Analysis and quantitation of fructooligosaccharides using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry

Inulin is a class of fructooligosaccharide (FOS) derived from plants, which is often used as a natural food ingredient. Inulin is currently used as an additive in baked goods, dairy products, infant formula, and dietary supplements as a result of its purported health-promoting properties. The growth of health-promoting lactobacilli and bifidobacteria is supported by FOS, giving it the classification of a prebiotic; however, its ability to selectivity stimulate only beneficial bacteria has not been demonstrated. In order to better understand the role of inulin and FOS as prebiotics, matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry has been used for qualitative and quantitative analysis on bacterial growth. A method using an internal standard has been developed to quantify the consumption of FOS by Bifidobacterium longum bv. infantis using a calibration curve. Due to the differential consumption of FOS, the calibration curve was modified to include intensity components for each polymer unit in order to achieve more accurate quantitation. The method described was designed to be more rapid, precise, and robust for quantitative analysis when compared to existing methods.     

Carbohydrate analysis by desorption electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

We report the use of desorption electrospray ionization hybrid Fourier transform ion cyclotron resonance mass spectrometry (DESI-FT-ICR-MS) for the analysis of carbohydrates. Spectra of neat carbohydrates are presented along with their mass measurement accuracies and limits of detection. Furthermore, a comparison is made between the analyses of O-linked glycans from mucin by DESI-FT-ICR-MS and matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. Finally, glycans from mucin are identified by using the high mass measurement accuracy and tandem MS capabilities afforded by the hybrid FT-ICR-MS platform.     

Capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for protein characterization

On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin beta chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study.     

Composition of explosives by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

Commercial explosives are complex mixtures that contain not only the active explosive agent(s) but also a host of other organic and inorganic compounds. The ultrahigh mass resolving power (m/delta m50% >200,000) and mass accuracy (<1 ppm) of electrospray ionization Fourier transform ion cyclotron resonance (ESI FTICR) mass spectrometry allow for definitive identification of various species in TNT, RDX, and HMX. We are thereby able to correct prior misassignments of the elemental compositions of the most abundant negative ions from electrospray of RDX and HMX. Although the (known) active agents of many explosives may be identified by low-resolution MS or MS/MS, it is the other characteristic components (indigenous or artificial additives) whose presence and elemental composition can potentially identify the source of the product. ESI FTICR mass spectrometry of smokeless powder, TNT, and Powermite resolves and identifies numerous nonactive ingredients, many of which are recovered in a postblast residue. In contrast, the residue recovered from an explosion of military C4 yielded several species derived from RDX but virtually none from other ingredients.     

High-resolution field desorption/ionization fourier transform ion cyclotron resonance mass analysis of nonpolar molecules

We report the first field desorption ionization broadband high-resolution (m/Deltam(50%) approximately 65 000) mass spectra. We have interfaced a field ionization/field desorption source to a home-built 9.4-T FT-ICR mass spectrometer. The instrumental configuration employs convenient sample introduction (in-source liquid injection) and external ion accumulation. We demonstrate the utility of this configuration by generating high-resolution positive-ion mass spectra of C(60) and a midboiling crude oil distillate. The latter contains species not accessible by common soft-ionization methods, for example, low-voltage electron ionization, electrospray ionization, and matrix-assisted laser desorption/ionization. The present work demonstrates significant advantages of FI/FD FT-ICR MS for analysis of nonpolar molecules in complex mixtures.     

