Wojciech Gabryelski

Rapid and sensitive differentiation of anomers, linkage, and position isomers of disaccharides using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS)

A challenging aspect of structural elucidation of carbohydrates is gaining unambiguous information for anomers, linkage, and position isomers. Such isomers with identical mass can’t be easily distinguished in mass spectrometry and a separation step is required prior to mass spectrometry identification. In our laboratory, gas-phase separation and differentiation of anomers, linkage, and position isomers of disaccharides was achieved using High-Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS). The FAIMS method responds to changes in ion mobility at high field rather than absolute values of ion mobility, and was shown to provide efficient separation and identification of disaccharide isomers at high sensitivity. Separation of analyzed disaccharide isomers can be accomplished at low nM level in a matter of seconds without sample purification or fractionation. Capability for examining a large population of ionic species of disaccharides by this method allowed for correlating structural details of disaccharide isomers with their separation properties in FAIMS. Results for disaccharide isomers indicate that this method could be applied to a larger group of carbohydrates.

PHOTO-INDUCED DISSOCIATION OF ELECTROSPRAY GENERATED IONS IN AN ION TRAP/TIME-OF-FLIGHT MASS SPECTROMETER

Laser photo-induced dissociation (PID) is an attractive alternative to collision-induced dissociation (CID) in probing structural features of biomolecules, such as peptides, by mass spectrometry. We report a new experimental setup for PID studies of biomolecules. It involves the use of an ion trap/time-of-flight mass spectrometer for the detection of PID products. The intact molecular ions are produced by electrospray ionization and introduced into a quadrupole ion trap. The ions stored in the trap are then dissociated by using a 266 nm laser beam from a pulsed Nd:yttrium–aluminium–garnet laser. After a short delay, all the fragment ions are extracted out of the trap and mass analyzed by a linear time-of-flight (TOF) mass spectrometer. The applications of this instrument for the PID studies of several peptides are demonstrated. The PID spectra are compared to those obtained by CID in the same instrument. It is shown that the amount of photoenergy deposited for fragmentation can be controlled by laser abso...

INVESTIGATION OF THE QUANTITATIVE CAPABILITIES OF AN ELECTROSPRAY IONIZATION ION TRAP/LINEAR TIME-OF-FLIGHT MASS SPECTROMETER

The quantitative analysis of pharmaceutical or environmental compounds represents an important application area of electrospray ionization (ESI) mass spectrometry (MS). Typically, these analyses are carried out using quadrupole mass analyzers. Recently, several other mass spectrometric configurations have been developed for ESI, including time-of-flight (TOF), which offers high sensitivity and fast data acquisition capabilities. This work investigates the capabilities and limitations of using ESI ion trap/TOFMS for quantitation. The analysis of sulfonamides, an important class of compounds used in the food industry, was selected for this study. It is demonstrated that a calibration curve exhibits a linear response for almost 3 orders of magnitude. The lower limit of the linear range is restricted by the chemical background noise while the upper limit is governed by the ESI source. The short term and long term reproducibility of the instrument are both demonstrated to be better than 8% relative standard deviation. The detection limits and linear dynamic range of this instrument are comparable with those obtained using a single quadrupole mass spectrometer for a single compound and superior for multicomponent analyses. For the analysis of five components, the detection limits using ESI ion trap/TOFMS are about three times lower. It is argued that improving the detection limits, through the use of tandem mass spectrometry, should further extend the linear dynamic range of the instrument for quantitation.

Improved gas chromatography methods for micro-volume analysis of haloacetic acids in water and biological matrices

A fast headspace solid-phase microextraction gas chromatography method for micro-volume (0.1 mL) samples was optimized for the analysis of haloacetic acids (HAAs) in aqueous and biological samples. It includes liquid-liquid microextraction (LLME), derivatization of the acids to their methyl esters using sulfuric acid and methanol after evaporation, followed by headspace solid-phase microextraction with gas chromatography and electron capture detection (SPME-GC-ECD). The derivatization procedure was optimized to achieve maximum sensitivity using the following conditions: esterification for 20 min at 80 degrees C in 10 microL methanol, 10 microL sulfuric acid and 0.1 g anhydrous sodium sulfate. Multi-point standard addition method was used to determine the effect of the sample matrix by comparing with internal standard method. It was shown that the effect of the matrix for urine and blood samples in this method is insignificant. The method detection limits are in the range of 1 microg L(-1) for most of the HAAs, except for monobromoacetic acid (MBAA) (3 microg L(-1)) and for monochloroacetic acid (MCAA) (16 microg L(-1)). The optimized procedure was applied to the analysis of HAAs in water, urine and blood samples. All nine HAAs can be separated in < 13 min for biological samples and < 7 min for drinking water samples, with total sample preparation and analysis time < 50 min. Analytical uncertainty can increase dramatically as the sample volume decreases; however, similar precision was observed with our method using 0.1 mL samples as with a standard method using 40 mL samples.

Nontarget analysis of urine by electrospray ionization-high field asymmetric waveform ion mobility-tandem mass spectrometry.

