Improve Digestion Efficiency Research Articles

Material flow analysis of an upgraded anaerobic digestion treatment plant with separated utilization of carbon and nitrogen of food waste

Anaerobic digestion of food waste can recover carbon in the form of biogas, while the high concentration of ammonia nitrogen in the digestion effluent becomes troublesome. Therefore, some new treatment plants use three-phase centrifugation to separate homogenized food waste into nitrogen-rich fine slag for insect cultivation and carbon-rich liquid for anaerobic digestion. To analyze the effects of the carbon–nitrogen separation, an upgraded plant’s material and elementary flows were investigated. The three-phase separation process redistributed carbon and nitrogen, and the biogas slurry was the primary output. The principal endpoint for C was the crude oil, capturing 57.1 ± 13.1 % of the total input; the find slag collected 48.3 ± 6.9 % of the total N input, and the biogas slag accepted 52.9 ± 4.4 % of the P input. The carbon–nitrogen separation strategy can improve digestion efficiency and increase treatment benefits significantly, marking a promising direction for future developments in food waste utilization.

Optimization and experimental study of variable frequency intermittent stirring strategy for anaerobic digestion based on CFD

The stirring strategy has a significant effect on the efficiency of anaerobic digestion (AD). In this study, the suspension velocity of multi-layer blade particles was characterized based on CFD simulation, and a variable frequency intermittent stirring strategy was proposed to study the stirring effect and mixing energy consumption. Firstly, the fermentation cycle was divided into three parts, and three stirring speeds and mixing times were set for each part to carry out an orthogonal combination test. Taking methane production and mixing energy consumption as optimization indexes, a net energy output equation was established to optimize the new stirring strategy. The results showed that the new variable frequency intermittent stirring strategy could effectively improve digestion efficiency. This provides a theoretical basis for setting a stirring strategy in a biogas project.

Accelerated stabilization of high solid sludge by thermal hydrolysis pretreatment in autothermal thermophilic aerobic digestion (ATAD) process.

Autothermal thermophilic aerobic digestion (ATAD) is a rapid biological treatment technology for sludge stabilization. To improve digestion efficiency and shorten stabilization time, thermal hydrolysis pretreatment was employed before ATAD of high solid sludge. The results showed that accelerated stabilization of high solid sludge (total solid=10.1%) was achieved by thermal hydrolysis pretreatment with volatile solid removal efficiency of 40.3% after 8 days of ATAD, 11 days earlier than unpretreated sludge. The enhanced release and hydrolysis of intracellular organics resulted in a solubilization degree of 45.3%. The reduced sludge viscosity and improved fluidity after thermal hydrolysis facilitated mixing, aeration and organics degradation during ATAD. Excitation emission matrix analysis indicated that the fluorescence intensity of soluble microbial byproduct and tyrosine-like protein increased markedly after thermal hydrolysis and decreased after ATAD. The proportion of high molecular weight (MW>10kDa) substances in the supernatant increased significantly after thermal hydrolysis, while the low MW (MW<1kDa) substances decreased after ATAD. The significant difference in microbial composition between the pretreatment and control groups elucidated the accelerated sludge stabilization under thermal hydrolysis. This work provides an efficient and practical strategy to achieve rapid stabilization of high solid sludge.

Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells.

Heterogeneity in composition is inherent in all cell populations, even those containing a single cell type. Single-cell proteomics characterization of cell heterogeneity is currently achieved by antibody-based technologies, which are limited by the availability of high-quality antibodies. Herein we report a simple, easily implemented, mass spectrometry (MS)-based targeted proteomics approach, termed cLC-SRM (carrier-assisted liquid chromatography coupled to selected reaction monitoring), for reliable multiplexed quantification of proteins in low numbers of mammalian cells. We combine a new single-tube digestion protocol to process low numbers of cells with minimal loss together with sensitive LC-SRM for protein quantification. This single-tube protocol builds upon trifluoroethanol digestion and further minimizes sample losses by tube pretreatment and the addition of carrier proteins. We also optimized the denaturing temperature and trypsin concentration to significantly improve digestion efficiency. cLC-SRM was demonstrated to have sufficient sensitivity for reproducible detection of most epidermal growth factor receptor (EGFR) pathway proteins expressed at levels ≥30 000 and ≥3000 copies per cell for 10 and 100 mammalian cells, respectively. Thus, cLC-SRM enables reliable quantification of low to moderately abundant proteins in less than 100 cells and could be broadly useful for multiplexed quantification of important proteins in small subpopulations of cells or in size-limited clinical samples. Further improvements of this method could eventually enable targeted single-cell proteomics when combined with either SRM or other emerging ultrasensitive MS detection.

