Coupling capillary electrochromatography with electrospray Fourier transform mass spectrometry for characterizing complex oligosaccharide pools

To deal with the complexity of the glycan mixtures released from glycoproteins, an efficient form of micro-column separations, capillary electrochromatography, has been combined with high-performance mass spectrometry (Fourier transform ion cyclotron resonance). Contour plots have been generated from the mixtures of O-linked oligosaccharides originated from bovine mucin and bile salt-stimulated lipase, a large glycoprotein enzyme.     

Electrospray ionization fourier transform ion cyclotron resonance mass spectrometry of human alpha-1-acid glycoprotein

The ultrahigh resolution and sensitivity of electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry have for the first time been exploited for the characterization of highly sialylated glycoproteins, using human alpha-1-acid glycoprotein as the model compound. An alternative approach to the widely used high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization (MALDI) assays is described. This new method does not require any enzymatic or chemical digestion (removal of sialyl groups or deglycosylation), chemical derivatization (introduction of chromophore groups), or preliminary chromatographic separation (HPLC or electrophoresis). Following ESI and accumulation of ions in a hexapole ion guide, ions are injected into the ICR cell. A selected mass window from the overall ion population is isolated and axialized prior to detection. After acquisition and Fourier transform of the transient signal the resulted spectrum is evaluated in order to determine the charge state of the detected ions and the isotope pattern of the measured protein glycoform. The presence of ions from the same glycoform with different charge states was confirmed. The advantages and limitations of the technique are discussed. Future prospects and possible applications are indicated.     

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.     

Experimental investigations of the internal energy of molecules evaporated via laser-induced acoustic desorption into a Fourier transform ion cyclotron resonance mass spectrometer

The internal energy of neutral gas-phase organic and biomolecules, evaporated by means of laser-induced acoustic desorption (LIAD) into a Fourier transform ion cyclotron resonance mass spectrometer, was investigated through several experimental approaches. The desorbed molecules were demonstrated not to undergo degradation during the desorption process by collecting LIAD-evaporated molecules and subjecting them to analysis by electrospray ionization/quadrupole ion trap mass spectrometry. Previously established gas-phase basicity values were remeasured for LIAD-evaporated organic molecules and biomolecules with the use of the bracketing method. No endothermic reactions were observed. The remeasured basicity values are in close agreement with the values reported in the literature. The amount of internal energy deposited during LIAD is concluded to be less than a few kilocalories per mole. Chemical ionization with a series of proton-transfer reagents was employed to obtain a breakdown curve for a protonated dipeptide, Val-Pro, evaporated by LIAD. Comparison of this breakdown curve with a previously published analogous curve obtained by using substrate-assisted laser desorption (SALD) to evaporate the peptide suggests that the molecules evaporated via LIAD have a similar internal energy as those evaporated via SALD.     

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.     

Routine Part-per-Million Mass Accuracy for High- Mass Ions: Space-Charge Effects in MALDI FT-ICR

The effect of ion space-charge on mass accuracy in Fourier transform ion cyclotron resonance mass spectrometry is examined. Matrix-assisted laser desorption/ionization is used to form a population of high-molecular-weight polymer ions with a wide mass distribution. The density of the ions in the analyzer cell is varied using ion remeasurement and suspended trapping techniques to allow the effect of ion space charge to be examined independently of other experimental influences. Observed cyclotron frequency exhibits a linear correlation with ion population. Mass errors of 100 ppm or more in externally calibrated mass spectra result when ion number is not taken into account. By matching the total ion intensities of calibrant and analyte mass spectra, the protonated ion of insulin B-chain, 3494.6513 Da, is measured with an accuracy of 0.07 ppm (average of 10 measurements, σ = 2.3 ppm, average absolute error 1.6 ppm) using a polymer sample as an external calibrant. Alternatively, the correction for space charge can be made by using a calibration equation that accounts for the total ion intensity of the mass spectrum. A calibration procedure is proposed and is tested with the measurement of the mass of insulin B-chain. A mass accuracy of 2.0 ppm (average of 20 measurements, σ = 4.2 ppm, average absolute error 3.5 ppm) is achieved. Space-charge-induced mass errors are more significant for samples with many components, such as a polymer, than for single-component samples such as purified peptides or proteins.     

