Ultrafast enzymatic digestion of proteins by microdroplet mass spectrometry

Abstract

Enzymatic digestion for protein sequencing usually requires much time, and does not always result in high sequence coverage. Here we report the use of aqueous microdroplets to accelerate enzymatic reactions and, in particular, to improve protein sequencing. When a room temperature aqueous solution containing 10 µM myoglobin and 5 µg mL^-1 trypsin is electrosonically sprayed (-3 kV) from a homemade setup to produce tiny (∼9 µm) microdroplets, we obtain 100% sequence coverage in less than 1 ms of digestion time, in sharp contrast to 60% coverage achieved by incubating the same solution at 37 °C for 14 h followed by analysis with a commercial electrospray ionization source that produces larger (∼60 µm) droplets. We also confirm the sequence of the therapeutic antibody trastuzumab (∼148 kDa), with a sequence coverage of 100% for light chains and 85% for heavy chains, demonstrating the practical utility of microdroplets in drug development.

Fig. 1. Schematic of the experimental apparatus for the online proteolysis by microdroplet chemistry coupled with mass spectrometry (ESSI-MS).

The inner capillary has an i.d. of 50 µm and an o.d. of 148 µm to which a high voltage (typically +3 kV or −3 kV) is applied.

Fig. 2. Mass spectra of 10-µM human ACTH (1–24) in 5-mM aqueous NH₄HCO₃ sprayed by the homemade sprayer.

a undigested (no trypsin); b–e digested with 5 µg mL⁻¹ trypsin at different travel distances between the sprayer tip and MS inlet for 2, 10, 20, and 50 mm, respectively. Magenta asterisks denote the peptide fragments, and blue #s mark undigested ACTH peaks.

Fig. 3. Comparison of ACTH digestion with various methods.

a commercial ESI-MS (LTQ Orbitrap Elite, Thermo Scientific), b bulk phase at 37 °C for 3 h, followed by analysis with commercial ESI-MS. The 16 peptide peaks found in b are listed in Supplementary Table 2. Magenta asterisks denote the peptide fragments, and blue #s mark undigested ACTH peaks. The unassigned peaks could be peptide adduct ions with solvent or background solvent peaks.

Fig. 4. Mass spectra of myoglobin digestion with various methods.

a Commercial ESI-MS (LTQ Orbitrap Elite, Thermo Scientific), b bulk phase at 37 °C for 14 h, followed by analysis with commercial ESI-MS, and ESSI-MS applied with c a positive high voltage (+3 kV), and d a negative high voltage (−3 kV). Magenta asterisks denote the peptide fragments, and blue #s mark undigested myoglobin peaks.

Fig. 5. Trypsin microdroplet digestion of proteins with ESSI-MS (+3 kV) after PAGE gel separation.

a PAGE gel showing the protein ladder and two protein bands stained with Coomassie blue. Mass spectra showing the microdroplet digestion of b α-casein and c cytochrome c. Magenta asterisks denote the peptide fragments.

Fig. 6. Trypsin digestion of a synthetic peptide with various methods.

a Peptide sequence, b mass spectrum of pure peptide. Mass spectra of the peptide digested with c commercial ESI-MS, d in bulk phase at 37 °C for 1 h, followed by analysis with commercial ESI-MS, and e ESSI-MS applied with a high voltage (+3 kV).

Fig. 7. Trypsin microdroplet digestion of the therapeutic antibody trastuzumab with ESSI-MS (−3 kV) after PAGE gel separation.

a PAGE gel showing the protein ladder, antibody’s light and heavy chain bands stained with Coomassie blue. Mass spectra showing the microdroplet digestion of b light chain and c heavy chain. d The heavy chain of trastuzumab, with the covered sequence marked in blue and the uncovered sequence in black. Magenta asterisks denote the peptide fragments.