Electrospray ionization Fourier transform ion

In previous work, we demonstrated the use of electrospray ionization to analyze small differences in size or sequence of relatively small polymerase chain reaction (PCR) products of 114 base pairs or less. The sequence information required to answer a biological question may be only a single nucleotide substitution or deletion. In many cases, the regions where these sequence variations can occur are several hundred base pairs in length, and the analysis of large PCR products is therefore desirable. Therefore, we have attempted to expand the size range of PCR products that can be analyzed by electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. Previous work has shown that the difficulties associated with PCR product analysis increase with product size. A revised cleanup scheme was employed to target the removal of detergents with ethanol wash or precipitation steps, followed by additional desalting. Additionally, an in-trap cleanup to collisionally induce dissociation of noncovalent salt adducts was employed. This approach was extended to a 223 base pair PCR product yielding mass measurement accuracy within 26 ppM. The mass measurement accuracy obtained illustrates that a single base substitution could be identified at this size of PCR product with a 7 tesla ESI-FTICR.     

CEPH family 1362 STR database: An online resource for characterization of PCR products using electrospray ionization mass spectrometry

An online database has been established in order to validate electrospray ionization mass spectrometry (ESI-MS) for genotyping and to publicize the procedures developed in our laboratory for the characterization of PCR products by ESI-MS. Genotypes derived from short tandem repeat (STR) loci that were obtained using ESI Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) have been posted for fifteen members of the CEPH family 1362 pedigree. The website provides specific information such as PCR parameters, PCR product cleanup approaches, and ESI solution compositions to enable other laboratories to reproduce our data. Links are provided to related websites in an effort to integrate information regarding the CEPH family, STR genotyping, and mass spectrometry. The database, currently available at http://www.people.vcu.edu/ -dcmuddim/genotype/ will be routinely updated with genotypes from additional STR loci including PCR parameters as well as PCR cleanup strategies as further developments are completed.     

Molecular mass measurement of intact ribonucleic acids via electrospray ionization quadrupole mass spectrometry

The use of electrospray ionization mass spectrometry for the accurate determination of molecular masses of polynucleotides and small nucleic acids is developed. The common problem of gas phase cation adduction that is particularly prevalent in the mass spectrometric analysis of nucleic acids is reduced through the use of ammonium acetate precipitations and by the addition of chemical additives that compete for adduct ions in solution. The addition of chelating agents such as trans-1,2-diaminocyclohexane-N,N,N,′,N′-tetraacetic acid to remove divalent metal ions and triethylamine to displace monovalent cations from the analyte, in conjunction with ammonium acetate precipitation, reduces cation adduction to levels that permit accurate mass analysis (mass errors of less than 0.01%) without further complex cleanup procedures. The potential utility of accurate mass measurements of small ribonucleic acids is discussed.     

Proteomic profiling of human urinary proteome using nano-high performance liquid chromatography/electrospray ionization tandem mass spectrometry

Urine, a blood filtrate produced by the urinary system, is an ideal bio-sample and a rich source of biomarkers for diagnostic information. Many components in urine are useful in clinical diagnosis, and urinary proteins can be strong indication for many diseases such as proteinuria, kidney, bladder and urinary tract diseases. To enhance our understanding of urinary proteome, the urine proteins were prepared by different sample cleanup preparation methods and identified by nano-high performance liquid chromatography electrospray ionization tandem mass spectrometry followed by peptide fragmentation pattern. The experimental results demonstrated that a total of 2283 peptides, corresponding to 311 unique proteins, were identified from human urine samples, in which 104 proteins with higher confidence levels. The present study was designed to establish optimal techniques to create a proteomic map of normal urinary proteins. Also, a discussion of novel approaches to urine protein cleanup and constituents is given.     

Microfluidic chips for mass spectrometry‐based proteomics

Microfluidic devices coupled to mass spectrometers have emerged as excellent tools for solving the complex analytical challenges associated with the field of proteomics. Current proteome identification procedures are accomplished through a series of steps that require many hours of labor‐intensive work. Microfluidics can play an important role in proteomic sample preparation steps prior to mass spectral identification such as sample cleanup, digestion, and separations due to its ability to handle small sample quantities with the potential for high‐throughput parallel analysis. To utilize microfluidic devices for proteomic analysis, an efficient interface between the microchip and the mass spectrometer is required. This tutorial provides an overview of the technologies and applications of microfluidic chips coupled to mass spectrometry for proteome analysis. Various approaches for combining microfluidic devices with electrospray ionization (ESI) and matrix‐assisted laser desorption/ionization (MALDI) are summarized and applications of chip‐based separations and digestion technologies to proteomic analysis are presented. Copyright © 2009 John Wiley & Sons, Ltd.     

