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.     

Automated liquid injection field desorption/ionization for Fourier transform ion cyclotron resonance mass spectrometry

We describe automation of liquid injection field desorption/ionization (LIFDI) for reproducible sample application, improved spectral quality, and high-throughput analyses. A commercial autosampler provides reproducible and unattended sample application. A custom-built field desorption (FD) controller allows data station or front panel control of source parameters including high-voltage limit/ramp rate, emitter heating current limit/ramp rate, and feedback control of emitter heating current based on ion current measurement. Automated LIFDI facilitates ensemble averaging of hundreds of Fourier transform ion cyclotron resonance mass spectra for increased dynamic range, mass accuracy, and S/N ratio relative to single-application FD experiments, as shown here for a South American crude oil. This configuration can be adapted to any mass spectrometer with an LIFDI probe.     

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-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.     

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.).     

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.     

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.     

Elimination of z-ejection in Fourier transform ion cyclotron resonance mass spectrometry by radio frequency electric field shimming

In Fourier transform ion cyclotron resonance (FT/ICR) mass spectrometry, coherent ion cyclotron orbital motion is produced by resonant radio frequency (rf) electric field excitation. However, because the excitation electrodes are of finite dimensions, the desired transverse (to the applied magnetic field) rf electric field is accompanied by an rf electric field component along the z- (magnetic field) direction, resulting in mass-dependent z-ejection and mass-dependent FT/ICR mass spectral peak relative magnitudes. Addition of several "guard wires" of voltage-divided rf amplitude allows the rf electric field to be "shimmed" to near-perfect uniformity. In this paper (see also the accompanying paper by Russell et al.), we introduce two types of rf-shimmed ion traps. In the first type, guard wires are placed only in front of the trapping electrodes. In the second type, guard wire rings are placed inside the detector and trapping electrodes. For either arrangement, simion simulations were used to adjust the rf voltages applied (by use of voltage dividers) to the guard wires or rings so as to produce an optimally uniform rf field within the trap. The virtual elimination of z-excitation is confirmed by plots of magnitude-mode relative peak height vs ICR orbital radius. Because the guard wires (or rings) tend to shield the ions from the trapping electrode potential, the shift in ICR frequency with trapping voltage is also reduced, but not as well as by a screened trap.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Analysis of saturated hydrocarbons by redox reaction with negative-ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

A novel technique was developed for characterization of saturated hydrocarbons. Linear alkanes were selectively oxidized to ketones by ruthenium ion catalyzed oxidation (RICO). Branched and cyclic alkanes were oxidized to alcohols and ketones. The ketones were then reduced to alcohols by lithium aluminum hydride (LiAlH(4)). The monohydric alcohols (O(1)) in the products obtained from the RICO and RICO-LiAlH(4) reduction reactions were characterized using negative-ion electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) for identification of iso-paraffins, acyclic paraffins and cyclic paraffins. Various model saturated compounds were used to determine the RICO reaction and ionization selectivity. The results from the FTICR MS analysis on the petroleum distillates derived saturated fraction were in agreement with those from field ionization gas chromatography time-of-flight mass spectrometry (FI GC-TOF MS) analysis. The technique was also used to characterize a petroleum vacuum residue (VR) derived saturates. The results showed that the saturated molecules in the VR contained up to 11 cyclic rings, and the maximum carbon number was up to 92.     

Unit mass baseline resolution for an intact 148 kDa therapeutic monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) provides the highest mass resolving power and mass measurement accuracy for unambiguous identification of biomolecules. Previously, the highest-mass protein for which FTICR unit mass resolution had been obtained was 115 kDa at 7 T. Here, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of noncovalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FTICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum.     

