(4)]5-methyltetrahydrofolate, [2H(4)]5-formyltetrahydrofolate and [2H(4)]10-formylfolic acid as internal standards, folates in spinach, apple juice and blood plasma were quantified by liquid chromatography coupled to tandem mass spectrometry. To liberate the pteroyl monoglutamates, sample extracts of foods were treated by rat plasma. Sample clean-up was achieved by solid-phase extraction on anion-exchange cartridges, which proved to be sufficient to obtain mass chromatograms devoid of matrix interferences. The bioavailability study was designed as a short-time protocol with three meals, the first consisting of 600 g spinach (meal A), the second consisting of 600 g apple sauce with additionally 400 microg synthetic folic acid (meal B) and the third consisting solely of 600 g apple sauce (meal C). Prior to the meals, the participating volunteer's tissue was saturated with folates to achieve a significant response of plasma folate to the meals. After consumption of meals A and B a significant rise in folate plasma level compared to meal C (mean level at 28 microg/ml) was observed. The relative bioavailability of folate following meal A exceeded significantly the suggested value of 50% for food folates by taking the dose-normalized area under the curve (AUC) following ingestion of meal B as reference.     

In-gel stable isotope labeling for relative quantification using mass spectrometry

Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.     

Stable isotope mass spectrometry in childhood lead poisoning

In order to better understand the relative importance of various sources of lead in childhood lead poisoning, high-precision, isotoperatio, solid-source-mass spectrometry of microgram-sized lead samples was applied to three hospitalized cases in Boston, ranging in age from 1.5 to 14 yr, that had blood-lead levels of 0.7–1.2 μg/g. The lead isotopes in the ambient Boston environment (air, soil, and dust) were also measured. In each case, the isotopic composition (IC) of the child's blood lead was identical with the IC of lead paint taken from the child's residence at a site accessible to the child. Fecal lead samples were also identical to that particular paint. Soil lead IC did not always match the IC of local paints. Paint samples vary widely in their IC's (206/204=17.5–19.4, about 200 times analytical reliability). Dust in homes that never had lead paint contained lead that resembled lead in urban soils. Dust lead IC did not necessarily have the same IC as current automobile lead emissions, but appeared to reflect the long-term accumulation of several sources of urban lead fallout. Limitations and implications of this data are discussed.     

Tryptic transpeptidation products observed in proteome analysis by liquid chromatography-tandem mass spectrometry

Commonly, prior to mass spectrometry based analysis of proteins or protein mixtures, the proteins are subjected to specific enzymatic proteolysis. For this purpose trypsin is most frequently used. However, the process of proteolysis is not unflawed. For example, some side activities of trypsin are known and have already been described in the literature (e.g., chymotryptic activity). Here, we describe the occurrence of transpeptidated peptides during standard proteome analysis using two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometric protein identification. Different types of transpeptidated peptides have been detected. The most frequently observed transpeptidation reaction is N-terminal addition of arginine or lysine to peptides. Furthermore, addition of two amino acids to the N-terminus of a peptide has also been detected. Another transpeptidation that we observed, is combination of two peptides, which were originally located in different regions of the analyzed protein. Currently, the full amount of peptides generated by transpeptidation is not clear. However, it should be recognized that protein information is presently lost as these effects are not detectable with available database search software.     

Quantitation of vitamin B6 in biological samples by isotope dilution mass spectrometry

Methods have been developed for the simultaneous quantitative analysis of vitamin B6 forms in biological samples by isotope dilution mass spectrometry using deuterated forms of pyridoxine, pyridoxal, pyridoxamine, and pyridoxic acid. The biological fluid or tissue sample was homogenized and then treated with a cocktail containing appropriate amounts of each deuterated vitamer, as well as the deuterated, phosphorylated vitamer forms. The individual vitamers were isolated from the homogenate by a complex high-performance liquid chromatographic procedure that provided separate fractions for each of the six vitamers found in biological samples. Aldehydic B6 vitamers were reduced to the alcohol form prior to acetylation and analysis by gas chromatography/mass spectrometry (GC/MS). The three resulting vitamers were analyzed by electron ionization GC/MS using a silicone capillary column. The methods have been applied to analysis of vitamin B6 in liver, milk, urine, and feces at levels as low as 0.02 nmol/ml.     