Covalent organic framework-coated magnetic graphene as a novel support for trypsin immobilization.

Deep and efficient proteolysis is the critical premise in mass spectrometry-based bottom-up proteomics. It is difficult for traditional in-solution digestion to meet the requirement unless prolonged digestion time and enhanced enzyme dosage are employed, which makes the whole workflow time-consuming and costly. The abovementioned problems could be effectively ameliorated by anchoring many proteases on solid supports. In this work, covalent organic framework-coated magnetic graphene (MG@TpPa-1) was designed and prepared as a novel enzyme carrier for the covalent immobilization of trypsin with a high degree of loading (up to 268μgmg-1). Profiting from the advantages of magnetic graphene and covalent organic frameworks, the novel trypsin bioreactor was successfully applied for the enzymatic digestion of a model protein with dramatically improved digestion efficiency, stability, and reusability. Complete digestion could be achieved in a time period as short as 2min. For the digestion of proteins extracted from Amygdalus pedunculata, a total of 2833 protein groups were identified, which was slightly more than those obtained by 12h of in-solution digestion (2739 protein groups). All of the results demonstrate that MG@TpPa-1-trypsin is an excellent candidate for sample preparation in a high-throughput proteomics analysis. Graphical abstract Covalent organic frameworks-coated magnetic graphene was prepared as novel carrier for highly efficient tryptic immobilization.

Application of Dual Protease Column for HDX-MS Analysis of Monoclonal Antibodies

A co-immobilized, dual protease column was developed and implemented to more efficiently digest IgG molecules for hydrogen/deuterium exchange mass spectrometry (HDX-MS). The low-pH proteolytic enzymes pepsin and type XIII protease from Aspergillus were packed into a single column to most effectively combine the complementary specificities. The method was optimized using an IgG2 monoclonal antibody as a substrate because they are known to be more difficult to efficiently digest. The general applicability of the method was then demonstrated using IgG1 and IgG4 mAbs. The dual protease column and optimized method yielded improved digestion efficiency, as measured by the increased number of smaller, overlapping peptides in comparison with pepsin or type XIII alone, making HDX-MS more suitable for measuring deuterium uptake with higher resolution. The enhanced digestion efficiency and increased sequence coverage enables the routine application of HDX-MS to all therapeutic IgG molecules for investigations of higher order structure, especially when posttranslational and storage-induced modifications are detected, providing further product understanding for structure–function relationships and ultimately ensuring clinical safety and efficacy.

Influence of steam explosion pretreatment on the anaerobic digestion of vinegar residue.

Vinegar residue is the by-product in the vinegar production process. The large amount of vinegar residue has caused a serious environmental problem owing to its acidity and corrosiveness. Anaerobic digestion is an effective way to convert agricultural waste into bioenergy, and a previous study showed that vinegar residue could be treated by anaerobic digestion but still had room to improve digestion efficiency. In this study, steam explosion at pressure of 0.8, 1.2, and 1.5 MPa and residence time of 5, 10, 15, and 20 min were used to pretreat vinegar residue to improve methane production, respectively. Scanning electron microscopy and X-ray diffraction analyses were applied to validate structural changes of vinegar residue after steam explosion. Results showed that steam explosion pretreatment could destroy the structure of lignocellulose by removing the hemicellulose and lignin, and improve the methane yield effectively. Steam explosion-treated vinegar residue at 0.8 MPa for 5 min produced the highest methane yield of 153.58 mL gVS (-1), which was 27.65% (significant, α < 0.05) more than untreated vinegar residue (120.31 mL gVS (-1)). The analyses of pH, total ammonia-nitrogen, total alkalinity, and volatile fatty acids showed that steam explosion did not influence the stability of anaerobic digestion. This study suggested that steam explosion pretreatment on vinegar residue might be a promising approach and it is worth further study to improve the efficiency of vinegar residue waste utilisation.