Size exclusion chromatography/mass spectrometry applied to the analysis of polysaccharides.

A novel method for analysing polysaccharide materials is described which employs size-exclusion chromatography (SEC) followed by detection by on-line electrospray ionisation mass spectrometry (ESI-MS) and off-line matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS). It is demonstrated through SEC/ESI ion trap mass spectrometry that the formation of multiply charged oligomer ions, which bind up to five sodium cations, allows the rapid analysis of polysaccharide ions with molecular weights in excess of 9 kDa. MALDI spectra generated from fractionation of the effluent collected from the same SEC separation are shown to be in good agreement with the ESI spectra with respect to molecular weight distributions and types of ions generated. ESI and MALDI mass spectra of samples obtained from sequential graded ethanol precipitation and SEC fractionation of acid and enzymatically digested arabinoxylan polysaccharides show important structural differences between polysaccharide fragments. In addition, a comparison is made between the mass spectra of native and permethylated SEC-separated fragments of acid and enzymatically treated arabinogalactan. Linkage information of the permethylated arabinogalactan oligomers can be rapidly established through the use of on-line SEC/ESI-MS( n) experiments.     

Multiresidue determination of pesticides in agricultural products by gas chromatography/mass spectrometry with large volume injection

A method is described for the rapid determination of pesticide residues in agricultural products. Pesticides were extracted from samples with acetonitrile. To remove pigments and fatty acids, an aliquot of the extract was cleaned up by a minicolumn that was packed both with graphitized carbon black and primary secondary amine. Analysis was performed by gas chromatography/ mass spectrometry with programmable temperature vaporizer-based large volume injection using a liner packed with phenylmethylsilicone chemically bonded silica. The method was evaluated for 114 pesticides by spiking into tomato, spinach, Japanese pear, grape, and brown rice at various concentrations of each pesticide (0.02-0.4 microg/g). The method, which gave good recovery (>60%) for 108 pesticides, is characterized by high cleanup efficiency and short cleanup time, and is useful as a rapid screening analysis.     

A rapid sample preparation method for mass spectrometric characterization of N-linked glycans

A rapid method for analysis of glycans of glycoproteins is presented. This method comprised deglycosylation, sample cleanup and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of glycans. The enzymatic deglycosylation of N-linked glycoproteins was enhanced in terms of speed and reproducibility using an enzyme-friendly surfactant. The released glycans were desalted using a micro-scale solid phase extraction (SPE) device packed with a hydrophilic interaction chromatography (HILIC) sorbent. Hydrophilic glycans were well retained by SPE, while salts and surfactants were removed from the sample. The glycans were eluted using 25-50 microL of solvent and analyzed directly without derivatization using MALDI-MS. MALDI quadrupole time-of-flight (Q-Tof) instrumentation was utilized for glycan profiling and structure characterization by tandem mass spectrometry (MS/MS). The presented method allows sensitive analysis of glycans benefiting from optimized deglycosylation reactions and efficient sample cleanup.     

A facile microdialysis interface for on-line desalting and identification of proteins by nano-electrospray ionization mass spectrometry

The adverse effect of salts, especially inorganic salts, on electrospray ionization mass spectrometry (ESI-MS) is one of the most serious obstacles that might limit its application. Among the numerous desalting approaches, the microdialysis technique is favorable for large molecules, such as proteins. In this work, employing a hollow fiber membrane of cellulose acetate (MWCO 3000 Da), a simple, facile and efficient microdialysis interface with the dead volume of less than 1 microL was constructed for the on-line desalting and identification of proteins dissolved in high salt concentration buffer by nano-ESI-MS. Furthermore, with counterflow added, the desalting procedure was accelerated, and could be finished within 1 min. This system was successfully applied to the analysis of myoglobin dissolved in either high concentration ammonium acetate or sodium chloride buffer. The experimental results showed that, by using such a microdialysis interface, the salt concentration, even as high as 1 M, could be decreased by at least 2 orders of magnitude, while sample loss was less than 10%, demonstrating the potential of such an interface in broadening the application of nano-ESI-MS in the analysis of large molecules.     

