Towards high-throughput metabolomics using ultrahigh-field Fourier transform ion cyclotron resonance mass spectrometry

With unmatched mass resolution, mass accuracy, and exceptional detection sensitivity, Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FTICR-MS) has the potential to be a powerful new technique for high-throughput metabolomic analysis. In this study, we examine the properties of an ultrahigh-field 12-Tesla (12T) FTICR-MS for the identification and absolute quantitation of human plasma metabolites, and for the untargeted metabolic fingerprinting of inbred-strain mouse serum by direct infusion (DI). Using internal mass calibration (mass error ≤1 ppm), we determined the rational elemental compositions (incorporating unlimited C, H, N and O, and a maximum of two S, three P, two Na, and one K per formula) of approximately 250 out of 570 metabolite features detected in a 3-min infusion analysis of aqueous extract of human plasma, and were able to identify more than 100 metabolites. Using isotopically-labeled internal standards, we were able to obtain excellent calibration curves for the absolute quantitation of choline with sub-pmol sensitivity, using 500 times less sample than previous LC/MS analyses. Under optimized serum dilution conditions, chemical compounds spiked into mouse serum as metabolite mimics showed a linear response over a 600-fold concentration range. DI/FTICR-MS analysis of serum from 26 mice from 2 inbred strains, with and without acute trichloroethylene (TCE) treatment, gave a relative standard deviation (RSD) of 4.5%. Finally, we extended this method to the metabolomic fingerprinting of serum samples from 49 mice from 5 inbred strains involved in an acute alcohol toxicity study, using both positive and negative electrospray ionization (ESI). Using these samples, we demonstrated the utility of this method for high-throughput metabolomics, with more than 400 metabolites profiled in only 24 h. Our experiments demonstrate that DI/FTICR-MS is well-suited for high-throughput metabolomic analysis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Increased protein identification capabilities through novel tandem MS calibration strategies

High mass measurement accuracy is critical for confident protein identification and characterization in proteomics research. Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is a unique technique which can provide unparalleled mass accuracy and resolving power. However, the mass measurement accuracy of FTICR-MS can be affected by space charge effects. Here, we present a novel internal calibrant-free calibration method that corrects for space charge-induced frequency shifts in FTICR fragment spectra called Calibration Optimization on Fragment Ions (COFI). This new strategy utilizes the information from fixed mass differences between two neighboring peptide fragment ions (such as y(1) and y(2)) to correct the frequency shift after data collection. COFI has been successfully applied to LC-FTICR fragmentation data. Mascot MS/MS ion search data demonstrate that most of the fragments from BSA tryptic digested peptides can be identified using a much lower mass tolerance window after applying COFI to LC-FTICR-MS/MS of BSA tryptic digest. Furthermore, COFI has been used for multiplexed LC-CID-FTICR-MS which is an attractive technique because of its increased duty cycle and dynamic range. After the application of COFI to a multiplexed LC-CID-FTICR-MS of BSA tryptic digest, we achieved an average measured mass accuracy of 2.49 ppm for all the identified BSA fragments.     

FTICR-mass spectrometry for high-resolution analysis in combinatorial chemistry

The diversity of compound collections required for finding lead structures in pharmaceutical research can be provided by means of combinatorial organic chemistry. The resultant enormous number of single compounds but also of compound mixtures represents a challenge for the analyst. With the introduction of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS or FT-MS), a new and, as yet, not widespread mass spectrometric technique (a means of analysis of such compound libraries with a very high mass resolution) high mass accuracy and high sensitivity has become available. Moreover, in combination with electrospray ionization (ESI), not only high-throughput measurements via flow-injection analysis (FIA) but also coupling with separation techniques such as high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) is possible. Structural verification by way of decomposing ions (MS(n); n > or = 2) using a variety of different dissociation techniques can be performed by FTICR-MS. This is the first review specifically covering applications of FTICR-MS in the field of combinatorial chemistry.     