Characterization of polyesters prepared from three different phthalic acid isomers by CID-ESI-FT-ICR and PSD-MALDI-TOF mass spectrometry

Polyesters prepared from the same diol, 2-butyl-2-ethyl-1,3-propanediol, but different phthalic acid isomers, phthalic, isophthalic, and terephthalic acid, were characterized by collision-induced dissociation electrospray ionization Fourier transform ion cyclotron resonance (CID-ESI-FT-ICR) and postsource-decay matrix-assisted laser desorption/ionization time-of-flight (PSD-MALDI-TOF) mass spectrometry. Sodiated dihydroxyl-terminated polyester oligomers containing five repeating units at m/z 1634 were selected as precursor ions for dissociation studies. Two main mechanisms occurred in the fragmentation of all of the polyesters, since dissociation of the oligomers was initiated by hydrogen rearrangement or transesterification reactions. Polyesters prepared from different phthalic acid isomers could be distinguished by their fragmentation behavior. Polyester prepared from phthalic acid was easily identified by using both CID-ESI-FT-ICR and PSD-MALDI-TOF mass spectrometry. However, distinguishing between the polyesters prepared from isophthalic and terephthalic acid succeeded marginally only with CID-ESI-FT-ICR mass spectrometry. Molecular dynamics calculations were used to obtain an idea of the fragmentation behavior of the polyesters. The low-energy structures of the precursor ions were determined, and the coordination of the oxygen atoms of the polyester oligomers to the sodium cation was examined more closely. Both the experimental and the theoretical studies showed that the sodium ion affinity of polyester changed with the phthalic acid isomer.     

Zeptomole-sensitivity electrospray ionization--Fourier transform ion cyclotron resonance mass spectrometry of proteins

Methods are being developed for ultrasensitive protein characterization based upon electrospray ionization (ESI) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). The sensitivity of a FTICR mass spectrometer equipped with an ESI source depends on the overall ion transmission, which combines the probability of ionization, transmission efficiency, and ion trapping in the FTICR cell. Our developments implemented in a 3.5 tesla FTICR mass spectrometer include introduction and optimization of a newly designed electrodynamic ion funnel in the ESI interface, improving the ion beam characteristics in a quadrupole-electrostatic ion guide interface, and modification of the electrostatic ion guide. These developments provide a detection limit of approximately 30 zmol (approximately 18,000 molecules) for proteins with molecular weights ranging from 8 to 20 kDa.     

Method for profiling mucin oligosaccharides from gastric biopsies of rhesus monkeys with and without Helicobacter pylori infection

A method for in vivo analysis of gastric mucin oligosaccharides was developed and applied to rhesus monkeys with and without Helicobacter pylori infection. Mucin-type O-linked oligosaccharides were directly released by reductive beta-elimination from gastric biopsies from rhesus monkeys. The released oligosaccharides were structurally characterized by matrix-assisted laser desorption/ionozation and electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. A diverse profile of neutral and acidic oligosaccharides was observed with these techniques. The most predominant core structure detected in all of the samples at relatively high abundance corresponded to core 2 (2HexNAc-1Hex, m/z = 611.227). The spectra generated from H. pylori-infected monkey samples showed fewer oligosaccharides collectively. Peaks corresponding to 1HexNAc-1Hex (m/z = 408.148) and 2HexNAc (m/z = 449.174), which most likely represent core structures, were absent in all infected monkeys studied, although present in all uninfected monkeys. Unsupervised cluster analysis demonstrated clear differences between the peaks detected in uninfected and naturally infected monkey samples. The results suggest that H. pylori infection is associated with lower relative abundance of oligosaccharides and loss of mucin-type core structures. This method can be applied to characterize the glycans associated with the mucin lining of live animals and allows for repeated analysis of the same animal over the course of infection.     

