Facile trypsin immobilization in polymeric membranes for rapid, efficient protein digestion

Abstract

Sequential adsorption of poly(styrene sulfonate) and trypsin in nylon membranes provides a simple, inexpensive method to create stable, microporous reactors for fast protein digestion. The high local trypsin concentration and short radial diffusion distances in membrane pores facilitate proteolysis in residence times of a few seconds, and the minimal pressure drop across the thin membranes allows their use in syringe filters. Membrane digestion and subsequent MS analysis of bovine serum albumin provide 84% sequence coverage, which is higher than the 71% coverage obtained with in-solution digestion for 16 h or the <50% sequence coverages of other methods that employ immobilized trypsin. Moreover, trypsin-modified membranes digest protein in the presence of 0.05 wt % sodium dodecyl sulfate (SDS), whereas in-solution digestion under similar conditions yields no peptide signals in mass spectra even after removal of SDS. These membrane reactors, which can be easily prepared in any laboratory, have a shelf life of several months and continuously digest protein for at least 33 h without significant loss of activity.

Figure 1

Membrane digestion efficiency for 0.1 mg/mL non-denatured α-casein solutions in 10 mM NH₄HCO₃. (a) SDS-PAGE of intact α-casein (Lanes 2 and 7) and α-casein after membrane digestion with different residence times (Lanes 3–6); (b) α-casein sequence coverages (revealed by MALDI-MS) obtained after membrane digestion.

Figure 2

MALDI-mass spectra of urea-denatured BSA (a) after membrane-digestion (miniaturized membrane reactor with an effective filtration area of 2 mm², residence time of 13 s, BSA concentration of 0.1 mg/mL for both digestion and MS analysis) and (b) after in-solution digestion for 16 h (BSA concentration of 0.5 mg/mL for digestion and 0.1 mg/mL for MS analysis).

Figure 3

MALDI-mass spectra of α-casein digested using both solution and membrane methods: (a–c) digestion in the presence of SDS; (d) addition of SDS only after digestion. (* shows identified peptides from α-casein.)

Figure 4

MALDI-MS sequence coverages of membrane-digested BSA as a function of the time employed for continuously passing urea-denatured BSA (0.1 mg/mL, 0.1 mL/h) through miniaturized nylon/PSS/trypsin and PVDF/trypsin membranes.

Figure 5

Mass spectra of the peptides enriched from a mixture containing urea-denatured BSA and α-casein (mass ratio of 10:1) digested with a membrane (a, 0.1 mg/mL total protein during digestion) and in solution (b, 0.5 mg/mL total protein during digestion). The phosphopeptides, which are labeled, were enriched on a MALDI plate modified with polymer-oxotitanium (1 µL of 0.3 mg/mL protein digest was spotted on the MALDI plate for enrichment).

Scheme 1

Conceptual representation of a membrane reactor for tryptic digestion.

Scheme 2

Conceptual drawing of the miniaturized membrane holder (top) and the Swinnex holder (bottom left), and a photograph (bottom right) of the 25-mm membrane disk and a miniaturized membrane that was exposed to dye while in the holder.