Method: Oxidative labeling MS

Conditions: pH 7.8; 22.0 Celsius; Probes: 33 peptides covering 93% of the sequence

Related publication:
 PMID 22940366

Experiment details: "α1AT was initially unfolded in 6 M GdnHCl at 22 °C for 2 hours (pH 7.8). Kinetic folding experiments with oxidative labeling were performed using a two-syringe continuous-flow mixing device. For the 0.5 s and 7 s time points, syringes 1 and 2 were advanced at 2.5 and 47.5 μl/min, respectively, using a syringe pump. Syringe 1 contained 200 μM α1AT denatured in 6 M GdnHCl, 10 mM phosphate buffer (pH 7.8), and 50 mM NaCl. Protein folding was triggered by combining this solution with phosphate buffer (pH 7.8) from syringe 2 at a homemade capillary mixer in a 1:19 volume ratio, resulting in a final denaturant concentration of 0.3 M (far below the unfolding midpoint) and a protein conc. of 10 μM. The outlet of the mixer was connected to a reaction capillary with an internal diameter of 100 μm. Syringe 2 also contained 15 mM glutamine and 0.1% (v/v) (ca 30 mM) H2O2. A KrF excimer laser (GAM EX 50, Orlando, FL) producing 18 ns pulses at 248nm, 72Hz, and 37mJ and an irradiation spot width of ca 1mm was used to generate -OH by photolysis of H2O2 within the reaction capillary. Glutamine acts as a radical scavenger that quenches the labeling reaction on a time scale of 1μs. Following initiation of folding, oxidative labeling was performed by irradiating the reaction mixture at different positions downstream of the mixer. Average reaction times of 0.5 s and 7 s correspond to distances between mixer and irradiation spot of 5.3 cm and 74.2 cm, respectively."

Protection threshold: completely refolded in 10 minutes

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Oxidative labeling MS

Conditions: pH 7.8; 22.0 Celsius; Probes: 33 peptides covering 93% of the sequence

Related publication:
 PMID 22940366

Experiment details: "α1AT was initially unfolded in 6 M GdnHCl at 22 °C for 2 hours (pH 7.8). Kinetic folding experiments with oxidative labeling were performed using a two-syringe continuous-flow mixing device. For the 0.5 s and 7 s time points, syringes 1 and 2 were advanced at 2.5 and 47.5 μl/min, respectively, using a syringe pump. Syringe 1 contained 200 μM α1AT denatured in 6 M GdnHCl, 10 mM phosphate buffer (pH 7.8), and 50 mM NaCl. Protein folding was triggered by combining this solution with phosphate buffer (pH 7.8) from syringe 2 at a homemade capillary mixer in a 1:19 volume ratio, resulting in a final denaturant concentration of 0.3 M (far below the unfolding midpoint) and a protein conc. of 10 μM. The outlet of the mixer was connected to a reaction capillary with an internal diameter of 100 μm. Syringe 2 also contained 15 mM glutamine and 0.1% (v/v) (ca 30 mM) H2O2. A KrF excimer laser (GAM EX 50, Orlando, FL) producing 18 ns pulses at 248nm, 72Hz, and 37mJ and an irradiation spot width of ca 1mm was used to generate -OH by photolysis of H2O2 within the reaction capillary. Glutamine acts as a radical scavenger that quenches the labeling reaction on a time scale of 1μs. Following initiation of folding, oxidative labeling was performed by irradiating the reaction mixture at different positions downstream of the mixer. Average reaction times of 0.5 s and 7 s correspond to distances between mixer and irradiation spot of 5.3 cm and 74.2 cm, respectively."

Protection threshold: refolded in 10-30 minutes

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
 CLICK TO DOWNLOAD SEQUENCE IN FASTA

Method: Pulse labeling HDX MS

Conditions: pH 7.8; 22.0 Celsius; Probes: monitors MS segments almost covering the full protein

Related publication:
 PMID 22392975

Experiment details: "To unfold the purified α1AT, 5 μg of the protein was incubated in 10 μL of 10 mM sodium phosphate (pH 7.8) and 50 mM NaCl containing 6 M GuHCl for 2 h at room temperature. The sample was diluted 10-fold with 10 mM sodium phosphate (pH 7.8) and 50 mM NaCl and refolded for increasing amounts of time. At various time points, refolding samples was deuterated for 10 s by an addition of 10 mM sodium phosphate (pD 7.8), 50 mM NaCl containing 0.6 M guanidine deuterochloride to a final volume of 1 mL. The deuteration reaction was quenched by adding HCl to lower pH to 2.3, and the sample was frozen and stored at −80°C until use."

Protection threshold: faster protection (without lag phase)

Sequence: EDPQGDAAQKTDTSHHDQDHPTFNKITPNLAEFAFSLYRQLAHQSNSTNIFFSPVSIATAFAMLSLGTKADTHDEILEGLNFNLTEIPEAQIHEGFQELLRTLNQPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYHSEAFTVNFGDTEEAKKQINDYVEKGTQGKIVDLVKELDRDTVFALVNYIFFKGKWERPFEVKDTEEEDFHVDQVTTVKVPMMKRLGMFNIQHCKKLSSWVLLMKYLGNATAIFFLPDEGKLQHLENELTHDIITKFLENEDRRSASLHLPKLSITGTYDLKSVLGQLGITKVFSNGADLSGVTEEAPLKLSKAVHKAVLTIDEKGTEAAGAMFLEAIPMSIPPEVKFNKPFVFLMIEQNTKSPLFMGKVVNPTQK
 CLICK TO DOWNLOAD SEQUENCE IN FASTA