On the Nature of Mass Spectrometer Analyzer Contamination

Sample throughput in electrospray ionization mass spectrometry (ESI-MS) is limited by the need for frequent ion path cleaning to remove accumulated debris that can lead to charging and general performance degradation. Contamination of ion optics within the vacuum system is particularly problematic as routine cleaning requires additional time for cycling the vacuum pumps. Differential mobility spectrometry (DMS) can select targeted ion species for transmission, thereby reducing the total number of charged particles entering the vacuum system. In this work, we characterize the nature of instrument contamination, describe efforts to improve mass spectrometer robustness by applying DMS prefiltering to reduce contamination of the vacuum ion optics, and demonstrate the capability of DMS to extend the interval between mass spectrometer cleaning. In addition, we introduce a new approach to effectively detect large charged particles formed during the electrospray ionization (ESI) process.

Open image in new window

Graphical Abstract ?

     

Maximizing Ion Transmission in Differential Mobility Spectrometry

We provide modeling and experimental data describing the dominant ion-loss mechanisms for differential mobility spectrometry (DMS). Ion motion is considered from the inlet region of the mobility analyzer to the DMS exit, and losses resulting from diffusion to electrode surfaces, insufficient effective gap, ion fragmentation, and fringing field effects are considered for a commercial DMS system with 1-mm gap height. It is shown that losses due to diffusion and radial oscillations can be minimized with careful consideration of residence time, electrode spacing, gas flow rate, and waveform frequency. Fragmentation effects can be minimized by limitation of the separation field. When these parameters were optimized, fringing field effects at the DMS inlet contributed the most to signal reduction. We also describe a new DMS cell configuration that improves the gas dynamics at the mobility cell inlet. The new cell provides a gas jet that decreases the residence time for ions within the fringing field region, resulting in at least twofold increase in ion signal as determined by experimental data and simulations.

Open image in new window

Graphical Abstract ?

     

Calibrant delivery for mass spectrometry

This article describes a means of sampling ions that are created at a location remote from the primary ion source used for mass spectral analysis. Such a source can be used for delivery of calibrant ions on demand. Calibrant ions are sprayed into an atmospheric pressure chamber, at a position substantially removed from the sampling inlet. A gas flow sweeps the calibrants towards the sampling inlet, and a new means for toggling the second ion beam into the instrument can be achieved with the use of a repelling field established by an electrode in front of the sampling inlet. The physical separation of two or more sources of ions eliminates detrimental interactions due to gas flows or fields. When using a nanoflow electrospray tip as the primary ion source, the potential applied to the tip completely repels calibrant ions and there is no compromise in terms of electrospray performance. When calibrant ions are desired, the potential applied to the nanoflow electrospray tip is lowered for a short period of time to allow calibrant ions to be sampled into the instrument, thus providing a means for external calibration that avoids the typical complications and compromises associated with dual spray sources. It is also possible to simultaneously sample ions from multiple ion beams if necessary for internal mass calibration purposes. This method of transporting additional ion beams to a sampling inlet can also be used with different types of atmospheric pressure sources such as AP MALDI, as well as sources configured to deliver ions of different polarity.     

Enantiomeric deoxycholic acid: total synthesis, characterization, and preliminary toxicity toward colon cancer cell lines

Deoxycholic acid (DCA) is an endogenous secondary bile acid implicated in numerous pathological conditions including colon cancer formation and progression and cholestatic liver disease. DCA involvement in these disease processes results partly from its ability to modulate signaling cascades within the cell, presumably through both direct receptor activation and general detergent mediated membrane changes. To further explore DCA induced changes in cell signaling, we completed a total synthesis of enantiomeric deoxycholic acid (ent-DCA) from achiral 2-methyl-1,3-cyclopentanedione. Using a modified method of the synthesis of ent-testosterone that proceeds through the (R)-(-)-Hajos-Parrish ketone, we have completed the successful synthesis of ent-DCA in 25 steps with a yield of 0.3% with all stereochemical assignments of the product confirmed by X-ray crystallography. Our studies toward this synthesis also uncovered the methodology for the development of a novel A,B-cis steroidal skeleton system containing a C3-C9 single bond as well as conditions to selectively ketalize the typically less reactive 12-carbonyl in poly-keto A,B-cis androgens. The critical micelle concentration (cmc) of ent-DCA, determined by a dye solubilization method, was identical to the cmc of natural DCA. Toxicity studies toward HT-29 and HCT-116 human colon cancer cell lines demonstrated that ent-DCA had similar effects on proliferation, yet showed a markedly decreased ability to induce apoptosis as compared to natural DCA.     

