In-line system containing porous polymer monoliths for protein digestion with immobilized pepsin, peptide preconcentration and nano-liquid chromatography separation coupled to electrospray ionization mass spectroscopy

Abstract

The use of two different monoliths located in capillaries for on-line protein digestion, preconcentration of peptides and their separation has been demonstrated. The first monolith was used as support for covalent immobilization of pepsin. This monolith with well-defined porous properties was prepared by in situ copolymerization of 2-vinyl-4,4-dimethylazlactone and ethylene dimethacrylate. The second, poly(lauryl methacrylate-co-ethylene dimethacrylate) monolith with a different porous structure served for the preconcentration of peptides from the digest and their separation in reversed-phase liquid chromatography mode. The top of the separation capillary was used as a preconcentrator, thus enabling the digestion of very dilute solutions of proteins in the bioreactor and increasing the sensitivity of the mass spectrometric detection of the peptides using a time-of-flight mass spectrometer with electrospray ionization. Myoglobin, albumin, and hemoglobin were digested to demonstrate feasibility of the concept of using the two monoliths in-line. Successive protein injections confirmed both the repeatability of the results and the ability to reuse the bioreactor for at least 20 digestions.

Figure 1

Scheme of the in-line system: (A) protein solution is injected in the immobilized pepsin reactor in 2% aqueous formic acid, digested, and peptides are trapped on the top of HPLC column; (B) the reactor is bypassed and peptides are separated in a gradient of acetonitrile.

Figure 2

SEM micrographs of the cross section of 100-µm I.D. monoliths in capillaries: (A) support for the enzymatic reactor prepared from 24% VAL, 16% EDMA, 34% 1-propanol, 26% 1,4-butanediol and 0.4% AIBN; (B) HPLC column prepared from 24% lauryl methacrylate, 16% ethylene dimethacrylate, 45% 1-propanol, 15% 1,4-butanediol and 0.4% AIBN; polymerizations were carried out at 70°C for 24 h.

Figure 3

Scheme of the immobilization reaction involving azlactone functionality of the monolithic poly(vinyl azlactone-co-ethylene dimethacrylate) support and pepsin.

Figure 4

Separation of peptides resulting from the in-line digestion of 2 pmol of myoglobin in 15 cm × 100 µm I.D. immobilized pepsin reactor using a 20 cm × 100 µm I.D. monolithic column. Myoglobin concentration in solution 1 µmol/L, injection volume 2 µL; Mobile phase: 2% formic acid in water for 25 min pumped through the complete system followed by switch of the valve and use of a 0–80% linear gradient of acetonitrile gradient in 2% aqueous formic acid in 40 min. Flow rate of 500 nL/min.

Figure 5

Mass spectra of 11 peaks obtained by on-line digestion/separation of myoglobin shown in Figure 4. Assignment of 15 peptide fragments associated with molecular masses are summarized in Table 1.

Figure 6

Separations resulting from the injection of 2 µL of 1 µmol/L hemoglobin (A) and albumin solution (C) in immobilized pepsin reactor and from the injection of these proteins in system in which the reactor is replaced with an empty capillary (B) and (D). Immobilized pepsin reactor 15 cm × 100 µm I.D., injection of 2 µL of 1 µmol/L protein solutions. For separation conditions see Figure 4.

Figure 7

Comparison of digestion/separation using myoglobin solutions with different concentration or different injected volume. Injection volume 2 µL; concentration 1 µmol/L (2 pmol injected) (A) , 400 nmol/L (800 fmol) (B), 100 nmol/L (200 fmol) (C); Ten successive injections of 2 µL of 100 nmol/L (2 pmol) myoglobin solution (D). For other conditions see Figure 4.

Figure 8

Repeated digestions of myoglobin in the enzymatic reactor. Inset: Mass spectrum of the peak eluting at 76.5 min after the 25th injection. For conditions see Figure 4.