High efficiency and quantitatively reproducible protein digestion by trypsin-immobilized magnetic microspheres.

ABSTRACT: Aldehyde- and NHS-activated magnetic microspheres were used to immobilize trypsin (CHO-trypsin and NHS-trypsin), and their performance for protein digestion was evaluated by reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometry using an LTQ Orbitrap Velos instrument. NHS-trypsin provided greater sequence coverage and identified more peptides for the digestion of bovine serum albumin. A 1-min digestion at room temperature using the immobilized trypsin also identified more peptides (96±6 vs. 48±1) and produced higher sequence coverage (90±2% vs. 75±2%) than traditional free trypsin digestion for 12h at 37 °C. Analysis of 15 nM (0.001 mg/mL) BSA digested by NHS-trypsin in 1 min at room temperature consistently yielded one detected peptide; 150 nM BSA generated

SUBMITTER: Sun L 

PROVIDER: S-EPMC3253205 | biostudies-literature | 2012 Jan

REPOSITORIES: biostudies-literature