Development of a sample preparation method for monitoring correct disulfide linkages of monoclonal antibodies by liquid chromatography–mass spectrometry

The number and positions of disulfide linkages in a therapeutic monoclonal antibody (mAb) play a crucial role in forming and stabilizing a correct mAb structure that is critical to its function. Peptide mapping by liquid chromatography–mass spectrometry (LC–MS) analysis of enzymatically digested mAb under nonreducing condition is a powerful method for disulfide linkage characterization to ensure mAb drug function and quality. However, the development of a robust sample preparation method with improved digestion efficiency and minimized disulfide scrambling for disulfide linkage analysis is essential but challenging. In this study, a sample preparation method for analysis of correct disulfide linkages in therapeutic mAbs was developed. Instead of common trypsin digestion, Lys-C plus trypsin was used in this approach to improve digestion efficiency. In addition, lower digestion temperature (25 °C) and lower digestion pH (pH 6.8) were also examined to minimize disulfide scrambling. Our results showed that Lys-C plus trypsin digestion at pH 6.8 and 25 °C is a better sample preparation condition for all therapeutic mAbs tested in this study because of a better digestion efficiency (all expected disulfide linkages can be confidently observed) and minimal disulfide scrambling.

DESIGN OF DOMESTIC SCALE BIO-DIGESTER ASSEMBLY, EXPERIMENTATION FOR BIOGAS PRODUCTION AND COMPARISON OF RESULTS WITH CONVENTIONAL DIGESTER

The work explored various digesters that are currently under implementation, explored their pros and cons and designed a digestion gas collection mechanism suitable for small scale applications. Internal mixing being uneconomical in the domestic and individual scale, it is not being practiced which has to be compensated with large hydraulic retention time to prevent sludge accumulation and scum formation. This in turn reduces the plant capacity for gas production. Our design configuration incorporates automatic and controlled pressure swing mechanisms incorporated by the gas production which acts as the driving force for slurry circulation in the digester and promotes gas recovery as well as improve digestion efficiency. The configuration was run in 20 l and 50 L scales and observed for the physical constraints encountered during operation, recorded for change in digestion parameters with changes in substrate and organic loading rates and also recorded the gas production which was compared with the results in the literature as well as conventionally operated domestic scale digesters.

An innovative microwave-assisted digestion method with diluted hydrogen peroxide for rapid extraction of trace elements in coal samples followed by inductively coupled plasma-mass spectrometry

The present work describes the development of a novel and efficient microwave-assisted digestion method based on the use of diluted hydrogen peroxide (H2O2) for rapid extraction of trace elements in coal samples prior to inductively coupled plasma-mass spectrometry (ICP-MS) determination. Diluted H2O2 was used to reduce acid wastes generated in analytical laboratories. After H2O2 digestion, the addition of diluted nitric acid was required to enhance the extraction of trace elements. Key parameters that affected the extraction efficiencies of the target analytes were examined using coal certified reference material (SARM 20). Improved digestion efficiency and quantitative trace element recoveries were obtained when 7molL−1, 200°C, 0.1g and 10min were used for [HNO3], temperature, coal amount and time, respectively. Agreement of 92–114% between the obtained results and the certified values was observed under the optimum conditions for most of the investigated trace elements in all three certified reference materials (SARMs 18, 19 & 20). Moderate extraction efficiencies (70–89%) were achieved for Sc, V, Cr, Y, and La, while Ti and Hf showed poor accuracy (10–50%). The use of H2O2 as the only digestion reagent resulted in colourless digest solutions with 0.084molL−1 of nitric acid. The diluted acid might have caused low method detection limits (MDLs) of 0.003–3.5μgg−1 and method quantification limits (MQLs) of 0.01–12.5μgg−1 for the current study due to less spectrometric interferences. These interferences are commonly caused by concentrated acids frequently used in classic acid digestion methods. The proposed microwave-assisted digestion method was comparable with literature reports and was successfully applied for trace element analysis in three South Africa coal samples.

Development and validation of a liquid chromatography tandem mass spectrometry assay for the quantitation of a protein therapeutic in cynomolgus monkey serum.

