Tandem Trapped Ion Mobility Spectrometry/Mass Spectrometry (tTIMS/MS) Reveals Sequence-Specific Determinants of Top-Down Protein Fragment Ion Cross Sections.

Top-down proteomics provides a straightforward approach to the level of proteoforms but remains technologically challenging. Using ion mobility spectrometry/mass spectrometry (IMS/MS) to separate top-down fragment ions improves signal/noise and dynamic range. Such applications, however, do not yet leverage the primary information obtained from IMS/MS, which is the characterization of the fragment ion structure by the measured momentum transfer cross sections. Here, we perform top-down analysis of intact proteins and assemblies using our tandem trapped ion mobility spectrometer/mass spectrometer (tTIMS/MS) and compile over 1400 cross section values of fragment ions. Our analysis reveals that most fragment ions exhibit multiple, stable conformations similar to those of intact polypeptides and proteins. The data further indicate that the conformational heterogeneity is strongly influenced by the amino acid sequences of the fragment ions. Moreover, time-resolved tTIMS/MS experiments reveal that conformations of top-down fragment ions can be metastable on the timescale of ion mobility measurements. Taken together, our analysis indicates that top-down fragment ions undergo a folding process in the gas phase and that this folding process can lead to kinetic trapping of intermediate states in ion mobility measurements. Hence, because the folding free energy surface of a polypeptide ion is encoded by its amino acid sequence and charge state, our analysis suggests that cross sections can be exploited as sequence-specific determinants of top-down fragment ions.

Structure Relaxation Approximation (SRA) for Elucidation of Protein Structures from Ion Mobility Measurements (II). Protein Complexes.

Characterizing structures of protein complexes and their disease-related aberrations is essential to understanding molecular mechanisms of many biological processes. Electrospray ionization coupled with hybrid ion mobility/mass spectrometry (ESI-IM/MS) methods offer sufficient sensitivity, sample throughput, and dynamic range to enable systematic structural characterization of proteomes. However, because ESI-IM/MS characterizes ionized protein systems in the gas phase, it generally remains unclear to what extent the protein ions characterized by IM/MS have retained their solution structures. Here, we discuss the first application of our computational structure relaxation approximation [Bleiholder, C.; et al. J. Phys. Chem. B 2019, 123 (13), 2756-2769] to assign structures of protein complexes in the range from ∼16 to ∼60 kDa from their "native" IM/MS spectra. Our analysis shows that the computed IM/MS spectra agree with the experimental spectra within the errors of the methods. The structure relaxation approximation (SRA) indicates that native backbone contacts appear largely retained in the absence of solvent for the investigated protein complexes and charge states. Native contacts between polypeptide chains of the protein complex appear to be retained to a comparable extent as contacts within a folded polypeptide chain. Our computations also indicate that the hallmark "compaction" often observed for protein systems in native IM/MS measurements appears to be a poor indicator of the extent to which native residue-residue interactions are lost in the absence of solvent. Further, the SRA indicates that structural reorganization of the protein systems in IM/MS measurements appears driven largely by remodeling of the protein surface that increases its hydrophobic content by approximately 10%. For the systems studied here, this remodeling of the protein surface appears to occur mainly by structural reorganization of surface-associated hydrophilic amino acid residues not associated with ß-strand secondary structure elements. Properties related to the internal protein structure, as assessed by void volume or packing density, appear unaffected by remodeling of the surface. Taken together, the structural reorganization of the protein surface appears to be generic in nature and to sufficiently stabilize protein structures to render them metastable on the time scale of IM/MS measurements.

Proteomic characterization of medicinal plants used in the treatment of diabetes.