Simplified pesticide multiresidue analysis of soybean oil by low-temperature cleanup and dispersive solid-phase extraction coupled with gas chromatography/mass spectrometry

A simple, fast, and economical method has been developed for the simultaneous determination of 28 various types of pesticides in soybean oil. Pesticides of low molecular mass were separated from the fat of the oil, which has a high molecular mass, by using low-temperature fat precipitation, followed by a cleanup process based on dispersive solid-phase extraction with primary secondary amine and C18 as sorbents and magnesium sulfate for the removal of residual water. The results for all pesticides determined by gas chromatography with mass spectrometry in the selected-ion monitoring mode were linear, and the matrix effect of the method was evaluated. Recoveries of most pesticides were acceptable at fortification levels of 0.02, 0.05, 0.2, and 1 mg/kg. The relative standard deviation was <20% even for determinations without internal standards. Limits of quantitation ranged from 20 to 250 microg/kg.     

On-line microdialysis for enhanced resolution and sensitivity during electrospray mass spectrometry of non-covalent complexes and competitive binding studies

Many proteins and macromolecules easily form metal adduct ions which impairs their analysis by mass spectrometry. The present study describes how the formation of undesired adducts can be minimized by on-line microdialysis for non-covalent binding studies of macromolecules with low molecular mass ligands with electrospray ionization mass spectrometry (ESI-MS). The technique was indispensable for protein-ligand studies due to reduction of unwanted adduct ions, and thus gave excellent resolution and a sensitivity improvement of at least 5 times. The core of the analytical system was a modified microdialysis device, which was operated in countercurrent mode. A novel technique based on microdialysis for competitive binding studies is also presented. The non-covalent complex between a protein and a ligand was formed in the sample vial prior to analysis. The complex was injected into an on-line microdialysis system where a competitive ligand was administered in the dialysis buffer outside of the fiber. The second ligand competitively displaced the first ligand through transport via the wall of the dialysis fiber, and the intact complexes were detected by ESI-MS.     

Electron-capture mass spectrometry: recent advances

Electron-capture (EC) is a sensitive and selective ionization technique for mass spectrometry (MS). In the most familiar form of EC, a susceptible analyte (electrophore) is detected after eluting from a gas chromatography (GC) column, where a low attomole detection limit for standards is routine. High-performance liquid chromatography can facilitate sample cleanup prior to detection by GC-EC-MS, but carryover and shifts in retention time for the "invisible" analyte can be difficulties. Solid-phase extraction avoids these difficulties, but the degree of cleanup and recovery can be problems. Alternative electrophoric derivatizing reagents are available to help deal with interferences, and new reagents such as "AMACE1" are emerging. Releasable forms of electrophores can be used as tags for labeling macromolecules, motivated by the desire to multiplex ligand-type assays. The conventional, gas-phase ion source for EC is not well-understood, especially the role of wall reactions. Using an electron monochromator to tune the electron energy adds to the selectivity and information provided by EC-MS. High-resolution and tandem EC-MS measurements are emerging. Electron-capture dissociation is a new technique to sequence small- to medium-sized peptides, having the advantage of providing more extensive sequence information relative to other MS techniques. Particle-beam EC-MS tends to be less sensitive than GC-EC-MS, but not always. Recently it was demonstrated that EC-MS can be accomplished on an ordinary laser desorption time-of-flight mass spectrometer, and also by using atmospheric pressure chemical ionization. Two applications are discussed here in detail: bile acids and oxidized phenylalanine. EC-MS is well-established as a useful technique for trace analysis in special cases, and the scope of its usefulness is broadening (qualitative analysis and detection of more polar and larger molecules), based on advances in both the chemical and instrumental aspects of this technique.     

Direct mass spectrometry of polymers. IX. Copoly(carbonate-urethanes) prepared by reorganization of polycarbonates with piperazine

A detailed analysis of the reaction products generated by reorganization of polycarbonates with piperazine, performed by direct mass spectrometry methods, has shown that this reaction actually follows the pathway postulated and that, at 50% piperazine incorporation level, urethanediphenol compounds are almost exclusively produced. This allowed us to obtain, by repolymerization with phosgene, alternating copoly(carbonate-urethanes) with thermal stabilities comparable to those of the parent polyurethanes. The primary fragmentation mechanisms in the thermal decomposition of these copolymers were studied by direct pyrolysis into the mass spectrometer. Ester exchange reactions predominate here, causing the formation of cyclic oligomers, which are subsequently cleaved to open-chain oligomers containing hydroxyl end groups.     