We have developed and fully validated a fast and simple LC-MS/MS assay to quantitate a therapeutic protein BMS-A in cynomolgus monkey serum. Prior to trypsin digestion, a recently reported sample pretreatment method was applied to remove more than 95% of the total serum albumin and denature the proteins in the serum sample. The pretreatment procedure simplified the biological sample prior to digestion, improved digestion efficiency and reproducibility, and did not require reduction and alkylation. The denatured proteins were then digested with trypsin at 60 °C for 30 min and the tryptic peptides were chromatographically separated on an Acquity CSH column (2.1 mm × 50 mm, 1.7 μm) using gradient elution. One surrogate peptide was used for quantitation and another surrogate peptide was selected for confirmation. Two corresponding stable isotope labeled peptides were used to compensate variations during LC-MS detection. The linear analytical range of the assay was 0.50-500 μg/mL. The accuracy (%Dev) was within ± 5.4% and the total assay variation (%CV) was less than 12.0% for sample analysis. The validated method demonstrated good accuracy and precision and the application of the innovative albumin removal sample pretreatment method improved both assay sensitivity and robustness. The assay has been applied to a cynomolgus monkey toxicology study and the serum sample concentration data were in good agreement with data generated using a quantitative ligand-binding assay (LBA). The use of a confirmatory peptide, in addition to the quantitation peptide, ensured the integrity of the drug concentrations measured by the method.

Optimization of Enzyme Digestion Conditions for Quantification of Glycated Hemoglobin Using Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

Glycated hemoglobin (HbA1c) is used as an index of mean glycemia over prolonged periods. This study describes anoptimization of enzyme digestion conditions for quantification of non-glycated hemoglobin (HbA0) and HbA1c as diagnosticmarkers of diabetes mellitus. Both HbA0 and HbA1c were quantitatively determined followed by enzyme digestion using isotopedilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) with synthesized N-terminal hexapeptides asstandards and synthesized isotope labeled hexapeptides as internal standards. Prior to quantification, each peptide was additionallyquantified by amino acid composition analysis using ID-LC-MS/MS via acid hydrolysis. Each parameter was consideredstrictly as a means to improve digestion efficiency and repeatability. Digestion of hemoglobin was optimized when using100 mM ammonium acetate (pH 4.2) and a Glu-C-to-HbA1c ratio of 1:50 at 37oC for 20 h. Quantification was satisfactorilyreproducible with a 2.6% relative standard deviation. These conditions were recommended for a primary reference method ofHbA1c quantification and for the certification of HbA1c reference material.

Global proteomic profiling of the membrane compartment of bovine testis cell populations

Spermatogonial stem cells hold enormous potential in mammalian reproductive medicine through the preservation of gametes, the restoration of fertility, enhancement of germ-lineage genetic manipulation and the improvement in our understanding of stem cell biology. Here we describe the protein profiles of the membrane compartment of bovine testicular cell isolates which were enriched for germ cells using differential plating. The isolated cells were characterised with antibodies to UCHL1 (previously known as PGP9.5) for type A spermatogonia; DDX4 (previously known as VASA) for germ cells and vimentin for Sertoli cells. Ultracentrifugation techniques were used to specifically isolate cell membranes, with membrane protein identifications significantly increased when compared to whole cell lysates. We utilised the filter-aided sample preparation protocol for improved digestion efficiency of membrane proteins. Using ESI-LC-MS/MS, we compared the proteins present in two cell populations. A total of 1,387 proteins were identified in bovine testis cell isolates, of which 39% were membrane-associated. A total of 64 proteins were differentially expressed in the non-adhered fraction (enriched for undifferentiated germ cells), of which 16 were unique to this cell population and the remaining 48 showed a two-fold change as judged by spectral counting. This analysis revealed a number of candidate germ cell markers including the known markers, DDX4 and UCHL1. The proteomic profiles generated in this study support and complement transcriptomics studies and reinforce the potential of proteomics in identifying and characterising the protein effectors of self-renewal and/or differentiation in stem cells. Normal 0 false false false EN-AU X-NONE X-NONE

Polyacrylamide Gel with Switchable Trypsin Activity for Analysis of Proteins

Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG-trypsin-aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline pH, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low pH. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis.

