Joseph H. Banoub

Mass Spectrometry, Review of the Basics: Electrospray, MALDI, and Commonly Used Mass Analyzers

Abstract Mass spectrometry (MS) has progressed to become a powerful analytical tool for both quantitative and qualitative applications. The first mass spectrometer was constructed in 1912 and since then it has developed from only analyzing small inorganic molecules to biological macromolecules, practically with no mass limitations. Proteomics research, in particular, increasingly depends on MS technologies. The ability of mass spectrometry analyzing proteins and other biological extracts is due to the advances gained through the development of soft ionization techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) that can transform biomolecules into ions. ESI can efficiently be interfaced with separation techniques enhancing its role in the life and health sciences. MALDI, however, has the advantage of producing singly charges ions of peptides and proteins, minimizing spectral complexity. Regardless of the ionization source, the sensitivity of a mass spectrometer is related to the mass analyzer where ion separation occurs. Both quadrupole and time of flight (ToF) mass analyzers are commonly used and they can be configured together as QToF tandem mass spectrometric instruments. Tandem mass spectrometry (MS/MS), as the name indicates, is the result of performing two or more sequential separations of ions usually coupling two or more mass analyzers. Coupling a quadrupole and time of flight resulted in the production of high-resolution mass spectrometers (i.e., Q-ToF). This article will historically introduce mass spectrometry and summarizes the advantages and disadvantages of ESI and MALDI along with quadrupole and ToF mass analyzers, including the technical marriage between the two analyzers. This article is educational in nature and intended for graduate students and senior biochemistry students as well as chemists and biochemists who are not familiar with mass spectrometry and would like to learn the basics; it is not intended for mass spectrometry experts.

Synthesis of cellulose fatty esters as plastics-influence of the degree of substitution and the fatty chain length on mechanical properties.

Alternative films: The effect of the chain length and the degree of substitution on the mechanical and hydrophobic properties of various cellulose fatty ester plastic films was studied. The results suggest that the cellulose ester plastic films are promising alternatives to petrochemical commodity plastics such as polyethylene.Cellulose-based plastic films were prepared by acylating cellulose in homogeneous media under microwave irradiation with fatty acyl chlorides containing either saturated or unsaturated chains of various lengths (C(12) to C(18)). The resultant cellulose esters were analysed by FTIR and (1)H NMR spectroscopy to confirm their structure and to determine their degree of substitution. Some of the cellulose fatty esters were then converted into polymer films by casting. The mechanical properties of these films were determined, including their elastic modulus, tensile strength and tensile strain level. The hydrophobicity of the polymer films was determined by contact angle measurement with water. The mechanical and hydrophobic properties of the plastic films were then compared to those of commodity plastics.

Protein free microcapsules obtained from plant spores as a model for drug delivery: ibuprofen encapsulation, release and taste masking

Sporopollenin exine capsules (SEC) extracted from Lycopodium clavatum spores were shown to encapsulate ibuprofen as a drug model, with 97 ± 1% efficiency as measured by recovery of the loaded drug and absence of the drug on the SEC surface by scanning electron microscopy (SEM). The encapsulated ibuprofen was shown to be unchanged from its bulk crystalline form by solid state NMR, FTIR and XRD. Essential for drug delivery applications, SEC were shown to be non-toxic to human endothelial cells and free of allergenic protein epitopes by MALDI-TOF-MS and ESI-QqToF-MS. Potential application for targeted release into the intestinal region of the gastrointestinal tract (GIT) was demonstrated by 88 ± 1% of the drug being retained in simulated gastric fluid (SGF) after 45 minutes and 85 ± 2% being released after 5 min in buffer (PBS; pH 7.4). The SEC were shown to provide significant taste masking of encapsulated ibuprofen in a double blind trial with 10 human volunteers.

Antifreeze glycoproteins: relationship between molecular weight, thermal hysteresis and the inhibition of leakage from liposomes during thermotropic phase transition.

Antifreeze glycoproteins (AFGP) were isolated and purified from the blood plasma of rock cod (Gadus ogac), using DEAE-Bio-gel ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The purified proteins were analyzed using polyacrylamide gel electrophoresis (PAGE), and electrospray mass spectrometry. The results indicated that rock cod synthesize seven size classes of glycoproteins, ranging from 2.6 to 24 kDa, with each size class containing multiple isoforms. Antifreeze activity, as determined by thermal hysteresis, indicated that the AFGP could be separated into two groups, with the larger size classes (molecular mass>13 kDa) having approximately 3-4 times the activity of the smaller, proline containing, size classes (molecular mass<10 kDa). All of the AFGP size classes prevented leakage from dielaidoylphosphatidylcholine (DEPC) liposomes as they were cooled through their phase transition temperature, with the larger size classes being approximately 4 times as effective as the smaller ones. It is hypothesized that AFGP prevent liposomes from leaking as they pass through the phase transition temperature by binding to the phospholipid membrane.

Structural investigation of bacterial lipopolysaccharides by mass spectrometry and tandem mass spectrometry

Mass spectrometric studies are now playing a leading role in the elucidation of lipopolysaccharide (LPS) structures through the characterization of antigenic polysaccharides, core oligosaccharides and lipid A components including LPS genetic modifications. The conventional MS and MS/MS analyses together with CID fragmentation provide additional structural information complementary to the previous analytical experiments, and thus contribute to an integrated strategy for the simultaneous characterization and correct sequencing of the carbohydrate moiety.

