The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real "snap-shot" of the situation in solution can be obtained. (J Am Soc Mass Spectrom 2010, 21, 286 -289) © 2010 American Society for Mass Spectrometry E lectrospray is an exceptionally soft ionization technique, which has allowed the observation of numerous noncovalent complexes, including protein-protein, protein-small molecule, and protein-DNA complexes [1,2]. A major question is still whether the information obtained from gas-phase results is representative of the species present in solution. The term "noncovalent bonding" in biochemistry generally summarizes three types of intermolecular forces: electrostatic interactions (e.g., salt bridges), dipolar interactions (e.g., hydrogen bonds), and van der Waals interactions (e.g., hydrophobic interactions) [3]. In addition, protein-water interactions often play a major role in complex stabilization. In solution, hydrophobic interactions are very weak compared with the hydrogen bonds between water molecules. Hence, it is not the hydrophobic forces that are the actual reason for the interaction of nonpolar binding partners, but rather the surrounding water molecules that "force" hydrophobic binding partners to aggregate. This solvent-driven process is called the hydrophobic effect, and plays a major role in protein folding, adhesion of lipid bilayers, partitioning effects of drugs and metabolites, and many other aggregation phenomena in chemistry and biology [4][5][6]. The hydrophobic effect is defined by the thermodynamics of mixing hydrophobic compounds with water, and is dominated by large, entropic factors [4,7]. Obviously, in the absence of a hydr...

The Sortase A (SrtA) enzyme from Staphylococcus aureus catalyses covalent attachment of protein substrates to pentaglycine cross-bridges in the Gram positive bacterial cell wall. In vitro SrtA-mediated protein ligation is now an important protein engineering tool for conjugation of substrates containing the LPXTGX peptide recognition sequence to oligo-glycine nucleophiles. In order to explore the use of alternative nucleophiles in this system, five different rhodamine-labelled compounds, with N-terminal nucleophilic amino acids, triglycine, glycine, and lysine, or N-terminal non-amino acid nucleophiles ethylenediamine and cadaverine, were synthesized. These compounds were tested for their relative abilities to function as nucleophiles in SrtA-mediated bioconjugation reactions. N-Terminal triglycine, glycine and ethylenediamine were all efficient in labelling a range of LPETGG containing recombinant antibody and scaffold proteins and peptides, while reduced activity was observed for the other nucleophiles across the range of proteins and peptides studied. Expansion of the range of available nucleophiles which can be utilised in SrtA-mediated bioconjugation expands the range of potential applications for this technology. As a demonstration of the utility of this system, SrtA coupling was used to conjugate the triglycine rhodamine-labelled nucleophile to the C-terminus of an Im7 scaffold protein displaying Aβ, a neurologically important peptide implicated in Alzheimer's disease. Purified, labelled protein showed Aβ-specific targeting to mammalian neuronal cells. Demonstration of targeting neuronal cells with a chimeric protein illustrates the power of this system, and suggests that SrtA-mediated direct cell-surface labelling and visualisation is an achievable goal.

In the wake of recent popular interest in physical fitness (Smith, 1983), there has been an increase in exerciserelated injuries. Although the vast majority are minor musculo-skeletal problems, care should be taken not to miss the more severe injury presenting with non-specific symptoms. The following report describes a stress fracture of the femoral neck occurring in a young marathon runner. CASE REPORTA 36-year-old male University Lecturer presented with severe right thigh pain, present since attempting a marathon 5 days previously. The patient began training one year prior to the race and gradually increased his activities to 50 miles per week; mostly on hard road surfaces and wearing purpose built shoes. Two weeks before the race he developed exercise-related pain localised to the medial aspect of the right thigh. He restricted his training, in the week prior to the race, to a 60-mile cycle ride and brisk walks. During the marathon attempt the initial 5 miles were accompanied by only minimal pain; but over the next 3 miles the pain increased to such a level that he was forced to retire. Medical advice was sought but no X-rays were taken and the injury was not diagnosed. After a further 5 days, medical advice was again sought and at this time examination revealed tenderness in the right groin and pain at the extremes of hip movement, passive flexion was accompanied by an audible click.

A deterioration in pulmonary function is a common problem in patients with cystic fibrosis. One strategy which might help to arrest this process is the administration of aminoglycosides by nebulizer in dosages which greatly exceed those which are normally given parenterally. Ten patients (4-19 years of age) with cystic fibrosis underwent evoked response audiometry at regular intervals following a 400 mg dose of nebulized tobramycin; the concentrations of tobramycin in samples of blood taken at the same time intervals were also determined. No significant abnormalities in the audiometric investigations were detected. The relationship between shifts in vestibulocochlear potentials and long-term ototoxic sequelae in patients receiving aminoglycosides via a nebulizer requires further evaluation.