Amino‐functionalized macroporous silica for efficient tryptic digestion in acidic solutions

Amino‐functionalized macroporous silica foam (NH₂‐MOSF) has been developed as a host reactor to realize highly efficient proteolysis in acidic solutions where normal tryptic reactions cannot occur. The digestion protocol consists simply of adding the functionalized NH₂‐MOSF into the protein and trypsin solutions without altering the bulk pH or preloading the enzymes on the materials. With this protocol, digestion of sample fractions from LC can be efficiently realized in the acidic solutions directly. Digestion of a protein fraction extracted from rat liver tissue after LC separation was performed to illustrate this principle, where 103 proteins were successfully identified at pH 3 after 1.5 h of tryptic digestion.     

High-throughput protein digestion by trypsin-immobilized monolithic silica with pipette-tip formula

Based on the monolithic silica gel materials with hierarchical pore structure and on the SPE devices (MonoTip) developed thereof, a trypsin-immobilized monolithic silica in a pipette tip (MonoTip Trypsin) suitable for digesting proteins has been newly developed. The surface of monolithic silica fixed into the tip was chemically modified with trypsin via an aminopropyl group. Trypsin-immobilized monolith successfully performed a rapid digestion of reduced and alkylated proteins with only a few times pipetting operation for the pre-treatment procedure of chromatographic analysis. The novel solid-phase digestion tool using monolithic silica allows a high-throughput trypsin proteolysis of bio-substances in proteomics.     

High-throughput protein digestion by trypsin-immobilized monolithic silica with pipette-tip formula

Based on the monolithic silica gel materials with hierarchical pore structure and on the SPE devices (MonoTip®) developed thereof, a trypsin-immobilized monolithic silica in a pipette tip (MonoTip® Trypsin) suitable for digesting proteins has been newly developed. The surface of monolithic silica fixed into the tip was chemically modified with trypsin via an aminopropyl group. Trypsin-immobilized monolith successfully performed a rapid digestion of reduced and alkylated proteins with only a few times pipetting operation for the pre-treatment procedure of chromatographic analysis. The novel solid-phase digestion tool using monolithic silica allows a high-throughput trypsin proteolysis of bio-substances in proteomics.     

Gold nanoparticle assembly microfluidic reactor for efficient on-line proteolysis

A microchip reactor coated with a gold nanoparticle network entrapping trypsin was designed for the efficient on-line proteolysis of low level proteins and complex extracts originating from mouse macrophages. The nanostructured surface coating was assembled via a layer-by-layer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles. The assembly process was monitored by UV-visible spectroscopy, atomic force microscopy, and quartz crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted Gamma(max) and K values were estimated to be 1.2 x 10(-7) mol/m(2) and 4.1 x 10(5) M(-1), respectively. An enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible gold nanoparticle network with a high loading capacity, preserved its bioactivity. The maximum proteolytic rate of the adsorbed trypsin was 400 mM/(min.microg). Trace amounts of proteins down to femtomole per analysis were digested using the microchip reactor, and the resulting tryptic products were identified by MALDI-TOF MS/MS. The protein mixtures extracted from the mouse macrophages were efficiently identified by on-line digestion and LC-ESI-MS/MS analysis.     

Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith

A metal‐ion chelate immobilized enzyme reactor (IMER) supported on organic–inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu²⁺, trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed‐phase liquid chromatography with ESI‐MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in‐solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu²⁺ via EDTA followed by trypsin immobilization with fresh Cu²⁺ introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ～5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in‐solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high‐throughput proteome profiling.     

Immobilization of trypsin on poly(urea-formaldehyde)-coated fiberglass cores in microchip for highly efficient proteolysis

Trypsin was covalently immobilized on poly(urea‐formaldehyde)‐coated fiberglass cores based on the condensation reaction between poly(urea‐formaldehyde) and trypsin for efficient microfluidic proteolysis in this work. Prior to use, a piece of the trypsin‐immobilized fiber was inserted into the main channel of a microchip under a magnifier to form a core‐changeable bioreactor. Because trypsin was not permanently immobilized on the channel wall, the novel bioreactor was regenerable. Two standard proteins, hemoglobin (HEM) and lysozyme (LYS), were digested by the unique bioreactor to demonstrate its feasibility and performance. The interaction time between the flowing proteins and the immobilized trypsin was evaluated to be less than 10 s. The peptides in the digests were identified by MALDI‐TOF MS to obtain PMF. The results indicated that digestion performance of the microfluidic bioreactor was better than that of 12‐h in‐solution digestion.     

