configuring and calibrating the tims software interface for proteomic analysis

Histonproteomisk analyse og karakterisering af post-translationel modifikation

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.

Forfatter Spotlight: Forbedret histon PTM-isomeridentifikation gennem LC-TIMS-ToF MS/MS og PASEF

Histonmodifikationsscreening ved hjælp af væskekromatografi, fanget ionmobilitetsspektrometri og time-of-flight-massespektrometri

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.

Konfiguration og kalibrering af TIMS-softwaregrænsefladen til proteomisk analyse

Åbn TIMS-styringssoftwaren. Klik på fanen Instrument, og skift til Betjen. Kontroller TIMS-parametrene, og kontroller derefter MS-indstillingerne, herunder Scan Begin, Scan End, Ion Polarity og Scan Mode.Dobbelttjek TIMS-indstillingerne, herunder tilstand, mobilitetsstart, mobilitetsslut, rampetid, akkumuleringstid, driftscyklus, rampehastighed, MS-hastighed, MS-gennemsnit og automatisk kalibrering. Skift derefter scanningstilstand til MS til kalibreringsformål. Når du har sikret, at instrumentet er i driftstilstand, skal du fra fanen Kilde aktivere Hamilton 500 mikroliter sprøjten, der indeholder tuningblanding.Gå til fanen Kalibrering. Klik på Mass, der skal oplades, og vælg kvadratisk tilstand under kalibreringstilstand. Kontroller zoom til plus eller minus 0,01 %Klik derefter på Kalibrer.Og når en score på 100% opnås, skal du klikke på Accepter. Gå derefter til fanen Mobilitet, og vælg Lineær tilstand under Kalibreringstilstand. Indstil detekteringsområdet til plus eller minus 5% og bredde til 0,1 dalton.Klik derefter på Kalibrer, og klik på Acceptér, når du modtager en score på mere end 98,5 %.

Research

JoVE Journal

Chemistry

Configuring and Calibrating the TIMS Software Interface for Proteomic Analysis

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 ...

LC-TIMS-PASEF-ToF MS/MS Method for Proteolytic Histone Peptide Analysis

LC-TIMS-PASEF-ToF MS / MS metode til proteolytisk histonpeptidanalyse