RESUMEN
Accurate reconstruction of metabolic pathways is an important prerequisite for interpreting metabolomics changes and understanding the diverse biological processes in disease models. A tracer-based metabolomics strategy utilizes stable isotope-labeled precursors to resolve complex pathways by tracing the labeled atom(s) to downstream metabolites through enzymatic reactions. Isotope enrichment analysis is informative and achieved by counting total labeled atoms and acquiring the mass isotopologue distribution (MID) of the intact metabolite. However, quantitative analysis of labeled metabolite substructures/moieties (MS2 fragments) can offer more valuable insights into the reaction connections through measuring metabolite transformation. In order to acquire the isotopic labeling information at the intact metabolite and moiety level simultaneously, we developed a method that couples hydrophilic interaction liquid chromatography (HILIC) with Zeno trap-enabled high-resolution multiple reaction monitoring (MRMHR). The method enabled accurate and reproducible MID quantification for intact metabolites as well as their fragmented moieties, with notably high sensitivity in the MS2 fragmentation mode based on the measurement of 13C- or 15N-labeled cellular samples. The method was applied to human-induced pluripotent stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID analysis of both intact metabolites and fragmented moieties, we validated the pathway reconstruction of de novo glutathione synthesis in mid-brain neurons. We discovered increased glutathione oxidization from both basal and newly synthesized glutathione pools under neuronal oxidative stress. Furthermore, the significantly decreased de novo glutathione synthesis was investigated and associated with altered activities of several key enzymes, as evidenced by suppressed glutamate supply via glucose metabolism and a diminished flux of glutathione synthetic reaction in the neuronal model of rotenone-induced neurodegeneration.
Asunto(s)
Metabolómica , Rotenona , Humanos , Isótopos de Carbono/química , Cromatografía Liquida/métodos , Metabolómica/métodos , Neuronas/metabolismo , Interacciones Hidrofóbicas e Hidrofílicas , Marcaje Isotópico/métodosRESUMEN
Time-of-flight MS systems for biopharmaceutical and protein characterization applications may play an even more pivotal role in the future as biotherapeutics increase in drug pipelines and as top-down MS approaches increase in use. Here, a recently developed TOF MS system is examined for monoclonal antibody (mAb) characterization from serum samples. After immunocapture, purified drug material spiked into monkey serum or dosed for an in-life study is analyzed by top-down MS. While characterization aspects are a distinct advantage of the MS platform, MS system and software capabilities are also shown regarding intact protein quantitation. Such applications are demonstrated to help enable comprehensive protein molecule quantitation and characterization by use of TOF MS instrumentation.
Asunto(s)
Anticuerpos Monoclonales , Espectrometría de Masas en Tándem , Electrones , Programas InformáticosRESUMEN
Protein post-translational modifications (PTMs) are crucial and dynamic players in a large variety of cellular processes and signaling. Proteomic technologies have emerged as the method of choice to profile PTMs. However, these analyses remain challenging due to potential low PTM stoichiometry, the presence of multiple PTMs per proteolytic peptide, PTM site localization of isobaric peptides, and neutral losses. Collision-induced dissociation (CID) is commonly used to characterize PTMs, but the application of collision energy can lead to neutral losses and incomplete peptide sequencing for labile PTM groups. In this study, we assessed the performance of an alternative fragmentation, electron activated dissociation (EAD), to characterize, site localize, and quantify peptides with labile modifications in comparison to CID, both operated on a recently introduced fast-scanning quadrupole-time-of-flight (QqTOF) mass spectrometer. We analyzed biologically relevant phosphorylated, succinylated, malonylated, and acetylated synthetic peptides using targeted parallel reaction monitoring (PRM or MRMHR) assays. We report that electron-based fragmentation preserves the malonyl group from neutral losses. The novel tunable EAD kinetic energy maintained labile modification integrity and provided better peptide sequence coverage with strong PTM-site localization fragment ions. Activation of a novel trap-and-release technology significantly improves the duty cycle and provided significant MS/MS sensitivity gains by an average of 6-11-fold for EAD analyses. Evaluation of the quantitative EAD PRM workflows revealed high reproducibility with coefficients of variation of â¼2-7%, as well as very good linearity and quantification accuracy. This novel workflow combining EAD and trap-and-release technology provides high sensitivity, alternative fragmentation information to achieve confident PTM characterization and quantification.
Asunto(s)
Electrones , Espectrometría de Masas en Tándem , Reproducibilidad de los Resultados , Proteómica/métodos , Proteínas/química , Procesamiento Proteico-Postraduccional , Péptidos/químicaRESUMEN
We report on the dissociation of singly protonated peptides by electrons using electron-activated dissociation (EAD), which comprises electron impact excitation of ions from organics (EIEIO), electronic-excitation dissociation (EED), and electron ionization dissociation (EIoD). Various singly protonated peptides were dissociated using a recently reported fast EAD device. The dissociation can be induced through two pathways: (i) vibrational dissociation similar to collision-activated dissociation (CAD, or collision-induced dissociation, CID) by relaxation from a molecular electronic excited state to high vibrational states; and (ii) radical-induced dissociation where molecular electronic excitation is followed by homolytic cleavage. EAD is complementary to CAD as additional molecular information can be obtained; e.g., fragile PTM moieties, such as glycosylation and sulfation, can be localized. Simultaneously, the energetic production of radical z⢠fragments enables Leu and Ile discrimination, like in a hot ECD process. Using the fast EAD device, LC-EIEIO-time-of-flight mass spectrometry was applied to a tryptic monoclonal antibody digest containing short singly protonated peptides.
Asunto(s)
Electrones , Péptidos , Iones/química , Espectrometría de Masas/métodos , Péptidos/química , Procesamiento Proteico-PostraduccionalRESUMEN
The possibility to record proteomes in high throughput and at high quality has opened new avenues for biomedical research, drug discovery, systems biology, and clinical translation. However, high-throughput proteomic experiments often require high sample amounts and can be less sensitive compared to conventional proteomic experiments. Here, we introduce and benchmark Zeno SWATH MS, a data-independent acquisition technique that employs a linear ion trap pulsing (Zeno trap pulsing) to increase the sensitivity in high-throughput proteomic experiments. We demonstrate that when combined with fast micro- or analytical flow-rate chromatography, Zeno SWATH MS increases protein identification with low sample amounts. For instance, using 20 min micro-flow-rate chromatography, Zeno SWATH MS identified more than 5000 proteins consistently, and with a coefficient of variation of 6%, from a 62.5 ng load of human cell line tryptic digest. Using 5 min analytical flow-rate chromatography (800 µl/min), Zeno SWATH MS identified 4907 proteins from a triplicate injection of 2 µg of a human cell lysate, or more than 3000 proteins from a 250 ng tryptic digest. Zeno SWATH MS hence facilitates sensitive high-throughput proteomic experiments with low sample amounts, mitigating the current bottlenecks of high-throughput proteomics.