A critique on the structural analysis of lignins and application of novel tandem mass spectrometric strategies to determine lignin sequencing

This review is devoted to the application of MS using soft ionization methods with a special emphasis on electrospray ionization, atmospheric pressure photoionization and matrix-assisted laser desorption/ionization MS and tandem MS (MS/MS) for the elucidation of the chemical structure of native and modified lignins. We describe and critically evaluate how these soft ionization methods have contributed to the present-day knowledge of the structure of lignins. Herein, we will introduce new nomenclature concerning the chemical state of lignins, namely, virgin released lignins (VRLs) and processed modified lignins (PML). VRLs are obtained by liberation of lignins through degradation of vegetable matter by either chemical hydrolysis and/or enzymatic hydrolysis. PMLs are produced by subjecting the VRL to a series of further chemical transformations and purifications that are likely to alter their original chemical structures. We are proposing that native lignin polymers, present in the lignocellulosic biomass, are not made of macromolecules linked to cellulose fibres as has been frequently reported. Instead, we propose that the lignins are composed of vast series of linear related oligomers, having different lengths that are covalently linked in a criss-cross pattern to cellulose and hemicellulose fibres forming the network of vegetal matter. Consequently, structural elucidation of VRLs, which presumably have not been purified and processed by any other type of additional chemical treatment and purification, may reflect the structure of the native lignin. In this review, we present an introduction to a MS/MS top-down concept of lignin sequencing and how this technique may be used to address the challenge of characterizing the structure of VRLs. Finally, we offer the case that although lignins have been reported to have very high or high molecular weights, they might not exist on the basis that such polymers have never been identified by the mild ionizing techniques used in modern MS.

Structural investigations on the core oligosaccharide of Aeromonas hydrophila (chemotype II) lipopolysaccharide

Abstract The core oligosaccharide of Aeromonas hydrophila (Chemotype III) lipopolysaccharide has been investigated. The studies involved the use of methylation analysis, oxidation with chromium trioxide, partial hydrolysis with acid, periodate oxidation, Smith degradation, and tagging of the reducing end-group. The core is unusual in having 3-acetamido-3,6-dideoxy- l -glucose as a constituent. As a result of these studies the following structure is proposed.

Characterization and de novo sequencing of Atlantic salmon vitellogenin protein by electrospray tandem and matrix-assisted laser desorption/ionization mass spectrometry

Vitellogenin (VTG) is a protein produced by the liver of oviparous animals in response to circulating estrogens. In the plasma of males and immature females, VTG is undetectable. VTG has been used as a biomarker for exposure to endocrine disruptors in many species. In the present study, characterization of intact Atlantic salmon VTG was effected using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI ToF MS). Tryptic digest peptides were analyzed by MALDI ToF MS to obtain a peptide mass fingerprint. De novo sequencing of the tryptic peptides used low-energy collisionally-induced dissociation (CID) in an electrospray ionization quadrupole-ToF orthogonal hybrid mass spectrometer (ESI Q-ToF MS/MS). The interpretation of the product-ion spectra obtained from the ESI Q-ToF MS/MS was done by Lutefisk, a computer-based software algorithm. The molecular mass of the intact protein was found to be 187335 Da. A total of 14 tryptic peptides were sequenced and compared with the complete rainbow trout VTG and the partial Atlantic salmon VTG sequences found in the Swiss-Prot database. De novo sequencing by CID MS/MS of 11 Atlantic salmon tryptic digest peptides with selected precursor ions at m/z 788.24, 700.20, 794.75, 834.31, 889.28, 819.79, 865.27, 843.81, 572.20, 573.66 and 561.68 showed high homology with the known sequence of rainbow trout VTG. The last two precursor peptide ions, found at m/z 573.66 and m/z 561.68, also specifically matched the known portion of the Atlantic salmon VTG sequence. Finally, three tryptic precursor peptide ions found at m/z 795.18, 893.28 and 791.05, provided product-ion spectra, which were exclusive to the unsequenced portion of the Atlantic salmon VTG.

Synthesis and structural characterization of macrocyclic carbohydrate derivatives obtained from catalytic metathesis reaction with chloro-aryloxide complexes of tungsten

Abstract Tungsten aryloxo-complex 1 catalyses the intramolecular metathesis reactions of di- and trisubstituted ω-unsaturated glucose, and glucosamine derivatives, yielding bicyclic carbohydrate-based compounds 12, 13, 14, 15 , containing 12-, and 14-atoms rings. Electrospray tandem mass spectrometry was used for the structural characterization of these novel bicyclic carbohydrate compounds.

Characterization and differentiation of isomeric self‐complementary DNA oligomers by electrospray tandem mass spectrometry

Two series of synthetic self-complementary isomeric DNA hexamers, namely d(CAGCTG), d(CGATCG) and d(CGTACG) (M(r) 1791) and d(CATATG), d(TGATCA) and d(TGTACA) (M(r) 1790) were analysed by negative electrospray mass spectrometry. As expected, these DNA hexamers exhibited identical series of multi-charged deprotonated molecular ions. Low-energy collision-induced dissociation tandem mass spectrometric analysis of the multi-charged oligonucleotide anions [M-3H]3- and [M-4H]4- provided characteristic and distinct finger-print patterns which permitted discrimination amongst the individual isomeric DNA hexamers and allowed complete direct sequence determination.