Packed capillary reversed-phase liquid chromatography with high-performance electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry for proteomics

In this study, high-efficiency packed capillary reversed-phase liquid chromatography (RPLC) coupled on-line with high-performance Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been investigated for the characterization of complex cellular proteolytic digests. Long capillary columns (80-cm) packed with small (3-micron) C18 bonded particles provided a total peak capacity of approximately 1000 for cellular proteolytic polypeptides when interfaced with an ESI-FTICR mass spectrometer under composition gradient conditions at a pressure of 10,000 psi. Large quantities of cellular proteolytic digests (e.g., 500 micrograms) could be loaded onto packed capillaries of 150-micron inner diameter without a significant loss of separation efficiency. Precolumns with suitable inner diameters were found useful for improving the elution reproducibility without a significant loss of separation quality. Porous particle packed capillaries were found to provide better results than those containing nonporous particles because of their higher sample capacity. Two-dimensional analyses from the combination of packed capillary RPLC with high-resolution FTICR yield a combined capacity for separations of > 1 million polypeptide components and simultaneously provided information for the identification of the separated components based upon the accurate mass tag concept previously described.     

C60 secondary ion Fourier transform ion cyclotron resonance mass spectrometry

Secondary ion mass spectrometry (SIMS) has seen increased application for high spatial resolution chemical imaging of complex biological surfaces. The advent and commercial availability of cluster and polyatomic primary ion sources (e.g., Au and Bi cluster and buckminsterfullerene (C(60))) provide improved secondary ion yield and decreased fragmentation of surface species, thus improving accessibility of intact molecular ions for SIMS analysis. However, full exploitation of the advantages of these new primary ion sources has been limited, due to the use of low mass resolution mass spectrometers without tandem MS to enable enhanced structural identification capabilities. Similarly, high mass resolution and high mass measurement accuracy would greatly improve the chemical specificity of SIMS. Here we combine, for the first time, the advantages of a C(60) primary ion source with the ultrahigh mass resolving power and high mass measurement accuracy of Fourier transform ion cyclotron resonance mass spectrometry. Mass resolving power in excess of 100?000 (m/Δm(50%)) is demonstrated, with a root-mean-square mass measurement accuracy below 1 part-per-million. Imaging of mouse brain tissue at 40 μm pixel size is shown. Tandem mass spectrometry of ions from biological tissue is demonstrated and molecular formulas were assigned for fragment ion identification.     

Instrumentation and method for ultrahigh resolution field desorption ionization fourier transform ion cyclotron resonance mass spectrometry of nonpolar species

We describe the construction and application of a 9.4-T FT-ICR mass spectrometer interfaced to a commercial field desorption ion source for high-resolution, high-mass accuracy measurements of nonpolar species. The FT-ICR MS instrument includes a liquid injection field desorption ionization source, octopole ion guides, external octopole ion trap capable of an axial potential gradient for ion ejection, capacitively coupled open cylindrical ion trap, and pulsed gas valve for ion cooling. Model compound responses with regard to various source and instrument conditions provide a basis for interpretation of broadband mass spectra of complex mixtures. As an example, we demonstrate broadband speciation of a Gulf Coast crude oil, with respect to numerous heteroatomic classes, compound types (rings plus double bonds), and carbon number distributions.     

Analysis of saturated hydrocarbons by using chemical ionization combined with laser-induced acoustic desorption/Fourier transform ion cyclotron resonance mass spectrometry

Laser-induced acoustic desorption (LIAD), combined with chemical ionization by the cyclopentadienyl cobalt radical cation (CpCo.+), is demonstrated to facilitate the analysis of saturated hydrocarbons by Fourier transform ion cyclotron resonance mass spectrometry. The LIAD/CpCo.+ method produces unique pseudomolecular ions for alkanes from C(24)H(50) to C(50)H(102). These alkanes were tested individually and in artificial mixtures of up to seven components. Only one product ion, [R + CpCo - 2H(2)].+, was detected for each alkane (R). The product ions' relative abundances correspond to the relative molar concentration of each alkane in mixtures. These findings provide a solid groundwork for the future application of this method for hydrocarbon polymer analyses.     