De novo sequencing and disulfide mapping of a bromotryptophan-containing conotoxin by Fourier transform ion cyclotron resonance mass spectrometry

T-1-family conotoxins belong to the T-superfamily and are composed of 10-17 amino acids. They share a common cysteine framework and disulfide connectivity and exhibit unusual posttranslational modifications, such as tryptophan bromination, glutamic acid carboxylation, and threonine glycosylation. We have isolated and characterized a novel peptide, Mo1274, containing 11 amino acids, that shows the same cysteine pattern, -CC-CC, and disulfide linkage as those of the T-1-family members. The complete sequence, GNWCCSARVCC, in which W denotes bromotryptophan, was derived from MS-based de novo sequencing. The FT-ICR MS/MS techniques of electron capture dissociation (ECD), infrared multiphoton dissociation, and collision-induced dissociation served to detect and localize the tryptophan bromination. The bromine contributes a distinctive isotopic distribution in all fragments that contain bromotryptophan. ECD fragmentation results in the loss of bromine and return to the normal isotopic distribution. Disulfide connectivity of Mo1274, between cysteine pairs 1-3 and 2-4, was determined by mass spectrometry in combination with chemical derivatization employing tris(2-carboxyethyl)phosphine, followed by differential alkylation with N-ethylmaleimide and iodoacetamide. The ECD spectra of the native and partially modified peptide reveal a loss of bromine in a process that requires the presence of a disulfide bond.     

Probing the interaction of cisplatin with cytochrome C by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

As one of the most important platinum drugs, the interactions of cisplatin with proteins play very important roles in its anticancer action and side effects. Here, the interaction of cisplatin with cytochrome c was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). On the basis of the high-resolution data, cytochrome c-Pt(NH(3))Cl was found to be the primary monoadduct. The platinated monoadducts were related to molar ratios of cytochrome c and cisplatin, and corresponding reaction pathways were proposed. Multiple binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without liquid chromatography (LC) separation. Four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified. Moreover, hydrogen/deuterium exchange (H/DX) combined with FTICR MS provides the sensitive method to insight the small conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes.     

A direct nanoflow liquid chromatography-tandem mass spectrometry system for interaction proteomics

One of the strategies of functional proteomics, research aiming to discover gene function at the protein level, is the comprehensive analysis of protein-protein interactions related to the functional linkage among proteins and analysis of functional cellular machinery to better understand the basis of cell functions. Here, we describe the direct nanoflow LC (DNLC) system, which is equipped with a fritless high-resolution electrospray interface column packed with 1-microm reversed-phase (RP) beads and a novel splitless nanoflow gradient elution system to operate the column. Using RP-DNLC at an extremely slow flow rate, <50 nL/min, combined with data-dependent collision-induced dissociation tandem MS (MS/MS) and computer-assisted retrieval of spectra, we identified approximately 100 protein components in a biological complex such as a premature mammalian ribosome pull-down from cultured cells when we used an epitope-tagged protein as bait. Because this analysis is most sensitive, requires approximately 0.2 microg of total protein, and is a fully automated 1-h process, we anticipated that it should be an excellent tool for analyzing a limited amount of functional multi-protein complexes in cells.     

Characterization of high-molecular-weight sulfur-containing aromatics in vacuum residues using Fourier transform ion cyclotron resonance mass spectrometry

Millions of tons of vacuum residues are produced in refineries every year and could be a potentially valuable resource for generating electricity and has possible application as heating and marine fuel. In this work, the polycyclic aromatic sulfur compounds (PASHs) from the aromatic fraction of vacuum residue before and after partial hydrodesulfurization (HDS) were derivatized by methylation to the methylsulfonium salts. Fourier transform ion cyclotron resonance mass spectrometry provided high-resolution data on these high-molecular-weight sulfur compounds. Compounds containing one and two S atoms were found to dominate, with masses up to ca. 900 Da. Classification according to hydrogen deficiency and the number of heteroatoms showed extensive series of homologues for double bond equivalents from 5 to 20. The sulfur-containing aromatics were separated using a palladium(II) complex as a liquid chromatographic phase into two compound groups: one containing compounds with an unconjugated thiophene ring and another with a condensed thiophene ring. This, combined with the mass spectrometry (MS) data, allows for the identification of several parent structures. Partial HDS removed primarily compounds with one S atom, whereas those with two S atoms were largely unaffected.     

