configuring and calibrating the tims software interface for proteomic analysis

Análise proteômica de histonas e caracterização de modificação pós-tradução

In eukaryotic cells, DNA is packaged as chromatin into functional units called nucleosomes. These units are composed of an octamer of four core histones (two each of H2A, H2B, H3, and H4)1,2,3,4. Histones are amongst the most abundant and highly conserved proteins in eukaryotes, which are largely responsible for gene regulation5. Histone posttranslational modifications (PTMs) play a large role in the regulation of chromatin dynamics and rigger various biological processes such as DNA transcription, replication, and repair6. PTMs occur primarily on the accessible surface of the N-terminal regions of histones that are in contact with DNA3,7. However, tail and core modifications influence chromatin structure, altering inter-nucleosome interactions and recruiting specific proteins3,8.
A current challenge during liquid chromatography-mass spectrometry (LC-MS)-based proteomics is the potential co-elution of analytes of interest. In the case of data-dependent analyses (DDA), this translates into the potential loss of several precursor ions during the MS/MS acquisition process9. Time-of-flight (ToF) instruments acquire spectra at very high frequency9,10 (up to tens of kHz)11; this makes them capable of rapidly scanning the total precursor ions within a complex sample (MS1), thus promising optimal sensitivity and MS/MS sequencing rates (up to 100 Hz)9 and making them ideal for biological sample analysis10. Nevertheless, the sensitivity available at these high scan rates is limited by the MS/MS rate9. The addition of trapped ion mobility spectrometry (TIMS) in combination with an orthogonal quadrupole time-of-flight (qToF) mass spectrometer was used to mitigate these limitations. In TIMS, all precursor ions are accumulated in tandem and eluted as a function of their mobility, rather than selecting single precursor masses with a quadrupole9. Parallel accumulation-serial fragmentation (PASEF) allows for hundreds of MS/MS events per second without any loss of sensitivity9.
The principal aim of this work was to show the recent developments of DDA using parallel accumulation in the mobility trap followed by sequential fragmentation and collision-induced dissociation (CID). Histone PTMs were confidently assigned based on their retention times (RTs), mobilities, and fragmentation patterns.

Autor em destaque: Identificação aprimorada de isômeros PTM de histonas por meio de LC-TIMS-ToF MS / MS e PASEF

Triagem de modificação de histona usando cromatografia líquida, espectrometria de mobilidade iônica aprisionada e espectrometria de massa por tempo de voo

A method is proposed using histone extraction protocol with 95%efficiency. Together with LC-TIMS-ToF MS/MS, in a short time, it is possible to obtain a screening for those PTMs present in the histone. And above all, the possibility of separating isomers.The separation of isomeric peptides though possible using tandem mass spectrometry alone is often difficult. By incorporating TIMS, we introduce an extra dimension of separation, simplifying many positional isomer identifications. In addition, passive technology increases the sensitivity of these experiments, further improving our ability to analyze epigenetic variations.With the increased ability to identify PTM positions, there comes the ability to further determine the correlation between various environmental or biological factors, and their effects on the epigenome. Methods that improve and facilitate analysis of histones can be applied to several research areas, including medical and environmental chemistry. The dynamics of protein confirmation in native or damaged states will be established from the implementation of TIMS MS/MS with HDX.Through LC-TIMS-ToF MS/MS and Nano LC-TIMS-ToF MS/MS, the differences in PTM existing in various biological sample will be charted.

Configurando e calibrando a interface do software TIMS para análise proteômica

Abra o software de controle TIMS. Clique na guia Instrumento e alterne para Operar. Verifique os parâmetros do TIMS e, em seguida, verifique as configurações do MS, incluindo o Scan Begin, o Scan End, o Ion Polarity e o Scan Mode.Verifique novamente as configurações do TIMS, incluindo o Modo, Início da mobilidade, Fim da mobilidade, Tempo de rampa, Tempo de acumulação, Ciclo de trabalho, Taxa de rampa, Taxa de MS, Média de MS e Calibração automática. Em seguida, altere o modo de varredura para MS para fins de calibração. Depois de garantir que o instrumento esteja em modo de operação, na guia Fonte, ative a seringa Hamilton 500 Microlitros contendo mistura de ajuste.Vá para a guia Calibração. Clique na Massa para carregar e, no modo Calibração, escolha o modo Quadrático. Controle o zoom para mais ou menos 0,01%Em seguida, clique em Calibrar.E quando uma pontuação de 100% for alcançada, clique em Aceitar. Em seguida, vá para a guia Mobilidade e, em Modo de calibração, selecione Modo linear. Defina o intervalo de detecção para mais ou para menos 5% e largura para 0,1 Daltons.Em seguida, clique em Calibrar e, ao receber uma pontuação superior a 98,5%, clique em Aceitar.

configuring-calibrating-tims-software-interface-for-proteomic

Research

JoVE Journal

Chemistry

Configuring and Calibrating the TIMS Software Interface for Proteomic Analysis

63 visualizações.  Florida International University. Abra o software de controle TIMS. Clique na guia Instrumento e alterne para Operar. Verifique os parâmetros do TIMS e, em seguida, verifique as configurações do MS, incluindo o Scan Begin, o Scan End, o Ion Polarity e o Scan Mode.Verifique novamente as configurações do TIMS, incluindo o Modo, Início da mobilidade, Fim da mobilidade, Tempo de rampa, Tempo de acumulação, Ciclo de trabalho, Taxa de rampa, Taxa de MS, Média de MS e Calibração automática. Em seguida, altere o mo...

Vídeo: Configurando e calibrando a interface do software TIMS para análise proteômica

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).