Unlabeled proteins, highly deuterated peptides and cytochrome c were analyzed using the UPLC system and conventional HPLC. In both LC-systems, labeled samples (50 µLs) were injected at a flow rate of 100 µL/min into a 2.1 mm × 50 mm stainless steel column that was packed with pepsin immobilized on POROS-20AL beads [prepared as described in8 (link), 9 (link)]. Under these conditions, the digestion time was approximately 30 seconds.
In the HPLC experiments, a Shimadzu HPLC (LC-10ADvp) system was used. Peptic peptides eluting from the online pepsin digestion step were trapped and desalted on a 1 mm × 8 mm C-18 peptide trap (Michrom Biosciences) and desalted for 3 min. The trap was placed inline with the analytical column, a Zorbax C-18, 3.5 µm 300 Å, 1.0 mm × 50 mm column (Agilent Technologies), and eluted into the mass spectrometer with a gradient of 15 to 30% acetonitrile in 6 min at a flow rate of 40 µL/min. HPLC mobile phases contained 0.05 % trifluoroacetic acid. The C-18 peptide trap and analytical column, as well as the injection and switching valves were placed in an ice-bath to maintain the required 0 °C. The mobile phases were kept in a separate ice-bath and then flowed through pre-cooling stainless steel loops (located before the gradient mixing tee) in the main ice-bath to ensure that they were cool prior to meeting deuterated sample. The pepsin column was held above the ice bath at approximately 15 °C9 (link).
In the UPLC experiments, peptic peptides from online pepsin digestion were trapped and desalted on a VanGuard Pre-Column (2.1 mm × 5 mm, ACQUITY UPLC BEH C18, 1.7 µm) for 3 min. The trap was placed in-line with an ACQUITY UPLC BEH C18 1.7 µm 1.0 × 100 mm column (Waters Corp.) and eluted into the mass spectrometer with a 8–40 % gradient of acetonitrile over 6 min at a flow rate of 40 µL/min. The volume of the system from the mixer to the head of the analytical column was ~ 30 µL which includes ~ 8 µL volume of the trap column in line. All mobile phases for the UPLC system contained 0.1 % formic acid.
Mass spectral analyses were carried out on a Waters LCT classic or QToF Premier. The LCT was used for initial validation of the cooled UPLC module chromatography and not for any analyses of deuterium incorporation. LCT classic instrument settings were: 3.2kV cone and 40 V capillary voltages. The LCT source and desolvation temperatures were 150 and 175 °C, respectively with a desolvation gas flow of 1024 L/hour and a cone gas flow of 99 L/hour. LCT mass spectra were acquired using a 0.50 sec scan time and 0.1 sec interscan delay time. QTof instrument settings were: 3.5kV cone and 40 V capillary voltages. The QTof source and desolvation temperatures were 80 and 175 °C, respectively with a desolvation gas flow of 600 L/hour. QTof mass spectra were acquired using a 0.450 sec scan time and 0.050 sec interscan time. All QTof data were collected in ESI (+) and V mode. Deuteration levels were calculated by subtracting the centroid of the isotopic distribution for peptide ions of undeuterated sample from the centroid of the isotopic distribution for peptide ions from the deuterium labeled sample. Deuterium levels were not corrected for back-exchange and are therefore reported as relative 1 (link).
In the HPLC experiments, a Shimadzu HPLC (LC-10ADvp) system was used. Peptic peptides eluting from the online pepsin digestion step were trapped and desalted on a 1 mm × 8 mm C-18 peptide trap (Michrom Biosciences) and desalted for 3 min. The trap was placed inline with the analytical column, a Zorbax C-18, 3.5 µm 300 Å, 1.0 mm × 50 mm column (Agilent Technologies), and eluted into the mass spectrometer with a gradient of 15 to 30% acetonitrile in 6 min at a flow rate of 40 µL/min. HPLC mobile phases contained 0.05 % trifluoroacetic acid. The C-18 peptide trap and analytical column, as well as the injection and switching valves were placed in an ice-bath to maintain the required 0 °C. The mobile phases were kept in a separate ice-bath and then flowed through pre-cooling stainless steel loops (located before the gradient mixing tee) in the main ice-bath to ensure that they were cool prior to meeting deuterated sample. The pepsin column was held above the ice bath at approximately 15 °C9 (link).
In the UPLC experiments, peptic peptides from online pepsin digestion were trapped and desalted on a VanGuard Pre-Column (2.1 mm × 5 mm, ACQUITY UPLC BEH C18, 1.7 µm) for 3 min. The trap was placed in-line with an ACQUITY UPLC BEH C18 1.7 µm 1.0 × 100 mm column (Waters Corp.) and eluted into the mass spectrometer with a 8–40 % gradient of acetonitrile over 6 min at a flow rate of 40 µL/min. The volume of the system from the mixer to the head of the analytical column was ~ 30 µL which includes ~ 8 µL volume of the trap column in line. All mobile phases for the UPLC system contained 0.1 % formic acid.
Mass spectral analyses were carried out on a Waters LCT classic or QToF Premier. The LCT was used for initial validation of the cooled UPLC module chromatography and not for any analyses of deuterium incorporation. LCT classic instrument settings were: 3.2kV cone and 40 V capillary voltages. The LCT source and desolvation temperatures were 150 and 175 °C, respectively with a desolvation gas flow of 1024 L/hour and a cone gas flow of 99 L/hour. LCT mass spectra were acquired using a 0.50 sec scan time and 0.1 sec interscan delay time. QTof instrument settings were: 3.5kV cone and 40 V capillary voltages. The QTof source and desolvation temperatures were 80 and 175 °C, respectively with a desolvation gas flow of 600 L/hour. QTof mass spectra were acquired using a 0.450 sec scan time and 0.050 sec interscan time. All QTof data were collected in ESI (+) and V mode. Deuteration levels were calculated by subtracting the centroid of the isotopic distribution for peptide ions of undeuterated sample from the centroid of the isotopic distribution for peptide ions from the deuterium labeled sample. Deuterium levels were not corrected for back-exchange and are therefore reported as relative 1 (link).
