Determination of testosterone and epitestosterone glucuronides in urine by ultra performance liquid chromatography-ion mobility-mass spectrometry.

UPLC-ion mobility spectrometry separations combined with mass spectrometry (UPLC-IM-MS) and tandem mass spectrometry (UPLC-IM-MS/MS) have been investigated for the simultaneous determination of testosterone and epitestosterone glucuronides in urine. The glucuronide epimers of testosterone and epitestosterone were separated by ion mobility spectrometry prior to mass analysis on the basis of differences in their collision cross sections, which have been measured in nitrogen. Combining ion mobility separation with UPLC/MS enhances the analysis of these low-abundance steroids in urine by selective interrogation of specific retention time, mass-to-charge and mobility regions. Detection limits for the UPLC-IM-MS/MS analysis of TG and ETG were 9.9 ng mL(-1) and 98 ng mL(-1) respectively, equivalent to 0.7 ng mL(-1) and 7.4 ng mL(-1) in urine, with linear dynamic ranges corresponding to 0.7-108 ng mL(-1) and 7.4-147 ng mL(-1) in urine. Repeatability (%RSD) for urine extracts was 0.64% and 2.31% for TG and ETG respectively.

Determination of Protein Haptenation by Chemical Sensitizers Within the Complexity of the Human Skin Proteome.

Skin sensitization associated with the development of allergic contact dermatitis occurs via a number of specific key events at the cellular level. The molecular initiating event (MIE), the first in the sequence of these events, occurs after exposure of the skin to an electrophilic chemical, causing the irreversible haptenation of proteins within skin. Characterization of this MIE is a key step in elucidating the skin sensitization adverse outcome pathway and is essential to providing parameters for mathematical models to predict the capacity of a chemical to cause sensitization. As a first step to addressing this challenge, we have exposed complex protein lysates from a keratinocyte cell line and human skin tissue with a range of well characterized sensitizers, including dinitrochlorobenzene, 5-chloro-2-methylisothiazol-3-one, cinnamaldehyde, and the non (or weak) sensitizer 6-methyl coumarin. Using a novel stable isotope labeling approach combined with ion mobility-assisted data independent mass spectrometry (HDMSE), we have characterized the haptenome for these sensitizers. Although a significant proportion of highly abundant proteins were haptenated, we also observed the haptenation of low abundant proteins by all 3 of the chemical sensitizers tested, indicating that within a complex protein background, protein abundance is not the sole determinant driving haptenation, highlighting a relationship to tertiary protein structure and the amino acid specificity of these chemical sensitizers and sensitizer potency.

On-line capillary column immobilised metal affinity chromatography/electrospray ionisation mass spectrometry for the selective analysis of histidine-containing peptides.

Capillary column immobilised metal affinity chromatography (IMAC) has been combined on-line with electrospray ionisation/quadrupole time-of-flight mass spectrometry for the fractionation of histidine-containing peptides. IMAC beads (Poros 20MC, 20 microm) containing imidodiacetate chelating groups on a cross-linked poly(styrene-divinylbenzene) support were packed into a fused silica column (250 microm i.d.), which was interfaced to the electrospray ion source of the spectrometer. A Cu(II) activated column was used to isolate histidine-containing peptides from tryptic and other peptide mixtures with an average breakthrough of 9.1%, to reduce the complexity of the mass spectral analysis. The analysis cycle time was reduced to less than 15 min, at an optimum flow rate of 7.5 microL/min, without sacrificing peptide selectivity. Direct coupling of capillary IMAC with MS allows on-line separation, using MS compatible loading and elution buffers, and detection in a high-throughput fashion when compared to off-line strategies.

Analysis of tryptic peptides using desorption electrospray ionisation combined with ion mobility spectrometry/mass spectrometry.

A novel method is reported for rapid protein identification by the analysis of tryptic peptides using desorption electrospray ionisation (DESI) coupled with hyphenated ion mobility spectrometry and quadrupole time-of-flight mass spectrometry (IMS/Q-ToF-MS). Confident protein identification is demonstrated for the analysis of tryptically digested bovine serum albumin (BSA), with no sample pre-treatment or clean-up. Electrophoretic ion mobility separation of ions generated by DESI allowed examination of charge-state and mobility distributions for tryptic peptide mixtures. Selective interrogation of singly charged ions allowed isobaric peptide responses to be distinguished, along with a reduction in spectral noise. The mobility-selected singly charged peptide responses were presented as a pseudo-peptide mass fingerprint (p-PMF) for protein database searching. Comparative data are shown for electrospray ionisation (ESI) of the BSA digest, without sample clean-up, from which confident protein identification could not be made. Implications for the robustness of the DESI method, together with potential insights into mechanisms for DESI of proteolytic digests, are discussed.