Analysis of mainstream and sidestream cigarette smoke particulate matter by laser desorption mass spectrometry

Laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (LDI-FTICRMS) was used to investigate particulate matter (PM) associated with mainstream (MSS) and sidestream cigarette smokes (SSS). The high mass resolution and the high mass measurement accuracy allowed a molecular formula for each detected signal in the 150-500 m/z range to be assigned. The high number of peaks observed in mass spectra required additional data processing to extract information. In this context, Kendrick maps and Van Krevelen diagrams were drawn. These postacquisition treatments were used to more easily compare different cigarette smokes: (i) MSS from different cigarettes and (ii) MSS and SSS from the same cigarette. In both ion detection modes, most of the detected species were found to be attributed to C(6-31)H(2-35)N(0-7)O(0-9) compounds. The compounds observed in the study of SSS appeared to be more unsaturated and less oxygenated than those observed when MSS of the same cigarette was investigated.     

Atmospheric pressure MALDI Fourier transform mass spectrometry of labile oligosaccharides

An atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) source coupled to Fourier transform ion cyclotron resonance mass spectrometry (FT ICR MS) under UV laser and solid matrix conditions has been demonstrated to analyze a variety of labile oligosaccharides including O-linked and N-linked complex glycans released from glycoproteins. Spectra were acquired by both AP MALDI and vacuum MALDI and directly compared. The results presented here confirm that AP MALDI can generate significantly less energetic ions than vacuum MALDI and is able to produce the intact molecular ions with little or no fragmentation in both positive and negative ion mode analyses. Under certain conditions, noncovalent complexes of sialylated oligosaccharides were observed. The sensitivity attainable by AP MALDI was found to be comparable to conventional MALDI, and tandem mass spectrometry of oligosaccharides ionized by AP MALDI was shown to allow detailed structural analysis. Analysis of N-glycan mixtures derived from human fibrinogen further demonstrated that AP MALDI-FT ICR MS is ideal for the study of complex glycan samples as it provides high-accuracy, high-resolution mass analysis with no difficulty in distinguishing sample constituents from fragment ions.     

MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue

A new approach is described for imaging mass spectrometry analysis of drugs and metabolites in tissue using matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR). The technique utilizes the high resolving power to produce images from thousands of ions measured during a single mass spectrometry (MS)-mode experiment. Accurate mass measurement provides molecular specificity for the ion images on the basis of elemental composition. Final structural confirmation of the targeted compound is made from accurate mass fragment ions generated in an external quadrupole-collision cell. The ability to image many small molecules in a single measurement with high specificity is a significant improvement over existing MS/MS based technologies. Example images are shown for olanzapine in kidney and liver and imatinib in glioma.     

High-mass accuracy of product ions produced by SORI-CID using a dual electrospray ionization source coupled with FTICR mass spectrometry

High-mass accuracy is demonstrated using internal calibration for product ions produced by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) of a 15-mer oligonucleotide, 5'-(CTG)5-3'. Internal calibration for this tandem MS experiment was accomplished using a dual electrospray ionization (ESI) source coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) utilizing hexapole accumulation and gated trapping. The pulse sequence entails injection, trapping, and gas-phase isolation of the precursor ion of interest followed by the SORI-CID of this ion and, subsequently, injection and trapping of the internal mass calibrant (i.e., poly(ethylene glycol) with a 1000 Da average mass). The product ions and the poly(ethylene glycol) ions are then simultaneously excited by a broadband frequency chirp excitation waveform and detected. This technique corrects for space-charge effects on the measurement of an ion's cyclotron frequency experienced when externally calibrated data are used. While external calibration for FTICR-MS can result in mass errors of greater than 100 ppm, this internal standardization method demonstrated significantly more consistent accurate mass measurements with average mass errors ranging from -1.2 to -3.2 ppm for the 15-mer oligonucleotide used in this study. This method requires limited modifications to ESI-FTICR mass spectrometers and is applicable for both positive and negative modes of ionization as well as other sample types (e.g., pharmaceuticals, proteins, 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.     

Mass defect labeling of cysteine for improving peptide assignment in shotgun proteomic analyses

A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2'-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology.     

