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HX-MS Workflow

Integrating Gas-Phase Fragmentation Into the Classical Bottom-Up HX-MS Workflow... [Pg.135]

Of the fragmentation techniques MALDI-ISD, ECD, and ETD, it is only the latter that is directly compatible with the requirements and timescale of an online HX-MS workflow. By combining the classic bottom-up HX-MS workflow with gas-phase fragmentation by ETD, detailed information on protein HX can be obtained [47,57]. In such a combined workflow, enzymatic solution-phase cleavage is followed by automated (data-dependent acquisition) or manual (targeted) selection of peptides for gas-phase cleavage by ETD [57] (Eigure 8.6). [Pg.135]

Figure 8.6 The bottom-up HX-ETD workflow. HX, quench and solution-phase cleavage with enzymes are analogous to the classical bottom-up workflow (see Figure 1.2). Briefly, HX is initiated by dilution in Dfi. At several time points, aliquots are quenched to pH 2.5 and 0°C and digested with an acid-stable protease. The m/z values of the peptides are detected in MS survey scans followed by ETD gas-phase fragmentation. In top-down HX-ETD workflows, gas-phase fragmentation is performed on the intact protein (see Chapter 9). Figure adapted with permission from [41] 2014 American Chemical Society. (See insert for color representation of the figure.)... Figure 8.6 The bottom-up HX-ETD workflow. HX, quench and solution-phase cleavage with enzymes are analogous to the classical bottom-up workflow (see Figure 1.2). Briefly, HX is initiated by dilution in Dfi. At several time points, aliquots are quenched to pH 2.5 and 0°C and digested with an acid-stable protease. The m/z values of the peptides are detected in MS survey scans followed by ETD gas-phase fragmentation. In top-down HX-ETD workflows, gas-phase fragmentation is performed on the intact protein (see Chapter 9). Figure adapted with permission from [41] 2014 American Chemical Society. (See insert for color representation of the figure.)...
In the online HX-MS/MS workflow, ETD fragmentation can be set up for all peptides in order to obtain as much site-specific HX information as possible, or alternatively, a targeted HX-ETD approach [47, 57] can be used to delineate site-specific HX only for selected peptides that display altered deuterium uptake upon ligand binding or across different protein states (Figure 8.9). [Pg.139]

Recent Applications of the Bottom-Up HX-MS/MS Workflow to Pinpoint the HX Properties of Proteins... [Pg.141]

Until several years ago, gas-phase fragmentation was viewed exclusively as a working tool of the top-down HX-MS measurements, even though sevraal attempts had been made ovct a decade ago to incorporate peptide ion fragmentation in the workflow of the hottom-up HX-MS measurements as well [34, 35]. However, earlier attempts to supplement proteolysis in solution with fragmentation in... [Pg.158]

The HX experiment can be automated by following two distinct experimental workflows. In one paradigm, the experimental design decouples automated digestion and LC-MS analysis from the HX sample preparation step (i.e., decoupled HX-MS). To enable the LC-MS analysis to be decoupled from the HX expraiment, samples are flash frozen in liquid nitrogen afta- the quench and stored at -80°C until required. Samples can be stored for days/weeks at -80°C with minimal loss of deuterium label. When the sample is ready for LC-MS analysis, it is thawed immediately prior to analysis with LC-MS by the autosampler. [Pg.216]

The first semiautomated HX-MS system was described by Virgil Woods in 2001 and was based around the decoupled experimental paradigm [44], Woods outlined an end-to-end HX workflow, titled DXMS, that began with (manual) sample preparation and freezing of samples in quench solution. When ready, samples were placed in a modified HPLC autosampler able to perform the thawing, digestion, and LC-MS analysis. Although this automation was never commercialized, it was in use for over a decade and was demonstrated to be extremely productive. [Pg.218]

Figure 15.2 Typical HX-MS experimental workflow for (a) protein adsorbed on solid surfaces, (b) protein In frozen solution, and (c) protein in lyophilized solid powders. The experimental conditions are shown in the figure... Figure 15.2 Typical HX-MS experimental workflow for (a) protein adsorbed on solid surfaces, (b) protein In frozen solution, and (c) protein in lyophilized solid powders. The experimental conditions are shown in the figure...

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