Determination of the glycation sites of Bacillus anthracis neoglycoconjugate vaccine by MALDI-TOF/TOF-CID-MS/MS and LC-ESI-QqTOF-tandem mass spectrometry

We present herein an efficient mass spectrometric method for the localization of the glycation sites of a model neoglycoconjugate vaccine formed by a construct of the tetrasaccharide side chain of the Bacillus anthracis exosporium and the protein carrier bovine serum albumin. The glycoconjugate was digested with both trypsin and GluC V8 endoproteinases, and the digests were then analyzed by MALDI-TOF/TOF-CID-MS/MS and nano-LC-ESI-QqTOF-CID-MS/MS. The sequences of the unknown peptides analyzed by MALDI-TOF/TOF-CID-MS/MS, following digestion with the GluC V8 endoproteinase, allowed us to recognize three glycopeptides whose glycation occupancies were, respectively, on Lys 235, Lys 420, and Lys 498. Similarly, the same analysis was performed on the tryptic digests, which permitted us to recognize two glycation sites on Lys 100 and Lys 374. In addition, we have also used LC-ESI-QqTOF-CID-MS/MS analysis for the identification of the tryptic digests. However, this analysis identified a higher number of glycopeptides than would be expected from a glycoconjugate composed of a carbohydrate-protein ratio of 5.4:1, which would have resulted in glycation occupancies of 18 specific sites. This discrepancy was due to the large number of glycoforms formed during the synthetic carbohydrate-spacer-carrier protein conjugation. Likewise, the LC-ESI-QqTOF-MS/MS analysis of the GluC V8 digest also identified 17 different glycation sites on the synthetic glycoconjugate.     

Identification of flavonoids using liquid chromatography with electrospray ionization and ion trap tandem mass spectrometry with an MS/MS library

Searchable MS/MS spectra libraries, constructed using the results of liquid chromatography coupled with electrospray ionization (ESI) tandem mass spectrometry (LC/MS/MS) with data-dependent acquisition on an ion trap mass spectrometer, are presented with regard to the identification and confirmation of a variety of closely related flavonoids in a set of biological samples. Flavonoids were found to exhibit a maximum amount of structurally specific MS/MS spectra at 45% of normalized collision energy on the instrument used, without wideband activation. These MS/MS spectra were then searched automatically against a 297-substance MS/MS library that contains many previously acquired spectra of standard flavonoids. The possible applications of this powerful technique to biological samples are also discussed. Daidzein and genistein were identified through the MS/MS spectra library while searching through LC/MS/MS data for plant and microbial extracts. Moreover, these compounds proved completely distinguishable from other flavonoids of closely related structures in the MS/MS spectra library, using the NIST MS search program. The applicability of the library-searchable spectra at low concentrations was demonstrated by successful identification of daidzein and genistein at 0.05 and 0.5 microg/mL, respectively.     

Identification of mammalian cell lines using MALDI-TOF and LC-ESI-MS/MS mass spectrometry

Direct mass spectrometric analysis of complex biological samples is becoming an increasingly useful technique in the field of proteomics. Matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) is a rapid and sensitive analytical tool well suited for obtaining molecular weights of peptides and proteins from complex samples. Here, a fast and simple approach to cellular protein profiling is described in which mammalian cells are lysed directly in the MALDI matrix 2,5-dihydroxybenzoic acid (DHB) and mass analyzed using MALDI-time of flight (TOF). Using the unique MALDI mass spectral "fingerprint" generated in these analyses, it is possible to differentiate among several different mammalian cell lines. A number of techniques, including MALDI-post source decay (PSD), MALDI tandem time-of-flight (TOF-TOF), MALDI-Fourier transform ion cyclotron resonance (FTICR), and nanoflow liquid chromatography followed by electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) were employed to attempt to identify the proteins represented in the MALDI spectra. Performing a tryptic digestion of the supernatant of the cells lysed in DHB with subsequent LC-ESI-MS/MS analysis was by far the most successful method to identify proteins.     

Determination of 48 fragrance allergens in toys using GC with ion trap MS/MS

This paper presents a method for the simultaneous determination of 48 fragrance allergens in four types of toys (plastic toys, play clays, plush toys, and paper toys) based on GC with ion trap MS/MS. Compared with single‐stage MS, MS/MS is superior in terms of the qualification and quantification of a large range of compounds in complicated matrices. Procedures for extraction and purification were optimized for each toy type. The method proved to be linear over a wide range of concentrations for all analytes with correlation coefficients between 0.9768 and 0.9999. Validation parameters, namely, LODs and LOQs, ranged from 0.005–5.0 and from 0.02–20 mg/kg, respectively. Average recoveries of target compounds (spiked at three concentration levels) were in the range of 79.5–109.1%. Intraday and interday repeatabilities of the proposed method varied from 0.7–10.5% and from 3.1–13.4%, respectively. The proposed method was used to monitor fragrance allergens in commercial toy products. Our findings indicate that this method is an accurate and effective technique for analyzing fragrance allergens in materials composed of complex components.     