Quantitative determination of formaldehyde in cosmetics using a combined solid-phase microextraction-isotope dilution mass spectrometry method

Solid-phase microextraction (SPME) in conjunction with isotope dilution mass spectrometry (ID-MS) was employed for the analysis of formaldehyde in cosmetic products. The formaldehyde is derivatized in situ with pentafluorophenyl hydrazine. The formed hydrazone is adsorbed over a poly(dimethylsiloxane)-divinylbenzene-coated fiber and analyzed using gas chromatography-mass spectrometry. The adsorption-time profiles and salting effect were studied. The quantitation was performed by using a stable isotope labeled analogue as an internal standard. The precision, recovery and detection limits were determined with spiked samples. The relative standard deviations from different spiked cosmetic samples were all less than 10% and the recoveries were between 89.00 and 101.23%. The limit of detection was of 0.39 microg/l. Compared with other techniques, the study shown here provides a simple, fast and reliable method for the analysis of formaldehyde in cosmetic products.     

Evaluation of the LTQ-Orbitrap mass spectrometer for the analysis of polymerase chain reaction products

We have investigated the potential and robustness of the off‐line coupling of polymerase chain reaction (PCR) with electrospray ionization mass spectrometry (ESI‐MS), for further applications in the screening of single‐nucleotide polymorphisms (SNPs). This was based on recently reported data demonstrating that anion‐exchange solid‐phase extraction was the most efficient technique for efficiently desalting PCR products, with a recovery of ～70%. Results showed that this purification approach efficiently removes almost all the chemicals commonly added to PCR buffers. ESI‐MS analysis of a model 114‐bp PCR product performed on the LTQ‐Orbitrap instrument demonstrated that detection limits in the nM range along with an average mass measurement uncertainty of 9.15 ± 7.11 ppm can be routinely obtained using an external calibration. The PCR/ESI‐MS platform was able to detect just a few copies of a targeted oligonucleotide. However, it was shown that if two PCR products are present in a mixture in a ratio higher than 10 to 1, the lower abundance one might not be reproducibly detected. Applications to SNPs demonstrated that an LTQ‐Orbitrap with a resolution of 30 000 (at m/z 400) easily identified a single (A ? G) switch, i.e. a 16 Da difference, in binary mixtures of ～ 35 kDa PCR products. Complementary experiments also showed that the combination of endonucleases and ESI‐MS could be used to confirm base composition and sequence, and thus to screen for unknown polymorphisms in specific sequences. For example, a single (T ? A) switch (9 Da mass difference) was successfully identified in a 114‐bp PCR product. Copyright © 2010 John Wiley & Sons, Ltd.     

Contributions of mass spectrometry to structural immunology

Mass spectrometry has made important contributions to the field of immunology in the past decade. A variety of mass spectrometric-based techniques have been applied to study the structures of macromolecules that play a vital role in the immune response. These include traditional molecular mass measurements to identify post-translational modifications and structural heterogeneity, mass mapping of proteolysis products, sequencing by tandem mass spectrometry and conformational analysis. Antigen-antibody and other immune complexes have been detected by mass spectrometry, providing an avenue to study macromolecular assemblies that are important to immune function. By virtue of the ability of mass spectrometry based techniques to analyze complex biological mixtures, mass spectrometry has also been employed to identify and sequence protein epitopes important in both the humoral and cellular immune responses. This has been achieved through a combination of immunoaffinity and mass spectrometric techniques, and the coupling of high-performance chromatographs to mass spectrometers. These approaches are important for the identification of pathogens and show promise for the early diagnosis of disease associated with viral and bacterial infection and malignancy. These investigations will enable the mechanisms associated with normal and impaired immune function to be elucidated. Mass spectrometry has been utilized to characterize the structure of peptide mimics, multiple antigenic peptides and other constructs in the design of synthetic immunogens. Information derived from these studies will aid in the development of novel therapeutics and vaccines.     

