Experimental Workflow

Myagi and Nakazawa have shown that the pseudo first-order rate constant, Jk, for hydrogen exchange at the C-2 positions of the histidine can be evaluated using Equation 10.3 [6]  

In Equation 10.6, is the value at the plateau on the alkaline side of the sigmoidal 

Knowing the values of individual histidine residues in an enzyme can be important for understanding the mechanism by which an enzyme carries out its catalytic function. The imidazole groups in the side chains of histidine residues in proteins generally have pK values in the range of 5-8. Thus, histidine residues are especially well equipped to participate in the many catalytic processes of enzymatic reactions that use general acid-base catalysis. 

The ESI-MS approach described previously has been used to study pK values of histidine residues in several diffeent protein systems including Escherichia coli dihydrofolate reductase (DHER) 

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Once the robotic system and procedure passed the optimization and reproducibility tests for a certain type of reaction, the researcher has the chance to move on to the most delightful part of a high-throughput experimentation workflow that is to follow the reaction kinetics of the reaction by withdrawing several samples under comparable conditions. The characterization of these samples allows the determination of the apparent rate constants and activation energies in a very reproducible way. As an example, the anionic polymerization of St in cyclohexane initiated by i-BuLi under different reaction conditions was investigated. Several samples were withdrawn during the reaction into small vials which were prefllled with 25 pL of... 

Principles and experimental workflow of fragment library screening... 

In a standard hydrogen exchange experiment (see Section 2.3.4 for variations in experimental workflow), the sample is deuterated for a predetermined time (milliseconds and longer times), and the reaction is slowed down several orders of magnitude by the addition of an acidic quench solution. Quenched reactions can be subjected to direct mass spectrometry analysis or immediately flash frozen in liquid nitrogen and stored at -80°C in multiple aliquots for future analysis. These flash-frozen samples need to be thawed prior to mass spectrometry. It is important to note that freeze-thaw cycles contribute to signal loss due to back-exchange (this is described further in Section 2.3.8), which... 

Figure 10.2 Schematic representation of the experimental workflow used in mass spectrometry-based experiments to determine the pK of histidine residues in proteins. The first step of the protocol involves incubation of the protein in a series of D,0 buffers at different pH values for at least 2 days before the hydrogen exchange reactions are quenched. The protein samples from each hydrogen exchange reaction are then digested with proteolytic enzyme(s), and the proteolytic fragments are analyzed by LC-TSI-MS to determine the deuterium content cjf histidine-ccjntaining peptides...

Figure 10.3 Typical mass spectral data obtained on a histidine-containing peptide generated in the experimental workflow shown in Figure 10.2. The data shown are from a histidine-containing peptide of sequence YTPHEETNNESF from the G protein-coupled receptor rhodopsin. The two mass spectra were obtained after the protein was exchanged for 0 and 72 h at pH 8.0. Reproduced with permission from Ref [26]. 2010, American Chemical Society...

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. 

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...

The relationship between the different protocols described here and their place in the overall experimental workflow is shown in Fig. 1. 

Identification of Taxon-Specific Biomarkers Experiences from our work and those of others strongly suggest that efficient strategies for identification of taxon-specific biomarkers should ideally involve measurements of a representative number of strains per taxonomic unit (Lasch and Naumann 2011). This is particularly important for accurate identification at and below the species level. To differentiate microorganisms below the species level, MALDI-TOF MS characterization should be ideally carried out by repetitive measurements from an adequate number of independent microbial cultures per strain. Therefore, the cmrent experimental workflow at the Robert Koch-Institute (RKI) encompasses measurements of four technical repUcate spectra per each individual culture, that is, per biological replicate. Since we normally generate three independent crrltrrres per strain subsequent statistical analyses can be carried out on the basis of at least 12 spectra per strain. In this way it is asstrred that the mass spectral database contains sufficient information with respect to repeatability and reproducibility. The expanded ntrmber of spectra serves as an improved basis for systematic statistical analyses to identify taxon-specific microbial biomarkers. 

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