Nearly a decade after first commercialization, high field asymmetric waveform ion mobility spectrometry (FAIMS) has yet to find its place in routine chemical analysis. Prototypes have been used to demonstrate the utility of this separation technique combined with mass spectrometry (MS). Unfortunately, first generation commercial FAIMS instruments have gone practically unused by early adopters. Here, we show this to be due to poor ion transmission in the FAIMS-MS source interface. We present simple instrumental modifications and optimization of experimental conditions to achieve good performance from the first generation commercial FAIMS device (the Ionalytics Selectra) coupled to a high resolution Q-TOF-MS. In combination with nanospray ionization, we demonstrate for the first time the nontarget analysis of urine by FAIMS with minimal sample preparation. We show the unique suitability of electrospray ionization (ESI)-FAIMS-MS for identification of low abundance species such as urinary biomarkers of damage of nucleic acids in a complex biological matrix. The elimination of electrospray noise and matrix components by FAIMS and the continuous flow of analytes through FAIMS for accurate and tandem mass analysis produce high quality spectral data suitable for structural identification of unknowns. These characteristics make ESI-FAIMS-MS ideal for nontarget identification, even when compared to high efficiency LC-ESI-MS.

Linear and nonlinear regimes of electrospray signal response in analysis of urine by electrospray ionization-high field asymmetric waveform ion mobility spectrometry-MS and implications for nontarget quantification.

Quantitative nontarget analysis is intended to provide a measurement of concentration of newly identified components in complex biological or environmental samples for which authentic or labeled standard do not exist. Electrospray ionization-high field asymmetric waveform ion mobility spectrometry-mass spectrometry (ESI-FAIMS-MS) has unique advantages that allowed us to develop a novel approach for quantification of nontarget analytes. In the nontarget analysis of urinary metabolites by ESI-FAIMS-MS, we find it practical and beneficial to analyze highly diluted urine samples. We show that urine extracts can be analyzed directly at very high dilutions (up to 20,000 times) by extending MS analysis times during slow FAIMS scanning. We explore the effects of sample dilution on ionization efficiency and ionization suppression in direct electrospray of complex sample matrixes. We consistently observe two distinct regimes in ESI operation related to the limited ionization capacity of this method. In the linear dynamic concentration range below the limiting ionization capacity, the analytical sensitivity of an analyte is constant and does not depend on matrix composition and concentration. Once the capacity of ESI is exceeded, all species exhibit log-log linearity in signal response. We show how quantification can be carried out using two different approaches, one for analytes which can be detected in the linear regime and another for those only detected in the suppression regime that overcomes the effects of ionization suppression. Our new insight into ionization suppression effects in ESI is of broad interest to anyone using ESI as an ionization technique for the MS analysis of complex samples.

The effect of using silicon based diffusion pump fluid on spectral quality in an electrospray ionization ion trap/time-of-flight mass spectrometer

The formation of adducts resulting from interactions between protein ions and residual gases in an electrospray ionization ion trap/time-of-flight mass spectrometer is reported. This instrument utilizes diffusion pumps to achieve high vacuum, and we found that when silicon based diffusion pump fluids were used, characteristic adduct peaks appeared in the mass spectra. These adduct peaks were not detected, however, when a polyphenyl ether (Santovac 5) was used as the diffusion pump fluid. In switching the diffusion pump fluid to Santovac 5, a rigorous cleaning procedure capable of removing residual silicon pump fluid from the instrument was a critical step. This cleaning procedure can also be used for routine cleaning of the ion trap and other source components.

Dissociation of protonated phenylthiohydantoin‐amino acids and phenylthiocarbamoyl‐dipeptides

The N-terminal phenylthiocarbamoyl (PTC) derivatives of peptides and the phenylthiohydantoin (PTH) derivatives of amino acids are the two major types of products generated in the Edman protein sequencing method. Understanding the fragmentation pathways of these species should facilitate structural elucidation and chemical identification based on the fragment ion mass spectra, particularly when mass spectrometry is combined with the Edman sequencer for the analysis of non-standard and modified amino acids. In this study, dissociation of the protonated PTH-X (where X=Thr, Ser, Trp and Tyr), PTH-Gly, PTC-X-Leu and PTC-Gly-Leu in electrospray ionization mass spectrometry was examined to investigate whether there is any isomerization of PTC to PTH derivatives in the gas phase during the fragmentation. It is shown that dissociation of the protonated PTH-X proceeds via hydrogen transfer from the side-chain of the amino acid to the PTH moiety with the elimination of the side-chain as a neutral species. The ions at m/z 193 formed from the source fragmentation of the protonated PTH-X are found to have the same structure and fragmentation pathways. The presence of this m/z 193 ion and its collisionally induced dissociation (CID) spectrum are unique for the PTH derivatives and they can be used to detect the presence of the PTH ions. It is shown that there is no isomerization of the thiazolone ions to the PTH ions during the dissociation of PTC-X-Leu (in this case, the b1 ions from PTC-X-Leu are believed to have the protonated thiazolone structure). In addition, comparative studies of CID spectra of PTH-X and PTH-Gly or PTC-X-Leu and PTC-Gly-Leu are presented. The proposed fragmentation mechanisms for the protonated PTH and PTC derivatives and the m/z 193 ions are given.

Characterization of an electrospray ionization ion trap/linear time-of-flight mass spectrometer for phenylthiohydantoin-amino acid analysis

Abstract An electrospray ionization ion trap/time-of-flight mass spectrometer was evaluated as a detector for identification of phenylthiohydantoin (PTH)-amino acids, the final products in the Edman sequencing process of peptides and proteins. Each of 22 PTH-amino acids studied can be characterized by well-defined mass spectral patterns. Fragmentation from source excitation followed by a long trapping period (250 ms) gave rise to unique and intense fragmentation products. This allowed for the differentiation of isomers of PTH-leucine/PTH-isoleucine and PTH-α–aminobutyric acid/PTH-α–aminoisobutyric acid. The interpretation of the fragmentation patterns is presented. The detection limits and relative detection sensitivity of 20 standard PTH-amino acids were determined. It is shown that all these compounds can be detected at the 100 fmol level.