Dual electrospray ionization source for confident generation of accurate mass tags using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has rapidly established a prominent role in proteomics because of its unparalleled resolving power, sensitivity and ability to achieve high mass measurement accuracy (MMA) simultaneously. However, space-charge effects must be quantitatively, routinely, and confidently corrected because they are known to profoundly influence MMA. We argue that the most effective way to account for space-charge effects is to introduce an internal mass calibrant (IMC) using a dual electrospray ionization (ESI) source where the IMC is added from a separate ESI emitter. The major disadvantage of our initial dual ESI source to achieve high MMA, and arguably the only one, was the time required to switch between the analyte emitter and IMC emitter (i.e., >300 ms). While this "switching time" was acceptable for direct infusion experiments, it did not lend itself to high-throughput applications or when conducting on-line liquid separations. In this report, we completely redesigned the dual ESI source and demonstrate several key attributes. First, the new design allows for facile alignment of ESI emitters, undetectable vibration, and the ability to extend to multiple emitters. Second, the switching time was reduced to <50 ms, which allowed the analyte and IMC to be accumulated "simultaneously" in the external ion reservoir and injected as a single ion packet into the ion cyclotron resonance cell, eliminating the need for a separate accumulation and ion injection event for the IMC. Third, by using a high concentration of the IMC, the residence time on this emitter could be reduced to approximately 80 ms, allowing for more time spent accumulating analyte ions of significantly lower concentration. Fourth, multiplexed on-line separations can be carried out providing increased throughput. Specifically, the new dual ESI source has demonstrated its ability to produce a stable ion current over a 45-min time period at 7 T resulting in mass accuracies of 1.08 ppm +/- 0.11 ppm (mean +/- confidence interval of the mean at 95% confidence; N = 160). In addition, the analysis of a tryptic digest of apomyoglobin by nanoLC-dual ESI-FT-ICR afforded an average MMA of -1.09 versus -74.5 ppm for externally calibrated data. Furthermore, we demonstrate that the amplitude of a peptide being electrosprayed at 25 nM can be linearly increased, ultimately allowing for dynamic analyte/IMC abundance modulation. Finally, we demonstrate that this source can reliably be used for multiplexing measurements from two (eventually more) flow streams.     

Probing proteomes using capillary isoelectric focusing-electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

Unlike the genome, the proteome is exquisitely sensitive to cellular conditions and will consist of proteins having abundances dependent upon stage in the cell cycle, cell differentiation, response to environmental conditions (nutrients, temperature, stress etc.), or disease state(s). Therefore, the study of proteomes under well-defined conditions can provide a better understanding of complex biological processes and inference of protein function. Thus, much faster, more sensitive, and precise capabilities for the characterization of cellular constituents are desired. We describe progress in the development and initial application of the powerful combination of capillary isoelectric focusing (CIEF) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry for measurements of the proteome of the model system Escherichia coli. Isotope depletion of the growth media has been used to improve mass measurement accuracy, and the comparison of CIEF-FTICR results for the analysis of cell lysates harvested from E. coli cultured in normal and isotopically depleted media are presented. The initial studies have revealed 400-1000 putative proteins in the mass range 2-100 kDa from total injections of approximately 300 ng of E. coli proteins in a single CIEF-FTICR analysis.     

High sensitivity Fourier transform ion cyclotron resonance mass spectrometry for biological analysis with nano-LC and microelectrospray ionization

Modifications to a 7 T nano-LC micro-ESI FT-ICR mass spectrometer, including a shorter octopole, approximately 100% duty cycle, improved nano-LC micro-ESI emitter tips, and reverse-phase HPLC resins that require no ion-pairing agent, combine to achieve attomole detection limit. Three peptides in a mixture totaling 500 attomoles (amol) each in water (10 microL, 50 amol/microL) are separated and detected, demonstrating detection from a mixture at low endogenous biological concentration. Two peptides in a mixture totaling 500 amol each in artificial cerebrospinal fluid (1 microL, 500 amol/microL) are separated and detected, demonstrating detection from a mixture at a biological concentration in a biological solvent. The highest sensitivity is attained with arg8-vasotocin, in which a total of 300 amol is detected in artificial cerebrospinal fluid (1 microL, 300 amol/microL) and a total of 100 amol in water (1 microL, 100 amol/microL). Arg8-vasotocin isolated from the pineal gland of rainbow trout is detected, demonstrating the ability of FT-ICR to detect and identify a true endogenous biological analyte.     