Dephosphorylation of intact glycoprotein to greatly improve digestion efficiency coupled with matrix-assisted laser desorption/ionization–Fourier transform ion cyclotron resonance mass spectrometric analysis

Sialylation is essential for a variety of cellular functions. Herein, we used bovine fetuin with three potential N-linked glycosylation sites containing complex-type glycan structures, four potential O-linked glycosylation sites and six potential phosphorylation sites as a model compound to develop a highly-efficient digestion strategy for sialylated glycoproteins and efficient enrichment strategy for sialylated glycopeptides using titanium dioxide. The former according to the process of alkaline phosphatase digestion followed by tryptic digestion and then proteinase K digestion could greatly improve the enzymatic efficiency on fetuin, and the latter could obviously enhance the enrichment efficiency for multisialylated glycopeptides using phosphoric acid solution as elution buffer. The mass spectra of the enriched glycopeptides derived from fetuin reveal that several series of the ion clusters with mass difference of 291Da correspond to the presence of multisialylated glycopeptides. In addition, the approach was applied to characterize the sialylated status of α2-macroglobulin and transferrin, respectively, from the sera of healthy subjects and sex- and age-matched patients with thyroid cancer, and their spectra indicate that the change in the amount of the glycoforms containing different number of sialic acid (SA) residues from one glycosylation site may be used to differentiate between healthy subjects and cancer cases.

Mechanism exploration of adsorption-immobilized enzymatic reactor using polymer-coated silica microbeads

A verified mechanism of adsorption-immobilized enzymatic reactor for enhanced proteolysis is presented. Silica microbeads coated with poly (diallyldimethylammonium chloride) (PDDA) or poly (styrene sulfonate) (PSS) were used to trap trypsin and proteins on the surface through electrostatic interactions in order to improve digestion efficiency. Charge states measured by zeta-potentials showed their positively and negatively charged respectively. We found that high proteolytic efficiency could be achieved only if both proteases and proteins were adsorbed by materials. Once the proteins and proteases were confined together in a nanoscopic area, the enrichment of the substrate could lead to a high performance proteolytic effect. Electrostatic interactions were considered as the predominant adsorption factor rather than hydrophilic/hydrophobic interactions. In less than 5min, in the presence of PSS-coated silica beads, 10 peptides digested from positively-charged cytochrome C were detected by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF), with the high sequence coverage up to 63%, while using PDDA-coated silica beads or conventional in-solution digestion yielded only 5 detectable peptides and 39% sequence coverage was obtained. Ovalbumin seemed incompatible with any kind of charged-material-aided tryptic digestion. The mechanism of adsorption-immobilized enzymatic processes has also been studied in detail. The adsorption equilibrium was proven to be attained in less than one minute, and the proteolytic procedure was regarded as the rate-determining step. This study provides a reasonable mechanism for an adsorption-material catalyzed proteolytic procedure and a promising guideline for designing the next generation of high-performance enzymatic reactors.

High rate mesophilic, thermophilic, and temperature phased anaerobic digestion of waste activated sludge: A pilot scale study

The paper reports the findings of a two-year pilot scale experimental trial for the mesophilic (35°C), thermophilic (55°C) and temperature phased (65+55°C) anaerobic digestion of waste activated sludge. During the mesophilic and thermophilic runs, the reactor operated at an organic loading rate of 2.2kgVS/m3d and a hydraulic retention time of 20days. In the temperature phased run, the first reactor operated at an organic loading rate of 15kgVS/m3d and a hydraulic retention time of 2days while the second reactor operated at an organic loading rate of 2.2kgVS/m3d and a hydraulic retention time of 18days (20days for the whole temperature phased system).The performance of the reactor improved with increases in temperature. The COD removal increased from 35% in mesophilic conditions, to 45% in thermophilic conditions, and 55% in the two stage temperature phased system. As a consequence, the specific biogas production increased from 0.33 to 0.45 and to 0.49m3/kgVSfed at 35, 55, and 65+55°C, respectively. The extreme thermophilic reactor working at 65°C showed a high hydrolytic capability and a specific yield of 0.33gCOD (soluble) per gVSfed. The effluent of the extreme thermophilic reactor showed an average concentration of soluble COD and volatile fatty acids of 20 and 9g/l, respectively. Acetic and propionic acids were the main compounds found in the acids mixture. Because of the improved digestion efficiency, organic nitrogen and phosphorus were solubilised in the bulk. Their concentration, however, did not increase as expected because of the formation of salts of hydroxyapatite and struvite inside the reactor.

