No abstract

Ion mobility and mass spectrometry techniques are coupled with a temperature-controlled electrospray ionization source to follow the structural transitions of ubiquitin in aqueous solution (pH = 3) at elevated solution temperatures (T = 26-96 °C). Changes in the charge state distribution are consistent with a two-state, cooperative unfolding transition having a melting temperature of T = 71 ± 2 °C, in agreement with prior measurements [ Wintrode , P. L. ; Makhatadze , G. I. ; Privalov , P. L. Proteins , 1994 , 18 , 246 - 253 ]. However, analysis of ion mobility distributions reveals the two-state transition is a composite of transitions involving at least nine unique species: three native or native-like structures; two that appear to be equilibrium intermediates (i.e., populations of new conformers that form at elevated temperatures but subsequently disappear at higher temperatures); and four products observed at high temperatures, including the well-characterized ubiquitin A state, and two solution species that are differentiated based on a cis- or trans-configured Glu-Pro peptide bond. These nine states vary in abundances by factors as large as ∼10 over the range of solution temperatures. Although experimental melting transitions are conceived as a loss of well-defined structure leading to a random distribution of unstructured, denatured forms, the results provide evidence for new conformers having at least some well-defined structural elements are stabilized as temperature is increased.

Matrix assisted ionization vacuum (MAIV) rapidly generates gas-phase analyte ions from subliming solid-phase matrix:analyte crystals for analysis by mass spectrometry (MS). Ionization from the solid-phase allows the use of a variety of surfaces for introducing matrix:analyte samples to the vacuum of a mass spectrometer, including common laboratory materials, such as disposable pipet tips, filter paper, tooth picks, and nylon mesh. MAIV is shown here to be capable of analyses as fast as 3 s per sample with achievable sensitivities in the low femtomole range. MAIV-MS coupled with ion mobility spectrometry (IMS)-MS and tandem mass spectrometry (MS/MS) is shown to be especially powerful for analysis and characterization of a wide range of molecules ranging from small molecules such as drugs and metabolites (∼300 Da) to intact proteins (25.6 kDa). Automated sample introduction is demonstrated on two different commercial mass spectrometers using a programmable XYZ stage. A MAIV high-throughput nontargeted MS(E) approach is also demonstrated utilizing IMS for rapid characterization of small molecules and peptides from standard solutions, as well as drug spiked human urine.

Matrix-assisted ionization (MAI) mass spectrometry (MS) is a simple and sensitive method for analysis of low- and high-mass compounds, requiring only that the analyte in a suitable matrix be exposed to the inlet aperture of an atmospheric pressure ionization mass spectrometer. Here, we evaluate the reproducibility of MAI and its potential for quantification using six drug standards. Factors influencing reproducibility include the matrix compound used, temperature, and the method of sample introduction. The relative standard deviation (RSD) using MAI for a mixture of morphine, codeine, oxymorphone, oxycodone, clozapine, and buspirone and their deuterated internal standards using the matrix 3-nitrobenzonitrile is less than 10% with either a Waters SYNAPT G2 or a Thermo LTQ Velos mass spectrometer. The RSD values obtained using MAI are comparable to those using ESI or MALDI on these instruments. The day-to-day reproducibility of MAI determined for five consecutive days with internal standards was better than 20% using manual sample introduction. The reproducibility improved to better than 5% using a mechanically assisted sample introduction method. Hydrocodone, present in a sample of undiluted infant urine, was quantified with MAI using the standard addition method.

Ion mobility and mass spectrometry techniques are used to investigate the stabilities of different conformations of bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg). At elevated solution temperatures, we observe a slow protonation reaction, i.e., [BK+2H]+H → [BK+3H], that is regulated by trans → cis isomerization of Arg-Pro, resulting in the Arg- cis-Pro- cis-Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg (all- cis) configuration. Once formed, the all- cis [BK+3H] spontaneously cleaves the bond between Pro-Pro with perfect specificity, a bond that is biologically resistant to cleavage by any human enzyme. Temperature-dependent kinetics studies reveal details about the intrinsic peptide processing mechanism. We propose that nonenzymatic cleavage at Pro-Pro occurs through multiple intermediates and is regulated by trans → cis isomerization of Arg-Pro. From this mechanism, we can extract transition state thermochemistry: Δ G = 94.8 ± 0.2 kJ·mol, Δ H = 79.8 ± 0.2 kJ·mol, and Δ S = -50.4 ± 1.7 J·mol·K for the trans → cis protonation event; and, Δ G = 94.1 ± 9.2 kJ·mol, Δ H = 107.3 ± 9.2 kJ·mol, and Δ S = 44.4 ± 5.1 J·mol·K for bond cleavage. Biological resistance to the most favored intrinsic processing pathway prevents formation of Pro-Gly-Phe-Ser- cis-Pro-Phe-Arg that is approximately an order of magnitude more antigenic than BK.