Precise relative ion abundances from Fourier transform ion cyclotron resonance magnitude-mode mass spectra

The area under a correctly phased absorption-mode spectral peak is a direct measure of the number of oscillators (ions, spins, molecules) in Fourier transform spectrometry (ion cyclotron resonance, magnetic resonance, interferometry absorbance). However, phase correction can prove difficult when (as in broad-band Fourier transform ion cyclotron resonance (FT/ICR] detection is considerably time-delayed after excitation. In the absence of noise, Huang, Rempel, and Gross showed that a "complex area" method yields the correct absorption-mode peak area, for an unphased noiseless spectrum. In this paper, we show that the number of oscillators may also be obtained from a least-squares fit to a magnitude-mode (i.e., phase-independent) spectrum. In the presence of noise and in the absence of peak overlap, the magnitude-mode method offers precision superior to that based on magnitude-mode peak height, "complex area", or even direct digital integration of a correctly phased absorption-mode peak, as demonstrated by both theoretical derivation and experimental FT/ICR results. The present method thus appears to offer the best available determination of the relative abundances of ions of different mass-to-charge ratio in FT/ICR mass spectrometry.     

Selective ionization of dissolved organic nitrogen by positive ion atmospheric pressure photoionization coupled with Fourier transform ion cyclotron resonance mass spectrometry

Dissolved organic nitrogen (DON) comprises a heterogeneous family of organic compounds that includes both well-known biomolecules such as urea or amino acids and more complex, less characterized compounds such as humic and fulvic acids. Typically, DON represents only a small fraction of the total dissolved organic carbon pool and therefore presents inherent problems for chemical analysis and characterization. Here, we demonstrate that DON may be selectively ionized by atmospheric pressure photionization (APPI) and characterized at the molecular level by Fourier transform ion cyclotron resonance mass spectrometry. Unlike electrospray ionization (ESI), APPI ionizes polar and nonpolar compounds, and ionization efficiency is not determined by polarity. APPI is tolerant to salts, due to the thermal treatment inherent to nebulization, and thus avoids salt-adduct formation that can complicate ESI mass spectra. Here, for dissolved organic matter from various aquatic environments, we selectively ionize DON species that are not efficiently ionized by other ionization techniques and demonstrate significant signal-to-noise increase for nitrogen species by use of APPI relative to ESI.     

Determination of structural building blocks in heavy petroleum systems by collision-induced dissociation Fourier transform ion cyclotron resonance mass spectrometry

Collision-induced dissociation Fourier Transform ion cyclotron resonance mass spectrometry (CID-FTICR MS) was developed to determine structural building blocks in heavy petroleum systems. Model compounds with both single core and multicore configurations were synthesized to study the fragmentation pattern and response factors in the CID reactions. Dealkylation is found to be the most prevalent reaction pathway in the CID. Single core molecules exhibit primarily molecular weight reduction with no change in the total unsaturation of the molecule (or Z-number as in chemical formula C(c)H(2c+Z)N(n)S(s)O(o)VNi). On the other hand, molecules containing more than one aromatic core will decompose into the constituting single cores and consequently exhibit both molecular weight reduction and change in Z-numbers. Biaryl linkage, C(1) linkage, and aromatic sulfide linkage cannot be broken down by CID with lab collision energy up to 50 eV while C(2)+ alkyl linkages can be easily broken. Naphthenic ring-openings were observed in CID, leading to formation of olefinic structures. Heavy petroleum systems, such as vacuum resid (VR) fractions, were characterized by the CID technology. Both single-core and multicore structures were found in VR. The latter is more prevalent in higher aromatic ring classes.