Creation and comparison of MS/MS spectral libraries using quadrupole ion trap and triple-quadrupole mass spectrometers

Searchable libraries of MS/MS spectra, obtained using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data-dependent scan mode switching on both quadrupole ion trap and triple-quadrupole mass spectrometers in conjunction with electrospray ionization, are presented. The effects on library search scores of changing the parameters for producing collision-induced dissociation (CID) on both instrument types are systematically evaluated. These observations serve as a basis for determining a universal set of conditions for building MS/MS libraries. A group of 19 closely related steroids was used. The ability to obtain library-searchable spectra at low concentrations is demonstrated for the analysis of a sample of progesterone spiked with hydroxyprogesterone impurities at 0.1 and 0.01%.     

MS/MS of ions in a low pressure linear ion trap using a pulsed gas

A pulsed valve was used to increase the pressure within the trapping region of a low-pressure linear ion trap by situating the pulsed valve close to the ion trapping region. The pressure was estimated to increase from a background pressure of 3.5e–5 Torr of nitrogen to 0.49 mTorr at the center of the trap. The increased pressure allowed excitation periods to be reduced from 100 to 25 ms without suffering losses in MS/MS efficiency during dipolar excitation. The reduction in excitation period translates into an increase in the overall duty cycle of the scan.     

Identification of two polyamides (PA11 and PA1012) using pyrolysis-GC/MS and MALDI-TOF MS

The main goal of this work is to identify two polyamides (PA11 and PA1012) by mass spectrometry including pyrolysis-GC/MS and MALDI-TOF MS. PA11 and PA1012 have similar properties and cannot be distinguished by many other methods. Using pyrolysis-GC/MS, the pyrograms of PA11 and PA1012 at 600 °C were compared. Specific pyrolyzates for PA11 and PA1012 were obtained, 2-azacyclotridecanone for PA11 and 1,10-diaminodecane, 1,10-dicyanodecane for PA1012, respectively, which was the basis to distinguish them. Meanwhile, MALDI-TOF MS can give specific repeat unit for these two polyamides, dehydrated 11-aminoundecanoic acid (M = 183) for PA11, acetylate of dodecanedioic acid and diaminodecane (M = 366) for PA1012, which can be another means of identifying them.     

Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS

Previously, we have reported a high-efficiency method of protein extraction from CBB-stained polyacrylamide gels for molecular mass measurement with MALDI-TOF MS [1]. In the present work, the alkaline extraction method was applied to CBB-stained 2-DE gels on which human plasma proteins were separated in the absence of denaturant. In order to examine the performance of the method, ten spots with apparent molecular masses (MMapp) in the range of 65 to 1000 kDa were selected and the proteins were extracted from the gel pieces. The extracts were subjected to whole-mass measurement by MALDI-TOF MS, with and without DTT treatment. In addition, the extracts were subjected to in-solution trypsin digestion followed by MALDI-TOF MS and PMF analysis. Successful extraction of proteins from the ten spots, up to MMapp 1000 kDa, has been ascertained by the significant PMF assignment (MASCOT) with high sequence coverage of the respective proteins or polypeptides. When direct mass measurement of the extracted proteins was attempted, three spots in MMapp range 65-100 kDa provided mass peaks. Five spots in MMapp range 150-400 kDa did not give mass peaks of the intact proteins, but showed those of the constituent polypeptides after the DTT treatment. Extraction of proteins prior to trypsin digestion enabled the procedure of PMF analysis to be much simpler than the conventional in-gel digestion method, providing comparable protein scores and sequence coverage. The technique presented here suggests a new strategy for the characterization of proteins separated by nondenaturing 2-DE.     