Alternative reagents for chemical noise reduction in liquid chromatography-mass spectrometry using selective ion-molecule reactions

Reduction of ionic chemical background noise based on selective gas-phase reactions with chosen neutral reagents has been proven to be a very promising approach in liquid chromatography—mass spectrometry (LC-MS). In this study further investigations on alternative reagents including the disulfides (dimethyl disulfide, diethyl disulfide, methyl propyl disulfide), dimethyl trisulfide, ethylene oxide, and butadiene monoxide, for example, have been carried out. Tandem mass spectrometric studies of ion/molecule reactions indicate that—besides dimethyl disulfide—ethylene oxide and butadiene monoxide also exhibit very efficient reactions with background ions. Furthermore, it is confirmed that the reactions are very selective according to the test with some analyte ions. In contrast to its rapid reactions with background ions, ethylene oxide does not react, or reacts much less, with these analytes. Therefore, it can be used as an alternative reagent for noise reduction. Although reactions of the other tested neutral reagents with background ions are evaluated, they are generally not suitable as reagents for this purpose because of lack of reactivity or dramatic ion losses during reactions.     

Design considerations for high speed quantitative mass spectrometry with MALDI ionization

A MALDI ion source on a triple quadrupole mass spectrometer constructed for the purpose of obtaining high speed quantitative measurements on drugs and other low molecular weight compounds is described. Particular attention is given to the ion generation and transport phenomena that affect analysis speed, throughput, and practical instrument robustness. In this regard parameters that affect desorption speed, beam spreading, ion flight times, sensitivity, signal-to-noise, ion fragmentation, sample carry-over, and instrument contamination are examined and experimental results are provided. MALDI and electrospray sensitivity is compared, to provide a practical frame of reference.     

A neurosteroid analogue photolabeling reagent labels the colchicine-binding site on tubulin: a mass spectrometric analysis

Previous studies have shown that the neurosteroid analogue, 6-Azi-pregnanolone (6-AziP), photolabels voltage-dependent anion channels and proteins of approximately 55 kDa in rat brain membranes. The present study used two-dimensional electrophoresis and nanoelectrospray ionization ion-trap mass spectrometry (nano-ESI-MS) to identify the 55 kDa proteins (isoelectric point 4.8) as isoforms of β-tubulin. This identification was confirmed by immunoblot and immunoprecipitation of photolabeled protein with anti-β-tubulin antibody and by the demonstration that 6-AziP photolabels purified bovine brain tubulin in a concentration-dependent pattern. To identify the photolabeling sites, purified bovine brain tubulin was photolabeled with 6-AziP, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization MS (MALDI). A 6-AziP adduct of TAVCDIPPR(m/z?= 1287.77), a β-tubulin specific peptide, was detected by MALDI. High-resolution liquid chromatography-MS/MS analysis identified that 6-AziP was covalently bound to cysteine 354 (Cys-354), previously identified as a colchicine-binding site. 6-AziP photolabeling was inhibited by 2-methoxyestradiol, an endogenous derivative of estradiol thought to bind to the colchicine site. Structural modeling predicted that neurosteroids could dock in this colchicine site at the interface between α- and β-tubulin with the photolabeling group of 6-AziP positioned proximate to Cys-354.     

Collision cross sections for protein ions

A method for the determination of cross sections for gas-phase protein ions, based on the energy loss of ions as they pass through a collision gas, is described. A simple model relates the energy loss to the number of collisions and hence the cross section. Results from a Monte Carlo model that support the validity of this approach are described. Experimental cross sections are reported for motilin, ubiquitin, cytochrome c, myoglobm, and bovine serum albumin. Cross sections range from approximately 800 Ų for motilin to approximately 14,000 Ų for bovine serum albumin and generally increase with the number of charges on the ion. Cytochrome c ions from aqueous solution show somewhat smaller cross sections than ions formed from solutions of higher organic content, suggesting that the gas-phase ions may retain some memory of their solution conformation.     

Neurosteroid Analogues. 13. Synthetic methods for the preparation of 2beta-hydroxygonane derivatives as structural mimics of ent-3alpha-hydroxysteroid modulators of GABA(A) receptors

Many different 3α-hydroxysteroids in the androstane and pregnane steroid series enhance the actions of γ-aminobutyric acid (GABA) at GABA type-A (GABA_A) receptors in the mammalian central nervous system. Recent studies have shown that (3α,5α)-3-hydroxyandrostan-17-one (androsterone) is less active at these receptors than its enantiomer ent-androsterone. Further structure-activity relationship (SAR) studies are needed to explore the structural features of ent-androsterone that are important for its enhanced action at these receptors. Molecular modeling shows that 2β-hydroxysteroids are similar in three-dimensional shape to the enantiomers of 3α-hydroxysteroids. The development of synthetic methods to gain access to C₁₇-substituted analogues of 2β-hydroxygonanes for SAR studies is demonstrated with the synthesis of (2β,5α,14β)-2-hydroxygonan-17-one.