Several plants have been studied for their medicinal properties, especially concerning the management of chronic diseases, such as diabetes, aiming at a more accessible form of treatment. In this context, the aim of this study was to characterize plant proteins used in folk medicine as hypoglycemic agents for the treatment of diabetes, namely "abajerú" (Chrysobalanus icaco) and "cow's paw" (Bauhinia forficata and Bauhinia variegata). The species were differentiated by proteome characterization. Proteins were in-solution digested using trypsin by the filter-assisted sample preparation (FASP) method. Peptides were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for protein characterization. In total, 131 proteins were identified. The main biological functions of these proteins were cellular respiration, transport, metabolism and photosynthesis. Insulin-like proteins were not detected, but proteins involved in controlling glucose levels were. The results are of value in the proteomic characterization of phytotherapeutic plants, and may serve as baseline for the assessed species in Brazil, where a lack of knowledge in this regard is observed.

Formation and identification of PAHs metabolites in marine organisms.

In this study, the development of the technique APCI(+) LC/MS/MS allowed the detection of phenanthrene, pyrene and metabolites of alkyl homologs in fish bile (in situ) and in urine of crabs. Laboratory experiments were carried out exposing crabs from an unpolluted mangrove (Barra de Guaratiba) to phenanthrene, and to the alkylated homologs 1-methyl phenanthrene and 2,6,9-trimethyl phenanthrene. Urine samples were collected at 0, 24, 48, 72, and 96 h. Fishes were captured from strategic sites from Guanabara Bay. Hydroxylated metabolites of phenanthrene, epoxides, orthoquinone and glucoside conjugates were identified in both samples. The method APCI(+) LC/MS/MS showed to be effective in a preliminary assessment of phenanthrene metabolite formation, although the low concentrations of 1-methyl phenanthene and 2,6,9-trimethyl phenanthrene did not allow a systematic evaluation of data. The method, however, proved to be excellent tool for studies of PAHs metabolites due to the high selectivity, sensitivity and separation attained.

Towards a personalized risk assessment for exposure of humans to toxic substances / Em direção a uma avaliação de risco personalizado para exposição de seres humanos a substâncias tóxicas

Abstract Great response variability caused by genetic and/or environmental factors has been observed among organisms exposed to hazardous chemicals. This subject has been a topic of intense discussion in the USA since President Obama announced support for an “era of precision medicine”, which consists in the inclusion of genetic data of patients in the treatment design, imposing a new approach to risk assessment. Personalized evaluation must consider the phenotypic factors of an individual. Among the markers that have been developed to evaluate any alteration in the structure or function of organisms, biomarkers of susceptibility are of great importance because they indicate the natural characteristics of a given organism which make it more sensitive to a specific adverse effect or disease, or more responsive to exposure to a specific chemical/drug. The ‘-omics’ technologies provide an insight into the relationship between chemical effects and molecular mechanisms of action. These technologies are the pillars for a personalized toxicology and precision medicine. Predictive toxicology requires a more comprehensive knowledge on specific individual factors or susceptibilities predisposing to diseases, enabling personalized risk assessment and adequate medical treatment.

Resumo Há uma grande variabilidade nas respostas observadas entre os organismos expostos a uma substância química perigosa. Essa variabilidade é causada por causas genéticas e / ou ambientais. Esse assunto tem sido intensamente discutido, mesmo nos Estados Unidos, desde que o presidente Obama anunciou o apoio a uma “era da medicina de precisão”, a qual consiste na inclusão de dados genéticos do paciente no projeto do tratamento, impondo uma nova abordagem para avaliação de risco. A avaliação personalizada deve considerar fatores fenotípicos de um indivíduo. Entre os biomarcadores que foram desenvolvidos para avaliar qualquer alteração da estrutura ou função do organismo, os biomarcadores de susceptibilidade têm uma grande importância, uma vez que indicam as características naturais de um dado organismo, que o tornam mais sensíveis a um efeito ou doença adversa específica ou em resposta a uma determinada exposição. As tecnologias “ômicas” permitem a compreensão da relação entre os efeitos químicos e dos mecanismos moleculares de ação. Essas tecnologias “ômicas” são os pilares para a toxicologia personalizada e para a medicina de precisão. Toxicologia preditiva exige uma melhor compreensão dos fatores ou susceptibilidades individuais específicas predisponentes a doenças, permitindo uma avaliação de riscos personalizada e um tratamento médico adequado.