Is post-feeding thermotaxis advantageous in elasmobranch fishes?

The effects of post-feeding thermotaxis on ileum evacuation and absorption rates were examined in the laboratory using two elasmobranch species, the Atlantic stingray Dasyatis sabina, which inhabits thermally variable environments, and the whitespotted bamboo shark Chiloscyllium plagiosum, a stenothermic fish living on Indo-Pacific reefs. Experiments at temperatures similar to those experienced in nature revealed temperature change had no significant effect on C. plagiosum absorption or evacuation rates, suggesting stenothermic sharks cannot exploit temperature differences as a means to improve digestion efficiency. On the other hand, D. sabina showed significantly lower evacuation and absorption rates at lower temperatures. The relative decrease was greater for evacuation (Q₁₀ = 3·08) than absorption rates (Q₁₀ = 2·20), resulting in a significant increase in total absorption, suggesting D. sabina can benefit from using shuttling behaviour to exploit thermal variability in their environment to maximize energetic uptake.

Enhanced digestion efficiency, peptide ionization efficiency, and sequence resolution for protein hydrogen/deuterium exchange monitored by Fourier transform ion cyclotron resonance mass spectrometry.

Solution-phase hydrogen/deuterium exchange (HDX) monitored by high-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry offers a rapid method to study protein conformations and protein-protein interactions. Pepsin is usually used to digest proteins in HDX and is known for lack of cleavage specificity. To improve digestion efficiency and specificity, we have optimized digestion conditions and cleavage preferences for pepsin and protease type XIII from Aspergillus saitoi. A dilution series of the proteases was used to determine the digestion efficiency for several test proteins. Protease type XIII prefers to cleave on the C-terminal end of basic amino acids and produced the highest number of fragments and the best sequence coverage compared to pepsin or protease type XVIII from Rhizhopus. Furthermore, protease type XIII exhibited much less self-digestion than pepsin and thus is superior for HDX experiments. Many highly overlapped segments from protease type XIII and pepsin digestion, combined with high-resolution FTICR mass spectrometry, provide high sequence resolution (to as few as one or two amino acids) for the assignment of amide hydrogen exchange rate. Our H/D exchange results correlate well with the secondary and tertiary structure of myoglobin. Such assignments of highly overlapped fragments promise to greatly enhance the accuracy and sequence resolution for determining conformational differences resulting from ligand binding or protein-protein interactions.

A bifunctional monolithic column for combined protein preconcentration and digestion for high throughput proteomics research

Enzymatic digestion of proteins is a key step in protein identification by mass spectrometry (MS). Traditional solution-based protein digestion methods require long incubation times and are limitations for high throughput proteomics research. Recently, solid phase digestion (e.g. trypsin immobilization on solid supports) has become a useful strategy to accelerate the speed of protein digestion and eliminate autodigestion by immobilizing and isolating the enzyme moieties on solid supports. Monolithic media is an attractive support for immobilization of enzymes due to its unique properties that include fast mass transfer, stability in most solvents, and versatility of functional groups on the surfaces of monoliths. We prepared immobilized trypsin monolithic capillaries for on-column protein digestion, analyzed the digested peptides through LC/FTICR tandem MS, and compared peptide mass fingerprinting by MALDI-TOF-MS. To further improve the digestion efficiency for low abundance proteins, we introduced C4 functional groups onto the monolith surfaces to combine on-column protein enrichment and digestion. Compared with immobilized trypsin monolithic capillaries without C4, the immobilized trypsin-C4 monolith showed improved digestion efficiency. A mechanism for increased efficiency from the combination of sample enrichment and on-column digestion is also proposed in this paper. Moreover, we investigated the effects of organic solvent on digestion and detection by comparing the observed digested peptide sequences. Our data demonstrated that all columns showed good tolerance to organic solvents and maintained reproducible enzymatic activity for at least 30 days.