Turnover rates of different collagen types measured by isotope ratio mass spectrometry.

The rates of collagen turnover in different tissues have been estimated in growing rats previously exposed to gaseous 18O2. The abundance of the stable isotope was measured using isotope ratio mass spectrometry following combustion of isolated collagen-derived hydroxyproline. Using this method, problems of label reutilization associated with radiolabelling methods are avoided. In general the results confirm the slow turnover rates with half-lives of total collagen in skin, muscle and gut of 74, 45 and 244 d, respectively. The use of cyanogen bromide digests of whole tissues followed by isolation of collagen type-specific peptides has allowed the comparison of turnover rates of collagen types I and III, indicating that collagen type III is turned over more rapidly than type I.     

Differential quantitative analysis of MHC ligands by mass spectrometry using stable isotope labeling

Currently, no method allows direct and quantitative comparison of MHC-presented peptides in pairs of samples, such as transfected and untransfected, tumorous and normal or infected and uninfected tissues or cell lines. Here we introduce two approaches that use isotopically labeled reagents to quantify by mass spectrometry the ratio of peptides from each source. The first method involves acetylation and is both fast and simple. However, higher peptide recoveries and a finer sensitivity are achieved by the second method, which combines guanidination and nicotinylation, because the charge state of peptides can be maintained. Using differential acetylation, we identified a beta catenin-derived peptide in solid colon carcinoma overpresented on human leucocyte antigen-A (HLA-A)(*)6801. Guanidination/nicotinylation was applied to keratin 18-transfected cells and resulted in the characterization of the peptide RLASYLDRV (HLA-A(*)0201), exclusively presented on the transfectant. Thus, we demonstrate methods that enable a pairwise quantitative comparison leading to the identification of overpresented MHC ligands.     

Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry

Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.     

Quantification of protein calibrants by amino acid analysis using isotope dilution mass spectrometry

This work demonstrates that amino acid analysis based on isotope dilution mass spectrometry (IDMS) can be applied to quantify proteins having different complexities and natures. Five proteins and one decapeptide were selected for the study: C-reactive protein (CRP), beta-2-microglobulin (B2 M), cystatine C (CysC), human serum albumin (HSA), Ara h1, and angiotensin I. The quantification was based on the determination of four amino acids, proline (Pro), isoleucine (Ile), valine (Val), and phenylalanine (Phe) within a working range between 5 and 100 pmol/injection of each amino acid, after 60 min digestion with HCl at 150 °C. The amino acids were selected taking into account their abundance in the protein sequence and to include the more difficult to break peptide bonds. Quantification of the protein amounts calculated from each amino acid is consistent, indicating that the method is working reliably. This consistency points to a complete hydrolysis of the proteins. The trueness of the method was proven when dry mass determination after dialysis was applied to HSA and CRP and the results were compared to those from amino acid analysis. Traceability to SI was assured by extensive characterisation of the amino acid calibrants by nuclear magnetic resonance, neutron activation analysis, and Karl Fischer titration.     

A new method for determination of plasma homocystine by isotope dilution and electrospray tandem mass spectrometry

A new analytical determination method of homocystine in human plasma has been developed. The method utilises liquid chromatography coupled to ionspray tandem mass spectrometry. Quantitative analysis was achieved using as an internal standard homocystine-d8. Mass spectrometer operated in the multiple reaction mode: homocystine and homocystine-d8 were detected through the transition from the precursor to the product ion (from m/z 269.3 to 90.0, and m/z 277.3 to 94.0, respectively). The method is extremely sensitive, with limit of detection in the range of 6 fmol/L. The interassay and intraassay coefficients of variation for homocystine were 6.22% and 3.4%, respectively. The accuracy for the added homocystine ranged from 85% to 110%. High specificity of tandem mass spectrometry coupled with a fast chromatographic process is suitable for a rapid and reliable assay of homocystine.