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| Sample preparation for time-dependent on-exchange HDXMS experiments. For all HDXMS experiments, three sets of samples will be prepared: timedependent on-exchange samples with multiple time points, fully deuterated (FD) control samples, and non-deuterated (ND) control samples. (a) Procedure to prepare time-dependent on-exchange samples. Protein mixture samples (e.g., 30 µl) are incubated with 3 volumes (e.g., 90 µl) of deuterium-exchange solution B (150 mM NaCl, 8.3 mM Tris, pH 7.2, in D 2 O) at specified temperature (0 °C or room temperature). Incubate for 10, 100, 1,000, 10,000, and 100,000 s. At each time point, transfer 24-µl aliquots to vials containing 36 µl of optimal quench solution at 0 °C to stop the exchange reaction. Mix gently by pipetting up and down four to five times. Divide the sample into three equal aliquots (20 µl in each vial) and transfer to vials A, B, and C labeled with the corresponding time points. (b) Procedure to prepare FD control samples. FD samples are used to estimate the extent of back-exchange. Add 6 µl of protein sample to 18 µl of deuterium-exchange solution C (0.5% (vol/vol) FA in 100% (vol/vol) D 2 O). Incubate the mixture at room temperature for 8-12 h, and then quench the reaction with 36 µl of optimal quench solution. Divide the FD sample into three equal aliquots (20 µl each vial). (c) Procedure to prepare ND control samples. ND samples are used to generate the list of peptides for HDX analysis. Add 6 µl of protein sample to 18 µl of deuterium-exchange solution A (150 mM NaCl, 8.3 mM Tris, pH 7.2, in H 2 O). No incubation is necessary, but allow the protein/buffer solution to settle for ~1 min before mixing with quench buffer. Divide the ND sample into three equal aliquots (20 µl in each vial).

| Sample preparation for time-dependent on-exchange HDXMS experiments. For all HDXMS experiments, three sets of samples will be prepared: timedependent on-exchange samples with multiple time points, fully deuterated (FD) control samples, and non-deuterated (ND) control samples. (a) Procedure to prepare time-dependent on-exchange samples. Protein mixture samples (e.g., 30 µl) are incubated with 3 volumes (e.g., 90 µl) of deuterium-exchange solution B (150 mM NaCl, 8.3 mM Tris, pH 7.2, in D 2 O) at specified temperature (0 °C or room temperature). Incubate for 10, 100, 1,000, 10,000, and 100,000 s. At each time point, transfer 24-µl aliquots to vials containing 36 µl of optimal quench solution at 0 °C to stop the exchange reaction. Mix gently by pipetting up and down four to five times. Divide the sample into three equal aliquots (20 µl in each vial) and transfer to vials A, B, and C labeled with the corresponding time points. (b) Procedure to prepare FD control samples. FD samples are used to estimate the extent of back-exchange. Add 6 µl of protein sample to 18 µl of deuterium-exchange solution C (0.5% (vol/vol) FA in 100% (vol/vol) D 2 O). Incubate the mixture at room temperature for 8-12 h, and then quench the reaction with 36 µl of optimal quench solution. Divide the FD sample into three equal aliquots (20 µl each vial). (c) Procedure to prepare ND control samples. ND samples are used to generate the list of peptides for HDX analysis. Add 6 µl of protein sample to 18 µl of deuterium-exchange solution A (150 mM NaCl, 8.3 mM Tris, pH 7.2, in H 2 O). No incubation is necessary, but allow the protein/buffer solution to settle for ~1 min before mixing with quench buffer. Divide the ND sample into three equal aliquots (20 µl in each vial).

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Figure 2 | Workflow for HDXMS, CXMS, and disulfide-trapping...
Figure 3 | Different types of cross-linkers with their reactive groups...
Figure 4 | Fragmentation tuning in HDXMS experiments. (a) Sample...
taBle 4 | | Troubleshooting table (continued).
+3 Figure 5 | Sample preparation for time-dependent on-exchange HDXMS...
nprot.2018.037
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  • Feb 2018
Many cellular functions necessitate structural assemblies of two or more associated proteins. The structural characterization of protein complexes using standard methods, such as X-ray crystallography, is challenging. Herein, we describe an orthogonal approach using hydrogen–deuterium-exchange mass spectrometry (HDXMS), cross-linking mass spectrome...
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... the quenched HDX samples should be maintained and handled at 0 °C with an optimized pH of 2.5.  crItIcal step Optimize enzymatic conditions to achieve best proteolytic peptide sequence coverage by varying protein-enzyme contact time, changing the size of the enzyme column, switching to other enzyme columns, or using a tandem-column setup. (Fig. 5a), (ii) FD control samples (Fig. 5b), (iii) ND control samples (Fig. 5c). Ideally, all samples should be prepared from the same batch of protein. For comparison studies such as apo protein versus holo protein, it is recommended to perform the experiments in parallel to minimize systematic variation. Prepare time-dependent on-exchange ...
