Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature.
We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.
Jeng
Jingyueh
J
Department of Biotechnology, Chia Nan University of Pharmacy & Science, 60, Erh-Jen RD., Sec.1, Jen-Te, Tainan 717, Taiwan.
Lin
Mon-Fang
MF
Cheng
Fong-Yu
FY
Yeh
Chen-Sheng
CS
Shiea
Jentaie
J
D004800:Enzymes, Immobilized / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020539:Sequence Analysis, Protein / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013696:Temperature; D014357:Trypsin / Q000737:chemistry*
D004800:Enzymes, Immobilized; D011506:Proteins; D014357:Trypsin