Capillary electrophoretic separation of organophosphonic acids using borate esterification and direct UV detection

One major challenge in the analysis of small ions by capillary zone electrophoresis (CZE) is detection. The most common commercially available detector for CZE is based on UV absorbance. For many small molecules, however, little UV absorbance occurs above 210 nm, limiting the usefulness of this detection method. Carbohydrates, alcohols and amides have been separated and directly detected by complexation with sodium borate. It has been shown that these complexes have UV absorbances in the range of 220 to 280 nm, whereas the analytes alone are UV transparent at energies less than 200 nm. Separation and direct detection of organophosphonic acids using sodium borate as both a buffer and derivatization agent is demonstrated. Detection limits on the order of nanograms are reported with separations that exhibit 10 000 to 1 740 000 theoretical plates. The ultraviolet, infrared, nuclear magnetic resonance and mass spectra of various borate/phosphonic acid esters are explored.     

Use of selective extraction and fast chromatographic separation combined with electrophoretic methods for mapping of membrane proteins

A model system for selective solubilization and fast separation of proteins from the rat liver membrane fraction and purified rat liver plasma membranes for their further proteomic analysis is presented. For selective solubilization, high-pH solutions and a concentrated urea solution, combined with different detergents, are used. After extraction, proteins are separated by anion-exchange chromatography or a combination of anion- and cation-exchange chromatography with convective interaction monolithic supports. This separation method enables fast and effective prefractionation of membrane proteins based on their hydrophobicity and charge prior to one-dimensional (1-D) and 2-D electrophoresis and mass spectrometry. By use of this sample preparation method, the less-abundant proteins can be detected and identified.     

Rapid screening and characterization of drug metabolites using a multiple ion monitoring-dependent MS/MS acquisition method on a hybrid triple quadrupole-linear ion trap mass spectrometer

A novel LC/MS/MS method that uses multiple ion monitoring (MIM) as a survey scan to trigger the acquisition of enhanced product ions (EPI) on a hybrid quadrupole-linear ion trap mass spectrometer (Q TRAP) was developed for drug metabolite identification. In the MIM experiment, multiple predicted metabolite ions were monitored in both Q1 and Q3. The collision energy in Q2 was set to a low value to minimize fragmentation. Results from analyzing ritonavir metabolites in rat hepatocytes demonstrate that MIM-EPI was capable of targeting a larger number of metabolites regardless of their fragmentation and retained sensitivity and duty cycle similar to multiple reaction monitoring (MRM)-EPI. MIM-based scanning methods were shown to be particularly useful in several applications. First, MIM-EPI enabled the sensitive detection and MS/MS acquisition of up to 100 predicted metabolites. Second, MIM-MRM-EPI was better than MRM-EPI in the analysis of metabolites that undergo either predictable or unpredictable fragmentation pathways. Finally, a combination of MIM-EPI and full-scan MS (EMS), as an alternative to EMS-EPI, was well suited for routine in vitro metabolite profiling. Overall, MIM-EPI significantly enhanced the metabolite identification capability of the hybrid triple quadrupole-linear ion trap LC/MS.     

Intact protein separation by chromatographic and/or electrophoretic techniques for top-down proteomics

Mass spectrometry used in combination with a wide variety of separation methods is the principal methodology for proteomics. In bottom-up approach, proteins are cleaved with a specific proteolytic enzyme, followed by peptide separation and MS identification. In top-down approach intact proteins are introduced into the mass spectrometer. The ions generated by electrospray ionization are then subjected to gas-phase separation, fragmentation, fragment separation, and automated interpretation of mass spectrometric and chromatographic data yielding both the molecular weight of the intact protein and the protein fragmentation pattern. This approach requires high accuracy mass measurement analysers capable of separating the multi-charged isotopic cluster of proteins, such as hybrid ion trap-Fourier transform instruments (LTQ-FTICR, LTQ-Orbitrap). Front-end separation technologies tailored for proteins are of primary importance to implement top-down proteomics. This review intends to provide the state of art of protein chromatographic and electrophoretic separation methods suitable for MS coupling, and to illustrate both monodimensional and multidimensional approaches used for LC-MS top-down proteomics. In addition, some recent progresses in protein chromatography that may provide an alternative to those currently employed are also discussed.     

Selective extraction, separation, and identification of anthocyanins from Hibiscus sabdariffa L. using solid phase extraction-capillary electrophoresis-mass spectrometry (time-of-flight /ion trap)

A method for selective extraction using SPE, electrophoretic separation at basic condition and the identification by using exact masses and fragmentation patterns has been developed in order to know the anthocyanins in dried calyces of Hibiscus sabdariffa L. A detailed and comparative study of several extraction procedures has been carried out to obtain the maximum number of anthocyanidins from the calyces and then a CE-TOF-MS method in positive mode using ESI has been developed for the separation and rapid identification of anthocyanins in H. sabdariffa L. Delphinidin-3-sambubioside, cyanidin-3-sambubioside have been detected as main components and cyanidin-3-O-rutinoside, delphinidin-3-O-glucoside and cyanidin-3,5-diglucoside, and chlorogenic acid as minor constituents. The confirmation of the anthocyanidins and chlorogenic acid was carried out using fragmentation ions with the IT-mass spectrometer (IT-MS).     

Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorption/ionization.

The molecular masses of electroblotted proteins were determined with a time-of-flight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.     

MALDI-TOF/MS Analysis of Non-Invasive Human Urine and Saliva Samples for the Identification of New Cancer Biomarkers

Cancer represents a group of heterogeneous diseases that are a leading global cause of death. Even though mortality has decreased in the past thirty years for different reasons, most patients are still diagnosed at the advanced stage, with limited therapeutic choices and poor outcomes. Moreover, the majority of cancers are detected using invasive painful methods, such as endoscopic biopsy, making the development of non-invasive or minimally invasive methods for the discovery and fast detection of specific biomarkers a crucial need. Among body fluids, a valuable non-invasive alternative to tissue biopsy, the most accessible and least invasive are undoubtedly urine and saliva. They are easily retrievable complex fluids containing a large variety of endogenous compounds that may provide information on the physiological condition of the body. The combined analysis of these fluids with matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF/MS), a reliable and easy-to-use instrumentation that provides information with relatively simple sample pretreatments, could represent the ideal option to rapidly achieve fast early stage diagnosis of tumors and their real-time monitoring. On this basis, the present review summarizes the recently reported applications relevant to the MALDI analysis of human urine and saliva samples.     

Capillary zone electrophoretic separation of basic proteins and drugs using guaran as a buffer modifier

Summary Guaran, a neutral polysaccharide, has been used as a buffer modifier to improve the separation of basic proteins and drugs. Migration reproductibility, peak shape and efficiency were improved when 0.1% guaran was added to the buffer. The concentration of guaran, ionic strength, and pH of buffer solution were optimized to obtain the optimum separation of proteins. Possible separation efficiencies of 700,000 plates per meter were obtained for test proteins. The relative standard deviation (% RSD) of the migration time of all test proteins was less than 0.5%. Improved separation of β-blockers was also observed when guaran was added to the buffer.     

Ion-permeable membrane for on-chip preconcentration and separation of cancer marker proteins

Cancer marker proteins have been electrophoretically concentrated and then separated in a microfluidic device. On-chip preconcentration was achieved using an ion-permeable membrane, consisting of acrylamide, N,N'-methylene-bisacrylamide and 2-(acrylamido)-2-methylpropanesulfonate. This negatively charged membrane was photopolymerized in the microdevice near the injection intersection. Anionic proteins were excluded from the porous membrane based on both size and charge, which concentrated target components in the injection intersection prior to separation by microchip capillary electrophoresis (μ-CE). Bovine serum albumin was used in the initial characterization of the system and showed a 40-fold enrichment in the μ-CE peak with 4 min of preconcentration. Adjustment of buffer pH enabled baseline resolution of two cancer biomarkers, α-fetoprotein (AFP) and heat shock protein 90 (HSP90), while fine control over preconcentration time limited peak broadening. Our optimized preconcentration and μ-CE approach was applied to AFP and HSP90, where enrichment factors of >10-fold were achieved with just 1 min of preconcentration. Overall, the process was simple and rapid, providing a useful tool for improving detection in microscale systems.     

Identification and quantitation of pyrethroid pesticide residues in vegetables by solid-phase extraction and liquid chromatography/electrospray ionization ion trap mass spectrometry

A quantitative method consisting of solid-phase extraction (SPE) followed by liquid chromatography/electrospray ionization ion trap mass spectrometry (LC/ESI-ITMS) analysis was developed for the identification and quantitation of ten pyrethroid pesticides commonly used in vegetables. The best HPLC separation was achieved using a gradient program of methanol/water mixture. For the vegetable samples, an SPE procedure to clean up the matrices was carried out prior to LC/MS analysis. Under the optimum conditions, the limits of quantification of the pyrethroid pesticides (tetramethrin, allethrin, fenpropathrin, lambda-cyhalothrin, cypermethrin, deltamethrin, fenvalerate, bioresmethrin, permethrin and bifenthrin) ranged from 0.03 to 0.1 mg kg-1 with relative standard deviations<20%, and the mean recoveries ranged from 69.5 to 102.5%. The proposed method has been successfully applied to the determination of pyrethroids in six vegetables with satisfactory results.