Computational protocol: Mono and Dual Cofactor Dependence of Human Cystathionine β-Synthase Enzyme Variants In Vivo and In Vitro

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Protocol publication

[…] Protease susceptibility of purified proteins was measured using thermolysin. The purified protein sample was diluted to approximately 10 mM in lysis buffer. The reaction was initiated by the addition of thermolysin at a final concentration of 10 μg/mL at room temperature. At defined time intervals a portion of the reaction was removed, and the protease digestion quenched by the addition of ethylenediamine tetraacetic acid at a final concentration of 12.5 mM. Samples were analyzed by 8% sodium dodecyl sulfate polyacrylamide gel electrophoresis.The MBP-CBS protein fusion, as well as the cleaved CBS and MBP fragments, were excised from the gel and their identity confirmed by mass spectrometry by the following procedure. A nano LC column packed with 10 cm of Polaris c18 5 μm packing material (Varian) was loaded with the protein sample by use of a pressure bomb and washed extensively with buffer A. The column was then directly coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 HPLC equipped with a split line, so as to deliver a flow rate of 30 nL/min, was used for chromatography. Peptides were eluted with a 90-min gradient from 100% buffer A to 60% buffer B. Buffer A was 5% acetonitrile/0.02% heptaflurobutyric acid; buffer B was 80% acetonitrile/ 0.02% heptaflurobutyric acid. The programs SEQUEST and DTASELECT were used to identify peptides and proteins from a database consisting of common contaminants and the engineered sequences of the proteins (; ). […]

Pipeline specifications

Software tools Comet, DTASelect
Application MS-based untargeted proteomics
Organisms Homo sapiens
Chemicals Amino Acids, Cystathionine, Heme, Homocysteine