Microfluidics with MALDI analysis for proteomics—A review

Various microfluidic devices have been developed for proteomic analyses and many of these have been designed specifically for mass spectrometry detection. In this review, we present an overview of chip fabrication, microfluidic components, and the interfacing of these devices to matrix-assisted laser desorption ionization (MALDI) mass spectrometry. These devices can be directly coupled to the mass spectrometer for on-line analysis in real-time, or samples can be analyzed on-chip or deposited onto targets for off-line readout. Several approaches for combining microfluidic devices with analytical functions such as sample cleanup, digestion, and separations with MALDI mass spectrometry are discussed.     

Determination of selected non-authorized insecticides in peppers by liquid chromatography time-of-flight mass spectrometry and tandem mass spectrometry

In this work, two analytical methods based on liquid chromatography coupled to electrospray time-of-flight mass spectrometry (LC/ESI-TOFMS) and tandem mass spectrometry (LC/ESI-MS/MS) are described for the identification, confirmation and quantitation of three insecticides non-authorized in the European Union (nitenpyram, isocarbophos and isofenphos-methyl) but detected in recent monitoring programmes in pepper samples. The proposed methodologies involved a sample extraction procedure using liquid-liquid partition with acetonitrile followed by a cleanup step based on dispersive solid-phase extraction. Recovery studies performed on peppers spiked at different fortification levels (10 and 50 microg kg(-1)) yielded average recoveries in the range 76-100% with relative standard deviation (RSD) (%) values below 10%. Identification, confirmation and quantitation were carried out by LC/TOFMS and LC/MS/MS using a hybrid triple quadrupole linear ion trap (QqLIT) instrument in multiple-reaction monitoring (MRM) mode. The obtained limits of quantitation (LOQs) were in the range 0.1-5 microg kg(-1), depending on each individual technique. Finally, the proposed methods were successfully applied to the analysis of suspected pepper samples.     

Chemical cross-linking and mass spectrometry for mapping three-dimensional structures of proteins and protein complexes

Chemical cross-linking of proteins, an established method in protein chemistry, has gained renewed interest in combination with mass spectrometric analysis of the reaction products for elucidating low-resolution three-dimensional protein structures and interacting sequences in protein complexes. The identification of the large number of cross-linking sites from the complex mixtures generated by chemical cross-linking, however, remains a challenging task. This review describes the most popular cross-linking reagents for protein structure analysis and gives an overview of the strategies employing intra- or intermolecular chemical cross-linking and mass spectrometry. The various approaches described in the literature to facilitate detection of cross-linking products and also computer software for data analysis are reviewed. Cross-linking techniques combined with mass spectrometry and bioinformatic methods have the potential to provide the basis for an efficient structural characterization of proteins and protein complexes.     

Online microdialysis-dynamic nanoelectrospray ionization-mass spectrometry for monitoring neuropeptide secretion

Although mass spectrometric approaches offer a sensitive method for identifying cell-cell signaling peptides, the high salt-containing environment of extracellular solutions often complicates characterization of these microscale samples. Accordingly, we have developed a miniature hollow-fiber microdialysis device optimized for desalting small-volume neuronal samples online, with the device directly connected to a modified dynamic nanoelectrospray ionization assembly interfaced with an ion trap mass spectrometer. Improvements over existing designs include placement of a capillary insert within the microdialysis fiber to minimize volume, as well as the use of a microinjector that enables 1 microl sample injections. We present detailed evaluation of peptide recoveries within the microdialysis fiber by liquid chromatography-electrospray ionization-ion trap-mass spectrometry analysis of tissue homogenate in artificial seawater with and without microdialysis. Analyte recoveries after microdialysis ranged from 6 to 78% with higher recoveries of more hydrophilic peptides, while little correlation between mass and percentage recovery was observed in the range studied (2000 to 6000 Da). Recoveries of peptides were the lowest for the analytes with the highest initial mass spectrometry signal intensity. Finally, we illustrate the utility of this microdialysis device for desalting neuropeptides secreted from preparations of the peptidergic bag cell neurons of the marine mollusk, Aplysia californica. Without microdialysis, the high concentration of salts ( approximately 0.5 M) prevented detection of peptides, whereas following online microdialysis-dynamic nanoelectrospray mass spectrometry of stimulated releasate, three peptides (acidic peptide, acidic peptide 1-24 and delta-bag cell peptide) were detected.