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Stabilities and structure(s) of proteins are directly coupled to their local environment or Gibbs free energy landscape as defined by solvent, temperature, pressure, and concentration. Solution pH, ionic strength, cofactors, chemical chaperones, and osmolytes perturb the chemical potential and induce further changes in structure, stability, and function. At present, no single analytical technique can monitor these effects in a single measurement. Mass spectrometry and ion mobility-mass spectrometry play increasingly essential roles in studies of proteins, protein complexes, and even membrane protein complexes; however, with few exceptions, the effects of the solution temperature on the stability and structure(s) of analytes have not been thoroughly investigated.Here, we describe a new variable-temperature electrospray ionization (vT-ESI) source that utilizes a thermoelectric chip to cool and heat the solution contained within the static ESI emitter. This design allows for solution temperatures to be varied from ∼5 to 98 °C with short equilibration times (<2 min) between precisely controlled temperature changes. The performance of the apparatus for vT-ESI-mass spectrometry and vT-ESI-ion mobility-mass spectrometry studies of cold-and heat-folding reactions is demonstrated using ubiquitin and frataxin. Instrument performance for studies on temperature-dependent ligand binding is shown using the chaperonin GroEL.

The use of charge-reducing reagents to generate lower-charge ions has gained popularity in the field of native mass spectrometry (MS) and ion mobility mass spectrometry (IM-MS). This is because the lower number of charged sites decreases the propensity for Coulombic repulsions and unfolding/restructuring, helping to preserve the native-like structure. Furthermore, lowering the charge state consequently increases the mass-to-charge values (m/z), effectively increasing spacing between signals originating from small mass differences, such as different proteoforms or protein–drug complexes. IM-MS yields collision cross section (CCS, Ω) values that provide information about the three-dimensional structure of the ion. Traveling wave IM (TWIM) is an established and expanding technique within the native MS field. TWIM measurements require CCS calibration, which is achieved via the use of standard species of known CCS. Current databases for native-like proteins and protein complexes provide CCS values obtained using normal (i.e., non-charge-reducing) conditions. Herein, we explored the validity of using “normal” charge calibrants to calibrate for charge-reduced proteins and show cases where it is not appropriate. Using a custom linear field drift cell that enables the determination of ion mobilities from “first principles”, we directly determined CCS values for 19 protein calibrant species under three solution conditions (yielding a broad range of charge states) and two drift gases. This has established a database of CCS and reduced-mobility (K 0) values, along with their associated uncertainties, for proteins and protein complexes over a large m/z range. TWIM validation of this database shows improved accuracy over existing methods in calibrating CCS values for charge-reduced proteins.

SUMMARY Jejunostomy losses of Na+ and water during enteral nutrition after massive intestinal resection may be severe. We have attempted to analyse this practical problem by using an in vivo perfusion technique in healthy volunteers to study Na+, water and nutrient absorption from a short (25 cm) segment of jejunum during perfusion of an isotonic solution of the elemental diet Vivonex. Further solutions made from the amino acid and carbohydrate components of Vivonex were also perfused in part I of the study in order to determine the causes of the marked Na+ and water secretion seen during Vivonex perfusion. Low initial Na concentration was found to be the major determinant of net Na+ secretion, initial Na+ concentration correlating significantly with Na+ absorption (r=095, n=7 p<0001). Water absorption correlated with net absorption of NaCl (r=0*82, n=7 p<0.01). There was, however, a better correlation with total absorption of NaCl plus amino acids (r=0.99, n=7, p<001). In part II of the study separate isotonic solutions of NaCl, glucose, and the polymeric diet, Ensure were also studied. Net sodium secretion occurred during glucose and Ensure perfusion, as predicted from their low Na+ concentration. Owing to rapid sucrose absorption from Ensure there was substantial luminal disappearance of osmotically active particles and hence marked water absorption, which was accurately predicted using the regression equation for water absorption derived in part I, substituting sucrose absorption for amino acid absorption. We conclude that the marked Na+ and water secretion observed during Vivonex perfusion is not a unique property of this amino acid based diet but is due to its low Na+ content.

Ultraviolet photodissociation (UVPD) has emerged as a useful technique for characterizing peptide, protein, and protein complex primary and secondary structure. We present here the modification of a commercial ion mobility (IM) mass spectrometer to incorporate 193 nm UVPD following IM separation, enabling IM-UVPD to fragment species selected by both their m/z (via quadrupole selection) and conformation (mobility-selection). File list (2) download file view on ChemRxiv UVPD_manuscript_submit.pdf (648.12 KiB) download file view on ChemRxiv UVPD_SI.pdf (1.22 MiB)