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

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 12 h 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 22 peptides. Peptide intensity and protein spectral count were used to evaluate the run-to-run digestion reproducibility of NHS-trypsin with a three-protein-mixture. Three high intensity peptides for each protein generated intensity ratios from 0.70 to 1.09 and spectral count ratios from 0.78 to 1.18. Finally, RAW 264.7 cell lysates were digested by NHS-trypsin for 10 min and 30 min at room temperature, 604 and 697 protein groups, respectively, were identified by RPLC–ESI-MS/MS, with a peptide false discovery rate of less than 1%. Digestion by solution phase trypsin for 12 h at 37 °C resulted in identification of 878 protein groups.

► Comprehensive comparison of chemistry for immobilization of trypsin to magnetic microbeads. ► Evaluation of precision of digestion in terms of peak intensity and spectral counts. ► A 1-min digestion with immobilized beads generated more peptides than 12 digestion using conventional solution-phase digestion. ► Obtained peptides from digestion of 15 nM protein.