To detect γ-carboxyglutamic acid in peptides by positive ion mode LC-ESI-MS/MS, samples were chemically derivatized essentially as previously described65 (link),66 (link), with some modifications. A total of 100 µL of day 13 bioreactor supernatant samples (concentrated in a 0.5 mL 10 kDa Amicon column) and 1 µg of purified rFIX and pdFIX were denatured, reduced, and precipitated overnight, as described above for in-solution digestion. Samples were completely dried in an acid resistant SpeedVac Savant SPD300DDA (Thermo Fisher), were resuspended in 25 µL of 3 M HCl in methanol (90964, Sigma), and incubated at 20 °C for 1 h. The methanol-HCl was removed by completely drying the samples in a SpeedVac Savant at 30 °C for 45 min followed by 40 °C for 20 min. Proteins were resuspended in 50 mM ammonium bicarbonate solution with 0.1 µg trypsin and incubated at 37 °C for 16 h in a Thermoshaker at 1500 r.p.m. Peptides were desalted with C18 ZipTips.
Speedvac savant
Manufactured by Merck Group
The SpeedVac Savant is a vacuum concentrator designed for the rapid evaporation of solvents from samples. It uses centrifugal force and controlled vacuum pressure to efficiently remove solvents, leaving the desired sample behind.
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Lab products found in correlation
2 protocols using speedvac savant
1
Detecting γ-Carboxyglutamic Acid in Peptides
2
Identification of g-Carboxyglutamic Acid in Peptides
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To detect g-carboxyglutamic acid in peptides by positive ion mode liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), samples were chemically derivatized essentially as previously described [56, 57] , with some modifications. 100 µL of day 13 bioreactor supernatant samples (concentrated in a 0.5 mL 10 kDa Amicon column) and 1 µg of purified rFIX and pdFIX were denatured, reduced, and precipitated overnight as described above for in solution digestion. Samples were completely dried in an acid resistant SpeedVac Savant SPD300DDA (ThermoFisher), were resuspended in 25 µL of 3 M HCl in methanol (90964, Sigma), and incubated at 20 °C for 1 h. The methanol-HCl was removed by completely drying the samples in a SpeedVac Savant at 30 °C for 45 min followed by 40 °C for 20 min. Proteins were resuspended in 50 mM ammonium bicarbonate solution with 0.1 µg trypsin and incubated at 37 °C for 16 h in a Thermoshaker at 1500 rpm. Peptides were desalted with C18 ZipTips.
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