Bioconjugation of ribonuclease A: a detailed chromatographic and mass spectrometric analysis of chemical modification by a cross-linking reagent

The modification of ribonuclease A with the heterobifunctional cross-linker, 4-succinimdyloxycarbonyl-methyl-alpha-[2-pyridyldithio]-toluene (SMPT) is described. RNase A has 11 potential sites of modification by the SMPT reagent. Tracking the two-dimensional separation and proteolytic digestion of SMPT-modified RNase A with ESI/FTICR-MS and HPLC/ESI/QIT-MS demonstrates the detailed information about number of SMPT modifications and sites of modification that can be obtained by application of these techniques. Analysis of native and modified RNase A tryptic digests by ESI/FTICR-MS resulted in the identification of the sites of modification. Semiquantitative results of the reactivity of certain lysine residues toward the coupling reagent SMPT are presented. Two sites (lysines 1 and 37) are highly reactive, while three sites (lysines 41, 61, and 104) appear to be unreactive toward SMPT under the conditions used. Experimental results demonstrate that quantitative comparison of relative intensities of peptide sequences of different charge states is not possible. No correlation was found between number of basic residues and sensitivity to detection. Digestion of the modified and unmodified RNase A by subtilisin followed by examination by HPLC/ESI/QIT-MS and MS(n) enabled further investigation of modification on lysines 1 and 7, including modification at the epsilon- and alpha-amino positions on lysine 1.     

Improving the success rate of proteome analysis by modeling protein-abundance distributions and experimental designs

Truly comprehensive proteome analysis is highly desirable in systems biology and biomarker discovery efforts. But complete proteome characterization has been hindered by the dynamic range and detection sensitivity of experimental designs, which are not adequate to the very wide range of protein abundances. Experimental designs for comprehensive analytical efforts involve separation followed by mass spectrometry-based identification of digested proteins. Because results are generally reported as a collection of identifications with no information on the fraction of the proteome that was missed, they are difficult to evaluate and potentially misleading. Here we address this problem by taking a holistic view of the experimental design and using computer simulations to estimate the success rate for any given experiment. Our approach demonstrates that simple changes in typical experimental designs can enhance the success rate of proteome analysis by five- to tenfold.     

Enhanced instrumental sensitivity and selectivity for aminopeptidase profiling

Laser-excited fluorimetry has been applied to the identification of bacteria and fungus. The instrumental sensitivity and selectivity of the aminopeptidase profiling method has been enhanced by the use of laser excitation in conjunction with improved spectral and temporal background rejection. The linear dynamic range for the aminopeptidase technique has been increased by achieving a reduced lower limit of detection of the fluorescent tag, beta-naphthylamine. Standard aminopeptidase methodology only provides a linear dynamic range of 1.5 orders of magnitude. The laser-based method expanded the range to three orders of magnitude allowing the inherent specificity of aminopeptidase enzymes within the pathogen to be observed. The enhanced linear dynamic range was observed in profiles of Agrobacterium tumefaciens rubi and Phytophthora megasperma var. sojae.     

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.     

Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry

We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches.     

The use of accurate mass tags for high-throughput microbial proteomics

We describe and review progress towards a global strategy that aims to extend the sensitivity, dynamic range, comprehensiveness, and throughput of proteomic measurements for microbial systems based upon the use of polypeptide accurate mass tags (AMTs) produced by global protein enzymatic digestions. The two-stage strategy exploits high accuracy mass measurements using Fourier transform ion cyclotron resonance mass spectrometry (FTICR) to validate polypeptide AMTs for a specific organism, from potential mass tags tentatively identified using tandem mass spectrometry (MS/MS), providing the basis for subsequent measurements without the need for routine MS/MS. A high-resolution capillary liquid chromatography separation combined with high sensitivity, and high-resolution accurate FTICR measurements is shown to be capable of characterizing polypeptide mixtures of more than 10(5) components, sufficient for broad protein identification using AMTs. Advantages of the approach include the high confidence of protein identification, its broad proteome coverage, and the capability for stable-isotope labeling methods for precise relative protein abundance measurements. The strategy has been initially evaluated using the microorganisms Saccharomyces cerevisiae and Deinococcus radiodurans. Additional developments, including the use of multiplexed-MS/MS capabilities and methods for dynamic range expansion of proteome measurements that promise to further extend the quality of proteomics measurements, are also described.     

Sensitivity measurement and compensation in spectral imaging.

