Electrospray Ion Trap Mass Spectrometry Research Articles

Determination of 1,4-benzodiazepines and their metabolites by capillary electrophoresis and high-performance liquid chromatography using ultraviolet and electrospray ionisation mass spectrometry

A study is presented for the separation and determination of fifteen 1,4-benzodiazepine drugs and metabolites by capillary electrophoresis (CE) compared with high-performance liquid chromatography (HPLC). A comparison is made between the CE determination of the compounds by conventional UV detection and LC determination with electrospray ionisation (ESI) ion-trap mass spectrometry. CE is shown to provide superior separation to HPLC but the MS–MS capability of the ion-trap allows for the specific detection and determination of four of the compounds, diazepam, N′-desmethyldiazepam, oxazepam and temazepam in the hair of a patient under clinical treatment with diazepam and temazepam. Selected mixtures of drugs and metabolites are determined by CE and LC and the determination of diazepam and its metabolites by CE–UV–ESI-MS–MS is also presented.

Electrospray ionization-ion trap mass spectrometry for structural analysis of complex N-linked glycoprotein oligosaccharides.

Electrospray ionization-ion trap mass spectrometry, with its capacity to perform multiple stages of fragmentation (MSn), is demonstrated as an effective method for the structural characterization of permethylated N-linked complex glycoprotein oligosaccharides. Complex glycan structural features, such as N-acetyllactosamine antenane, neuraminic acids, and nonreducing terminal GlcNAc monosaccharides, commonly suppress cross-ring and core saccharide cleavages in traditional MS/MS experiments. Using ion trap mass spectrometry, removal of these substituents permits determination of branching patterns and intersaccharide linkages by MS3 and MS4. Both sequence and linkage data are obtained for N-acetyllactosamine and sialyl-N-acetyllactosamine oligosaccharide antennae from biantennary glycans using MS3, and the location of a bisecting GlcNAc residue is also established after exposing the core pentasaccharide. Higher-order experiments further illustrate the potential of electrospray ionization-quadrupole ion trap mass spectrometry for carbohydrate analysis, as MS8 is used to produce significant and otherwise unobtainable branching information for an oligosaccharide from chicken ovalbumin. These studies constitute further evidence of the unique role that ion trap mass spectrometry can assume in the area of oligosaccharide analysis.

Mass Spectral Identification and Positional Mapping of Aflatoxin B 1–Guanine Adducts in Oligonucleotides

The biological consequences of a carcinogen–DNA adduct are defined by the structure of the lesion and its position within the genome. Electrospray ionization ion trap mass spectrometry (ESI-ITMS) is shown here to be a sensitive and rapid approach capable of defining both of these parameters. Three isomeric oligonucleotides of the sequence 5′-CCGGAGGCC modified by the potent human carcinogen aflatoxin B 1 (AFB 1) at different guanines were analyzed by ESI-ITMS. All three samples possessed the same molecular ion confirming the presence of an intact aflatoxin moiety in each oligonucleotide. In addition, each sample displayed a characteristic fragmentation pattern that permitted unambiguous identification of the site of modification within the sequence. Furthermore, an AFB 1-modified oligonucleotide was converted under alkaline conditions to its more stable formamidopyrimidine (FAPY) derivative. Analysis of this sample revealed the presence of a molecular ion corresponding to the presence of the FAPY adduct and a distinctive fragmentation pattern that paralleled the known chemical stability of the FAPY metabolite. This approach should be of general use in the determination of not only the nature and site of covalent modifications, but also the chemical stability of DNA adducts.

Interfacing of CE in a PVP matrix to ion trap mass spectrometry: analysis of isomeric and structurally related (N-acetylamino)fluorene-modified oligonucleotides.

This work demonstrates the interfacing of capillary electrophoresis in a poly(N-vinylpyrrolidone) (PVP) solution to electrospray ionization ion trap mass spectrometry (ESI-ITMS). This methodology was used for on-line analysis of modified and unmodified oligonucleotides. Oligonucleotides were covalently modified using the model carcinogen 2-(N-acetoxy-N-acetylamino)fluorene. In the presence of PVP, separation was achieved for a set of isomeric (N-acetylamino)fluorene (AAF)-modified oligonucleotides differing only in their base sequences, while open-tube control experiments showed no separation between these compounds. The resolved analytes were identified by ESI-ITMS with negative ion detection. Online acquisition of MS and MS/MS data allowed unambiguous identification of all structural isomers. Baseline separation was also accomplished for a 10-compound mixture containing a series of five nonisomeric AAF-modified oligonucleotides and their unmodified parent oligonucleotides.

Characterization of rat brain stathmin isoforms by two-dimensional gel electrophoresis-matrix assisted laser desorption/ionization and electrospray ionization-ion trap mass spectrometry.

Stathmin is a regulatory phosphoprotein that is a target for both cell cycle and cell surface receptor-regulated phosphorylation events. There are at least 14 isoforms of stathmin that migrate on two-dimensional electrophoresis (2-DE): two unphosphorylated, and 12 increasingly phosphorylated proteins. Following extracellular stimuli, stathmin is phosphorylated on four serines (Ser16, Ser25, Ser38, and Ser63) by several kinases, such as mitogen-activated protein (MAP), cdc2 kinase, protein kinase A, and Ca2+/calmodulin-dependent kinase-Gr. While all forms of stathmin are derived from the same protein encoded by a single mRNA, the precise nature of the post-translational modifications has not been clear. In this study we have characterized three rat brain stathmin isoforms, #1, #3 and #4, which electrophorese on 2-DE with apparent molecular weight (Mr)/isoelectric point (pI) values of 15,500/6.2, 15,000/6.1, and 15,000/6.0, respectively. The phosphorylation status of these isoforms was determined using a combination of peptide mapping, matrix-assisted laser desorption/ionization mass spectrometry and electrospray-ionization ion trap mass spectrometry. Stathmin isoform #1 was not phosphorylated, stathmin isoform #3 was phosphorylated on Ser38 only, and stathmin isoform #4 was phosphorylated on Ser38; however, the phosphorylation status of Ser63 could not be determined. In addition, three proteins which electrophorese near stathmin were identified in order to more accurately define the Mr/pI locus of this region of the 2-DE gel map. These include: phosphatidyl ethanolamine binding protein (Mr approximately 18,000/pI 6.0), synuclein forms 2 and 3 (Mr approximately 14,000/pI 5.4), and synuclein form 2 (Mr approximately 15,000/pI 5.0).

Analysis of neuropeptides by perfusion liquid chromatography/electrospray ion‐trap mass spectrometry

Perfusion high-performance liquid chromatography (HPLC) combined with electrospray ion trap mass spectrometry (ITMS) was evaluated for the determination of neuropeptides in plasma. Perfusion HPLC offers the capability of resolving neuropeptides spiked into plasma in 5 min compared to the 30-60 min separations performed on packed capillary C18 columns. Electrospray combined with the ITMS provides the ability to ionize these neuropeptides and mass analyze them with high sensitivity and specificity. Sub-picomole quantities of neuropeptides injected on-column could be specifically detected in a plasma matrix. The electrospray-ITMS mass spectrum of each neuropeptide showed multiply charged ions which could be used to determine or confirm their molecular weights.