An automated matrix-assisted laser desorption/ionization quadrupole Fourier transform ion cyclotron resonance mass spectrometer for "bottom-up" proteomics

Here we describe a new quadrupole Fourier transform ion cyclotron resonance hybrid mass spectrometer equipped with an intermediate-pressure MALDI ion source and demonstrate its suitability for "bottom-up" proteomics. The integration of a high-speed MALDI sample stage, a quadrupole analyzer, and a FT-ICR mass spectrometer together with a novel software user interface allows this instrument to perform high-throughput proteomics experiments. A set of linearly encoded stages allows sub-second positioning of any location on a microtiter-sized target with up to 1536 samples with micrometer precision in the source focus of the ion optics. Such precise control enables internal calibration for high mass accuracy MS and MS/MS spectra using separate calibrant and analyte regions on the target plate, avoiding ion suppression effects that would result from the spiking of calibrants into the sample. An elongated open cylindrical analyzer cell with trap plates allows trapping of ions from 1000 to 5000 m/z without notable mass discrimination. The instrument is highly sensitive, detecting less than 50 amol of angiotensin II and neurotensin in a microLC MALDI MS run under standard experimental conditions. The automated tandem MS of a reversed-phase separated bovine serum albumin digest demonstrated a successful identification for 27 peptides covering 45% of the sequence. An automated tandem MS experiment of a reversed-phase separated yeast cytosolic protein digest resulted in 226 identified peptides corresponding to 111 different proteins from 799 MS/MS attempts. The benefits of accurate mass measurements for data validation for such experiments are discussed.     

Hartley transform ion cyclotron resonance mass spectrometry

The Hartley transform offers a useful alternative to the Fourier transform for the conversion of a time-domain ion cyclotron resonance (ICR) signal into its corresponding frequency-domain mass spectrum. The Hartley transform has the advantage that it eliminates the need for complex variables, when (as for linearly polarized signals) the time-domain signal can be represented by a mathematically real function. Moreover, the Hartley transform produces the same spectra (absorption mode, dispersion mode, magnitude mode) as does the Fourier transform. In particular, the discrete fast Hartley transform (FHT) produces the same spectrum at twice the speed of a complex fast Fourier transform (FFT), making the FHT equivalent in speed to a "real" FFT. Hartley and Fourier transform methods in ICR mass spectrometry are compared and demonstrated experimentally. Essentially the same advantages and computational methods should apply to the use of the Hartley transform in place of the Fourier transform in other forms of spectrometry (e.g., nuclear magnetic resonance, infrared, etc.).     

Laser-induced acoustic desorption/fourier transform ion cyclotron resonance mass spectrometry for petroleum distillate analysis

Laser-induced acoustic desorption (LIAD) coupled with a 3-T Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR) allows the simultaneous analysis of both the nonpolar and polar components in petroleum distillates. The LIAD/FT-ICR method was validated by examining model compounds representative of the various classes of polar and nonpolar hydrocarbons commonly found in petroleum. LIAD successfully desorbs all the compounds as intact neutral molecules into the FT-ICR. Electron ionization (EI) at low energies (10 eV) and chemical ionization using cyclopentadienyl cobalt radical cation (CpCo*+) were employed to ionize the desorbed molecules. The EI experiments lead to extensive fragmentation of many of the hydrocarbon compounds studied. However, the CpCo*+ ion ionizes all the hydrocarbon compounds by producing only pseudomolecular ions without other fragmentation, with the exception of one compound (*CH3 loss occurs). Examination of two different petroleum distillate samples revealed hundreds of compounds. The most abundant components have an even molecular weight; i.e., they are likely to contain no (or possibly an even number of) nitrogen atoms.     