BACKGROUND: The ImageStream system combines advances in CCD technologies with a novel optical architecture for high sensitivity and multispectral imaging of cells in flow. The sensitivity and dynamic range as well as a methodology for spectral compensation of imagery is presented. METHODS: Multicolored fluorescent beads were run on the ImageStream and a flow cytometer. Four single color fluorescent control samples of cells were run to quantify spectral overlap. An additional sample, labeled with all colors was run and compensated in six spectral channels. RESULTS: Analysis of empirical data for sensitivity and dynamic range matched theoretical predictions. The ImageStream system demonstrated fluorescence sensitivity comparable to a PMT-based flow cytometer. A methodology for addressing spectral overlap, individual pixel anomalies, and multiple imaging modalities was demonstrated for spectral compensation of K562 cells. Imagery is shown pre- and post-compensation. CONCLUSIONS: Unlike intensity measurements made with conventional flow cytometers, object size impacts both dynamic range and fluorescence sensitivity in systems that utilize pixilated detection. Simultaneous imaging of alternate modalities can be employed to increase fluorescent sensitivity. Effective compensation of complex multimode imagery spanning six spectral bands is accomplished in a semi-automated manner.     

Evolutionary optimization of fluorescent proteins for intracellular FRET

Fluorescent proteins that exhibit Forster resonance energy transfer (FRET) have made a strong impact as they enable measurement of molecular-scale distances through changes in fluorescence. FRET-based approaches have enabled otherwise intractable measurements of molecular concentrations, binding interactions and catalytic activity, but are limited by the dynamic range and sensitivity of the donor-acceptor pair. To address this problem, we applied a quantitative evolutionary strategy using fluorescence-activated cell sorting to optimize a cyan-yellow fluorescent protein pair for FRET. The resulting pair, CyPet-YPet, exhibited a 20-fold ratiometric FRET signal change, as compared to threefold for the parental pair. The optimized FRET pair enabled high-throughput flow cytometric screening of cells undergoing caspase-3-dependent apoptosis. The CyPet-YPet energy transfer pair provides substantially improved sensitivity and dynamic range for a broad range of molecular imaging and screening applications.     

Comparison of ovalbumin quantification using forward-phase protein microarrays and suspension arrays

We employed ovalbumin (a simulant used for ricin and botulism toxins in biodefense applications) and its high affinity polyclonal antibody as a model system to examine the sensitivity, dynamic range, linearity, and reproducibility of forward-phase array results in comparison to suspension arrays. It was found that protein microarrays had a dynamic range of 4 orders of magnitude and a sensitivity of less than 1 pg/mL, respectively. The dynamic range and sensitivity of suspension arrays were close to 2 orders of magnitude and 0.25 ng/mL, respectively. The sensitivity we observed for the suspension arrays is comparable to that reported for enzyme-linked immunosorbent assays (ELISAs) in the literature. We used ovalbumin samples with two different purities, 38.0% and 76.0% (w/w), as determined by polyacrylamide gel electrophoresis (PAGE). These samples were used to evaluate the effect of impure samples on detection. The data obtained from the forward-phase protein arrays gave values that were consistent with the PAGE data. The data from the suspension arrays were not as consistent and may indicate that this format may not give as reliable data with impure samples. Knowledge of the advantages and disadvantages of the two proteomic methods would allow their more rational use in clinical diagnosis.     

Making blind robots see: the synergy between fluorescent dyes and imaging devices in automated proteomics

Proteomics investigations endeavor to provide a global understanding of gene product synthesis rate, degradation rate, functional competence, posttranslational modification, subcellular distribution and physical interactions with other cell components. Protein expression encompasses an enormous dynamic range. Since rare proteins cannot be amplified by any type of PCR method, sensitive detection is critical to proteome projects. Fluorescence methods deliver streamlined detection protocols, superior detection sensitivity, broad linear dynamic range and excellent compatibility with modern microchemical identification methods such as mass spectrometry. Two general approaches to fluorescence detection of proteins are currently practiced: the covalent derivatization of proteins with fluorophores or noncovalent interaction of fluorophores either via the SDS micelle or through direct electrostatic interaction with proteins. One approach for quantifying fluorescence is to use a photomultiplier tube detector combined with a laser light scanner. In addition, fluorescence imaging is performed using a charge-coupled device camera combined with a UV light or xenon arc source. Fluorescent dyes with bimodal excitation spectra may be broadly implemented on a wide range of analytical imaging devices, permitting their widespread application to proteomics studies and incorporation into semiautomated analysis environments.     

The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response

The cloning of the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) cDNAs provides a basis for understanding the actions of glucocorticoids in the central nervous system. Structural evidence is presented for the identity of the type I corticosteroid binding site as the MR expressed in the brain. This identification is supported by the anatomical distribution of MR mRNA, determined by in situ hybridization histochemistry, which parallels the steroid autoradiographic localization of the type I sites. An in vitro assay for MR and GR function demonstrates that these receptors respond to different levels of glucocorticoid, suggesting that together they confer a larger dynamic range of sensitivity to this hormone. These studies lead to a new hypothesis for glucocorticoid action in the central nervous system.     