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... be maintained and handled at 0 °C with an optimized pH of 2.5.  crItIcal step Optimize enzymatic conditions to achieve best proteolytic peptide sequence coverage by varying protein-enzyme contact time, changing the size of the enzyme column, switching to other enzyme columns, or using a tandem-column setup. (Fig. 5a), (ii) FD control samples (Fig. 5b), (iii) ND control samples (Fig. 5c). Ideally, all samples should be prepared from the same batch of protein. For comparison studies such as apo protein versus holo protein, it is recommended to perform the experiments in parallel to minimize systematic variation. Prepare time-dependent on-exchange samples (Figs. 2a and 5a). To do this, ...
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... with an optimized pH of 2.5.  crItIcal step Optimize enzymatic conditions to achieve best proteolytic peptide sequence coverage by varying protein-enzyme contact time, changing the size of the enzyme column, switching to other enzyme columns, or using a tandem-column setup. (Fig. 5a), (ii) FD control samples (Fig. 5b), (iii) ND control samples (Fig. 5c). Ideally, all samples should be prepared from the same batch of protein. For comparison studies such as apo protein versus holo protein, it is recommended to perform the experiments in parallel to minimize systematic variation. Prepare time-dependent on-exchange samples (Figs. 2a and 5a). To do this, calculate the total number of ...
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... the dry ice into thin pieces with a hammer for quick freezing of quenched vials. , mix gently by pipetting up and down four to five times, and incubate the mixture for 10, 100, 1,000, 10,000, and 100,000 s ( Fig. 5a (i)).  crItIcal step We perform most HDX experiments at 0 °C. If proteins and protein complexes are thermo-stable, the HDX experiments can also be performed at room ...
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... each time point, stop the exchange reaction by transferring 24 µl of the HDX mixture to microcentrifuge tubes preloaded with 36 µl of optimal quench solution at 0 °C ( Fig. 5a (ii)). Mix gently by pipetting up and down four to five times, avoiding bubbles. Leave on ice for 1 min.  crItIcal step These steps must be completed within the time-point constraints. Make sure to keep track of the time! For the 10-s time point, it is recommended to have pipettes set up beforehand to meet the time ...
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... Divide the sample into three 20-µl aliquots and transfer them to vials A, B, and C within 1 min, freeze the samples on dry ice, and transfer them to a −80 °C freezer (Fig. 5a (iii)).  pause poInt Samples can be divided into aliquots and stored for several months at − 80 ...
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... preparation of FD control samples • tIMInG 2 d  crItIcal It is best to prepare the FD control samples (FD samples) separate from the on-exchange set, preferably the night before (Fig. 5b). The measurements below follow the protein/buffer/quench ratio of 1:3:6. Prepare samples in triplicate. Results from FD samples are used to calculate the back-exchange rates during on-line pepsin digestion and LC/MS ...
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... Add 6 µl of protein to 18 µl of deuterium-exchange solution C (0.5% (vol/vol) FA in 100% (vol/vol) D 2 O, stored at 4 °C) ( Fig. 5b (i)) and incubate at room temperature for 8-12 h.  crItIcal step If the sample is not stable at room temperature for longer time points, speed up the labeling process by instead using GuDCl (deuterated guanidine hydrochloride) to completely denature the protein or by incubating the protein at an elevated temperature for a shorter amount ...
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... After incubation, add 36 µl of optimal quench solution at 0 °C on ice (Fig. 5b (ii)). Keep the sample on ice for ~1 ...
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... Prepare 20-µl aliquots of FD sample in three pre-labeled autosampler vials (Fig. 5b (iii)). Cap the vials and freeze on dry ice.  pause poInt Aliquots can be prepared and stored for several months at − 80 °C. protocol nature protocols | VOL.13 NO.6 | 2018 | 1417 24| Prepare ND control samples (Fig. 5c) by following the same procedure as described above, making sure to use deuterium-exchange solution A, rather than ...
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... Prepare 20-µl aliquots of FD sample in three pre-labeled autosampler vials (Fig. 5b (iii)). Cap the vials and freeze on dry ice.  pause poInt Aliquots can be prepared and stored for several months at − 80 °C. protocol nature protocols | VOL.13 NO.6 | 2018 | 1417 24| Prepare ND control samples (Fig. 5c) by following the same procedure as described above, making sure to use deuterium-exchange solution A, rather than deuterium-exchange solution C. No incubation is necessary, but allow the protein/buffer solution to settle for ~1 min before mixing with quench buffer. Triplicate samples are recommended.  crItIcal step ND samples are used ...