Automated gain control and internal calibration with external ion accumulation capillary liquid chromatography-electrospray ionization Fourier transform ion cyclotron resonance

When combined with capillary LC separations, electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR MS) has demonstrated capabilities for advanced characterization of proteomes based upon analyses of proteolytic digests. Incorporation of external (to the ICR cell) multipole devices with FTICR for ion selection and ion accumulation has enhanced the dynamic range, sensitivity, and duty cycle of measurements. However, the highly variable ion production rate from an LC separation can result in "overfilling" of the external trap during the elution of major peaks and result in m/z discrimination and fragmentation of peptide ions. Excessive space charge trapped in the ICR cell also causes significant shifts in the detected ion cyclotron frequencies, reducing the achievable mass measurement accuracy (MMA) and making protein identification less effective. To eliminate m/z discrimination in the external ion trap, further increase duty cycle, and improve MMA, we have developed the capability for data-dependent adjustment of ion accumulation times in the course of an LC separation, referred to as automated gain control (AGC). This development has been implemented in combination with low kinetic energy gated ion trapping and internal calibration using a dual-channel electrodynamic ion funnel. The overall system was initially evaluated in the analysis of a tryptic digest of bovine serum albumin. In conjunction with internal calibration, the capillary LC-ESI-AGC-FTICR instrumentation provided a approximately 10-fold increase in the number of identified tryptic peptides compared to that obtained using a fixed ion accumulation time and external calibration methods.     

High-performance mass spectrometry: Fourier transform ion cyclotron resonance at 14.5 Tesla

We describe the design and current performance of a 14.5 T hybrid linear quadrupole ion trap Fourier transform ion cyclotron resonance mass spectrometer. Ion masses are routinely determined at 4-fold better mass accuracy and 2-fold higher resolving power than similar 7 T systems at the same scan rate. The combination of high magnetic field and strict control of the number of trapped ions results in external calibration broadband mass accuracy typically less than 300 ppb rms, and a resolving power of 200,000 (m/Delta m50% at m/z 400) is achieved at greater than 1 mass spectrum per second. Novel ion storage optics and methodology increase the maximum number of ions that can be delivered to the FTICR cell, thereby improving dynamic range for tandem mass spectrometry and complex mixture applications.     

Characterization of pyrogenic black carbon by desorption atmospheric pressure photoionization Fourier transform ion cyclotron resonance mass spectrometry

We present a new method for molecular characterization of intact biochar directly, without sample preparation or pretreatment, on the basis of desorption atmospheric pressure photoionization (DAPPI) coupled to Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Conventional ionization methods (e.g., electrospray or atmospheric pressure photoionization) for characterization of natural organic matter have limited utility for the characterization of chars due to incomplete solubility in common solvents. Therefore, direct ionization techniques that do not require sample dissolution prior to analysis are ideal. Here, we apply DAPPI FTICR mass spectrometry to enable the first molecular characterization of uncharred parent oak biomass and after combustion (250 °C) or pyrolysis (400 °C). Parent oak is primarily composed of cellulose-, lignin-, and resin-like compounds. Oak combusted at 250 °C contains condensed aromatic compounds with low H/C and O/C ratios while retaining compounds with high H/C and O/C ratios. The bimodal distribution of aromatic and aliphatic compounds observed in the combusted oak sample is attributed to incomplete thermal degradation of lignin and hemicellulose. Pyrolyzed oak constituents exhibit lower H/C and O/C ratios: approximately three-quarters of the identified species are aromatic. DAPPI FTICR MS results agree with bulk elemental composition as well as functional group distributions determined by elemental analysis and solid state (13)C NMR spectroscopy. Complete molecular characterization of biomass upon thermal transformation may provide insight into the biogeochemical cycles of biochar and future renewable energy sources, particularly for samples currently limited by solubility, separation, and sample preparation.