An autonomous system for identifying and governing a cell's state in yeast

We present an approach for an autonomous system that detects a particular state of interest in a living cell and can govern cell fate accordingly. Cell states could be better identified by the expression pattern of several genes than of a single one. Therefore, autonomous identification can be achieved by a system that measures the expression of these several genes and integrates their activities into a single output. We have constructed a system that diagnoses a unique state in yeast, in which two independent pathways, methionine anabolism and galactose catabolism, are active. Our design is based on modifications of the yeast two-hybrid system. We show that cells could autonomously report on their state, identify the state of interest, and inhibit their growth accordingly. The system's sensitivity is adjustable to detect states with limited dynamic range of inputs. The system's output depends only on the activity of input pathways, not on their identity; hence it is straightforward to diagnose any pair of inputs. A simple model is presented that accounts for the data and provides predictive power. We propose that such systems could handle real-life states-of-interest such as identification of aberrant versus normal growth.     

Flight of a cytidine deaminase complex with an imperfect transition state analogue inhibitor: mass spectrometric evidence for the presence of a trapped water molecule

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K(d) ～ 10(-12) M), capturing all but ～5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K(tx) ～ 10(-16) M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme-inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364-11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E(2)Zn(2)W(2) (dimeric Zn-CDA with two water molecules), E(2)Zn(2)W(2)Fz, and E(2)Zn(2)W(2)Fz(2), where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E(2)Zn(2) is the only dimeric species detected, with no additional water molecules. Experiments conducted in H(2)(18)O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E(2)Zn(2)) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k(off) > 0.14 s(-1)) much more rapidly than is 5-fluorozebularine (k(off) = 0.014 s(-1)), despite the greater thermodynamic stability of the zebularine complex.     

Trends in mass spectrometry instrumentation for proteomics

Mass spectrometry has become a primary tool for proteomics because of its capabilities for rapid and sensitive protein identification and quantitation. It is now possible to identify thousands of proteins from microgram sample quantities in a single day and to quantify relative protein abundances. However, the need for increased capabilities for proteome measurements is immense and is now driving both new strategies and instrument advances. These developments include those based on integration with multi-dimensional liquid separations and high accuracy mass measurements and promise more than order of magnitude improvements in sensitivity, dynamic range and throughput for proteomic analyses in the near future.     

Low abundance protein enrichment for discovery of candidate plasma protein biomarkers for early detection of breast cancer

Molecular biomarkers of early stage breast cancer may improve the sensitivity and specificity of diagnosis. Plasma biomarkers have additional value in that they can be monitored with minimal invasiveness. Plasma biomarker discovery by genome-wide proteomic methods is impeded by the wide dynamic range of protein abundance and the heterogeneity of protein expression in healthy and disease populations which requires the analysis of a large number of samples. We addressed these issues through the development of a novel protocol that couples a combinatorial peptide ligand library protein enrichment strategy with isobaric label-based 2D LC-MS/MS for the identification of candidate biomarkers in high throughput. Plasma was collected from patients with stage I breast cancer or benign breast lesions. Low abundance proteins were enriched using a bead-based combinatorial library of hexapeptides. This resulted in the identification of 397 proteins, 22% of which are novel plasma proteins. Twenty-three differentially expressed plasma proteins were identified, demonstrating the effectiveness of the described protocol and defining a set of candidate biomarkers to be validated in independent samples. This work can be used as the basis for the design of properly powered investigations of plasma protein expression for biomarker discovery in larger cohorts of patients with complex disease.     

Precision mapping of the metabolome

The global study of the structure and dynamics of metabolic networks has been hindered by a lack of techniques that identify metabolites and their biochemical relationship in complex mixtures. The recent application of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to metabolomic analysis suggests a way to tackle the problem. A lower-cost alternative to high-field FTICR-MS, the Orbitrap mass analyzer, promises accelerated activity in this area. Here, we show how the ultra-high mass accuracy and resolution provided by this new generation of mass spectrometers can help to identify metabolites and connect them into metabolic networks. Data from perturbation studies and isotope-tracking experiments can complement this information to create metabolic maps de novo and chart unexplored areas of metabolism.     

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9) GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.