The proteome of the recently discovered bacterium Methylocella silvestris has been characterized using three profiling and comparative proteomics approaches. The organism has been grown on two different substrates enabling variations in protein expression to be identified. The results obtained using the experimental approaches have been compared with respect to number of proteins identified, confidence in identification, sequence coverage and agreement of regulated proteins. The sample preparation, instrumental time and sample loading requirements of the differing experiments are compared and discussed. A preliminary screen of the protein regulation results for biological significance has also been performed.
Three ionisation techniques that require no sample preparation or extraction prior to mass analysis have been used for the rapid analysis of pharmaceutical tablets and ointments. These methods were (i) the novel direct analysis in real time (DART), (ii) desorption electrospray ionisation (DESI), and (iii) desorption atmospheric pressure chemical ionisation (DAPCI). The performance of the three techniques was investigated for a number of common drugs. Significant differences between these approaches were observed. For compounds of moderate to low polarity DAPCI produced more effective ionisation. Accurate DESI and DAPCI tandem mass spectra were obtained and these greatly enhance the selectivity and information content of the experiment. The detection from human skin of the active ingredients from ointments is reported together with the detection of ibuprofen metabolites in human urine.
Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two ␣-and two ␣ -globin  chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo-and holo-forms of ␣-and ␣ -monomers and  heterodimers; ␣-and ␣ -monomers in both apo-and holo-forms were found to have similar  cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-␣-apo-␣  is not an essential intermediate within the The typical electrospray ionization (ESI) mass spectrum of a protein consists of an envelope of peaks attributed to a series of multiply charged gas-phase ions that can indicate the stability and compactness of its structure in the gas phase [4,5]. Multiply charged ions are produced by proton attachment, predominantly to exposed basic sites on the protein [6], and those of lowest charge are thought to be most representative of native structure [7]. A highly compact protein would have fewer exposed basic sites than those of an unfolded conformation of the same protein and thus would accept fewer charges [4,8].Ion mobility spectrometry is a shape-selective technique, based on the time taken for an ion to traverse a mobility cell containing an inert gas under the influence of a weak electric field [9]. This time is related to the rotationally averaged collision cross section, mass, and charge of the ion. The coupling of ion mobility separation with mass spectrometry has provided a powerful method for the analysis of complex mixtures and for the determination of molecular structure [10].Ion mobility mass spectrometry has emerged as a complementary technique to the well-established methods of X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy for three-dimensional protein structure analysis [11]. There is now substantial evidence to support...
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