Rapid and efficient protein digestion using trypsin-coated magnetic nanoparticles under pressure cycles

Abstract

Trypsin-coated magnetic nanoparticles (EC-TR/NPs), prepared via a simple multilayer random crosslinking of the trypsin molecules onto magnetic nanoparticles, were highly stable and could be easily captured using a magnet after the digestion was complete. EC-TR/NPs showed a negligible loss of trypsin activity after multiple uses and continuous shaking, whereas the conventional immobilization of covalently attached trypsin on NPs resulted in a rapid inactivation under the same conditions due to the denaturation and autolysis of trypsin. A single model protein, a five-protein mixture, and a whole mouse brain proteome were digested at atmospheric pressure and 37°C for 12 h or in combination with pressure cycling technology at room temperature for 1 min. In all cases, EC-TR/NPs performed equally to or better than free trypsin in terms of both the identified peptide/protein number and the digestion reproducibility. In addition, the concomitant use of EC-TR/NPs and pressure cycling technology resulted in very rapid (∼1 min) and efficient digestions with more reproducible digestion results.

Figure 1

(a) Schematic illustrations for the preparation of trypsin coating onto magnetic nanoparticles. (b) Magnetic capture of EC-TR/NPs. (c) Transmission electron microscopy (TEM) image of EC-TR/NPs (scale bar, 100 nm).

Figure 2

(a) Schematic for the trypsin-catalyzed hydrolysis of BAPNA. (b) Enzyme stabilities of CA-TR/NP (empty circles), EC-TR/NP (filled circles) and Free trypsin (empty triangles) under recycled uses and shaking at 200 rpm at room temperature. The activity was measured by the hydrolysis of BAPNA at each time point, and the relative activity was calculated from the ratio of residual activity at each time point to the initial activity of each sample.

Figure 3

Time course analyses of iterative BSA digestions using EC-TR/NPs as a function of unique identified peptides (empty circles) or protein coverage (filled circles).

Figure 4

Comparison of digestion performances for EC-TR/NPs and free trypsin overnight, and EC-TR/NPs pressure assisted digestion. A five standard protein mixture consisting of BSA, ovalbumin, myoglobin, carbonic anhydrase and lactoglobulin were used for the experiment. (a) Histograms comparing number of unique peptides and percentage of protein coverage for three technical replicates. (b) Venn diagrams showing the peptide overlap between three technical digestion replicates. (c) Pie charts showing the percentage of missed cleavages for the different digestion methods.

Figure 5

(a) The workflow of analyzing whole mouse brain proteome utilizing EC-TR/NP. After reduction and alkylation in 3 min using a sonoreactor, the proteome samples are subjected to three different digestions: conventional overnight digestion using free enzyme, overnight digestion using EC-TR/NP, and 1 min digestion using EC-TR/NP with PCT treatment. (b) Representative chromatograms obtained from EC-TR/NPs-PCT digestion of mouse brain samples after gas-phase fractionations. While imperfect, gas-phase fractionations clearly helped alleviating under sampling of whole proteome analyses. (c) Scatter plots of the corrected scores for charge states +2 and +3 obtained after SEQUEST database search from the generated MS/MS spectra using the three samples using different digestion methods. (d) Histograms comparing the number of unique identified peptides (empty bars), total identified peptides (diagonal filled bars) and percentage of protein coverage for the three different digestion methods. (e) Venn diagram showing the protein overlap among the three different protocols.