Integration of On-Line Protein Digestion, Peptide Separation, and Protein Identification Using Pepsin-Coated Photopolymerized Sol−Gel Columns and Capillary Electrophoresis/Mass Spectrometry

Abstract

A miniaturized pepsin reactor was prepared inside a fused-silica capillary (i.d. 75 microm) by coating a pepsin-containing gel on a photopolymerized porous silica monolith. The pepsin-encapsulated film was prepared by a sol-gel method. The sol-gel reaction was optimized so that the sol solution containing pepsin forms a thin film on the photopolymerized sol-gel (PSG) monolith that was initially fabricated at the inlet of the capillary. Pepsin was encapsulated into the gel matrix without losing its activity. The large surface area of the PSG monolith enabled the immobilized pepsin to achieve a high catalytic turnover rate, and the porous nature of the PSG promotes penetration of large molecular proteins into the column. The immobilized pepsin-digested peptides and proteins, and the resulting mixture of peptide fragments, could be directly separated in the portion of the capillary where no PSG monolith exists. The durability and repeatability of the fabricated pepsin-coated column was tested and found to be satisfactory. An acidic solution consisting of 0.5 M formic acid was used as the running buffer, because it suppresses the adsorption of proteins or peptides on the inner surface of the capillary as well as enables direct connection of the output of the capillary electrophoresis column to a mass spectrometer. The on-line digestion of insulin chain beta and lysozyme provides identification of the proteolytic peptides. Recovery was achieved for 100% of the insulin chain beta amino acid sequence and 73% of the lysozyme amino acid sequence.

Development of an automated digestion and droplet deposition microfluidic chip for MALDI-TOF MS

“…Note that this value is appropriate for confirmation of cytochrome c based on Mowse score. The fraction at 100 s did not produce significant differences from the fraction at 60 s. In contrast with in-solution digestion which cannot be reused, the solid-phase bioreactor could be reused without noticeable loss of activity of immobilized enzyme [7,33,45]. During three independent experimental runs similar to that shown in Figure 4 using the same bioreactor, the activity remained relatively constant after rinsing the bioreactor with 50 mM ammonium bicarbonate between experimental runs.…”

“…Typically, the reaction time required for homogeneous solution tryptic digestions is tens of hours to achieve a sufficient population of peptide fragments for effective PMF because the enzyme-to-substrate ratio must be kept low to avoid interferences from autodigested trypsin [26]. To overcome this problem, enzymeimmobilization on solid supports for solid-phase bioreactors has been introduced [26,[31][32][33]. Many of these solid-phase bioreactors use immobilization of proteolytic enzymes through covalent attachment to supports or encapsulation within gel matrices.…”

“…Functionalized 3-D support networks can also be made in situ [31][32][33][34], which overcomes the difficulty of packing beads into the microchannels. Typically, these in situ methods use a polymerization reaction of a monomer solution inside a channel to modify the support.…”

“…Peterson et al [31] developed a porous organic polymer monolithic bioreactor as in situ approach that allowed trypsin to be immobilized covalently within the monolith using the azlactone functional groups of the monolith [32]. Kato et al [33] reported a monolithic photopolymerized sol-gel column that was used to immobilize pepsin within a fused silica capillary for integration of protein digestion and identification by CE/MS. Recently, Huang et al [34] described a similar device that utilized trypsin immobilized into a modified PMMA chip with zeolite nanoparticles using a silica sol-gel method.…”

Monolithic stationary phases for fast ion chromatography and capillary electrochromatography of inorganic ions

“…The nature of the photoinitiated polymerisation allowed the confinement of the monolith in the irradiated part of the capillary column and furthermore has been demonstrated in the successful preparation within microchip channels [52]. Derivatisation of the RP sol-gel monoliths was demonstrated for the analysis of peptides [53] and proteins [54], thus validating the viability of these sol-gel monoliths as IC stationary phases. However, besides SEM images of the microglobular structure, the authors did not conduct any morphology studies for these sol-gel polymerised monoliths.…”