Baseline mass resolution of peptide isobars: a record for molecular mass resolution

Baseline resolution of two peptides, RVMRGMR and RSHRGHR, of neutral monoisotopic mass, approximately 904 Da, has been achieved by microelectrospray ionization Fourier transform ion cyclotron resonance mass spectrometry at a mass resolving power of approximately 3 300 000. The elemental compositions of these molecules differ by N40 vs. S2H8 (0.000 45 Da), which is less than one electron's mass (0.000 55 Da)! This result establishes a new record for the smallest resolved mass difference between any two molecules. This achievement is made possible by a combination of high magnetic field (9.4 T), large-diameter (4-in.) Penning trap, and low ion density. The implications for proteomics based on accurate mass measurements are discussed briefly.     

Enhanced detection and identification of glycopeptides in negative ion mode mass spectrometry

A combined mass spectrometry (MS) and tandem mass spectrometry (MS/MS) approach implemented with matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI FTICR MS) in the negative ion mode is described for enhanced glycopeptide detection and MS/MS analysis. Positive ion mode MS analysis is widely used for glycopeptide characterization, but the analyses are hampered by potential charge-induced fragmentation of the glycopeptides and poor detection of the glycopeptides harboring sialic acids. Furthermore, tandem MS analysis (MS/MS) via collision-induced dissociation (CID) of glycopeptides in the positive ion mode predominantly yields glycan fragmentation with minimal information to verify the connecting peptide moiety. In this study, glycoproteins such as, bovine lactoferrin (b-LF) for N-glycosylation and kappa casein (k-CN) for O-glycosylation were analyzed in both the positive- and negative ion modes after digestion with bead-immobilized Pronase. For the b-LF analysis, 44 potential N-linked glycopeptides were detected in the positive ion mode while 61 potential N-linked glycopeptides were detected in the negative ion mode. By the same token, more O-linked glycopeptides mainly harboring sialic acids from k-CN were detected in the negative ion mode. The enhanced glycopeptide detection allowed improved site-specific analysis of protein glycosylation and superior to positive ion mode detection. Overall, the negative ion mode approach is aimed toward enhanced N- and O-linked glycopeptide detection and to serve as a complementary tool to positive ion mode MS/MS analysis.     

Exact masses and chemical formulas of individual Suwannee River fulvic acids from ultrahigh resolution electrospray ionization Fourier transform ion cyclotron resonance mass spectra

Molecular formulas have been assigned for 4626 individual Suwannee River fulvic acids based on accurate mass measurements from ions generated by electrospray ionization and observed by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Formula assignments were possible because of the mass accuracy of FTICR MS at high field (9.4 T) and the regular mass spacing patterns found in fulvic acid mixtures. Sorting the 4626 individually observed ions according to Kendrick mass defect and nominal mass series (z* score) revealed that all could be assigned to 1 of 266 distinct homologous series that differ in oxygen content and double bond equivalence. Tandem mass spectrometry based on infrared multiphoton dissociation identified labile fragments of fulvic acid molecules, whose chemical formulas led to plausible structures consistent with degraded lignin as a source of Suwannee River fulvic acids.     

Determination of Block Length Distributions of Poly(oxypropylene) and Poly(oxyethylene) Block Copolymers by MALDI-FTICR Mass Spectrometry

Matrix-assisted laser desorption/ionization (MALDI) was performed on an external ion source Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS) to analyze the block length distributions of triblock polymers of poly(oxypropylene) and poly(oxyethylene). The first series of results presented demonstrate that the apparent molecular weight distributions are distorted. This distortion is induced by the flight-time-induced mass discrimination inherent in the experimental technique, the variation of isotopic patterns over the measured mass range, and the overlap of peaks in the spectrum. Subsequently, a method for the treatment of molecular weight distributions measured by MALDI on an external ion source FTICR-MS is developed to yield the actual molecular weight distribution and, from that, the individual block length distributions. For the first time, detailed and accurate molecular weight data were obtained on a complex sample using this methodology, which independently validates the data provided by the manufacturer. The experimentally verified random coupling hypothesis proves the validity of the methodology.