Alternating current-assisted on-plate proteolysis for MALDI-TOF MS peptide mapping

In this report, alternating current-assisted on-plate proteolysis has been developed for rapid peptide mapping. Protein solutions containing trypsin were allowed to digest directly on the spots of a stainless steel MALDI plate with the assistance of low-voltage alternating current electricity. Alternating current (AC) was allowed to pass through the protein solutions via the MALDI plate and a platinum disc electrode. The feasibility and performance of the novel proteolysis approach were investigated by the digestion of BSA and cytochrome c (Cyt-c). It was demonstrated that AC substantially enhanced the efficiency of proteolysis and the digestion time was significantly reduced to 5 min. The digests were identified by MALDI-TOF MS with sequence coverages of 42% (BSA) and 77% (Cyt-c) that were comparable to those obtained by using conventional in-solution tryptic digestion. The present proteolysis strategy is simple and efficient, offering great promise for MALDI-TOF MS peptide mapping.     

Efficient on-chip proteolysis system based on functionalized magnetic silica microspheres

An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.     

Optimization of mass spectrometry-compatible surfactants for shotgun proteomics

An optimization and comparison of trypsin digestion strategies for peptide/protein identifications by microLC-MS/MS with or without MS compatible detergents in mixed organic-aqueous and aqueous systems was carried out in this study. We determine that adding MS-compatible detergents to proteolytic digestion protocols dramatically increases peptide and protein identifications in complex protein mixtures by shotgun proteomics. Protein solubilization and proteolytic efficiency are increased by including MS-compatible detergents in trypsin digestion buffers. A modified trypsin digestion protocol incorporating the MS compatible detergents consistently identifies over 300 proteins from 5 microg of pancreatic cell lysates and generates a greater number of peptide identifications than trypsin digestion with urea when using LC-MS/MS. Furthermore, over 700 proteins were identified by merging protein identifications from trypsin digestion with three different MS-compatible detergents. We also observe that the use of mixed aqueous and organic solvent systems can influence protein identifications in combinations with different MS-compatible detergents. Peptide mixtures generated from different MS-compatible detergents and buffer combinations show a significant difference in hydrophobicity. Our results show that protein digestion schemes incorporating MS-compatible detergents generate quantitative as well as qualitative changes in observed peptide identifications, which lead to increased protein identifications overall and potentially increased identification of low-abundance proteins.     

High-throughput peptide mass mapping using a microdevice containing trypsin immobilized on a porous polymer monolith coupled to MALDI TOF and ESI TOF mass spectrometers

An enzymatic microreactor with a volume of 470 nL has been prepared by immobilizing trypsin on a 10 cm long reactive porous polymer monolith located in a 100 microm i.d. fused silica capillary. This reactor affords suitable degrees of digestion of proteins even after very short residence times of less than 1 min. The performance is demonstrated with the digestion of eight proteins ranging in molecular mass from 2848 to 77 754. The digests were analyzed using mass spectrometry in two modes: off-line MALDI and in-line nanoelectrospray ionization. The large numbers of identified peptides enable a high degree of sequence coverage and positive identification of the proteins. The extent of sequence coverage decreases as the molecular mass of the digested protein increases.     

A combination method of chemical with enzyme reactions for identification of membrane proteins

A simple method for effective analysis of various proteins has been developed, including membrane proteins, with LC-MS/MS, using CNBr and acetic acid cleavage in one reaction for the digestion of both the M/ and /D/ positions within the target proteins. This dual chemical reaction has been compared with traditional CNBr or an acid cleavage method using a rat kidney membrane fraction and it showed an advantage of the dual reaction with respect to a high number of peptides detected and a high protein recovery. Furthermore, when this dual chemical reaction was combined with trypsin digestion, the number of proteins surprisingly increased approximately 3.0 times more than in the cases with the trypsin digestion only. It was also 1.9 times more than in cases dealing with Tube-Gel trypsin digestion, which is one of the most efficient digestion methods. In addition, it was shown that this dual chemical reaction could be applied to an in-gel digestion. Using the combination of the chemical and enzyme reaction, 172 proteins including 95 membrane proteins were identified. This indicated that this method is one of the efficient systems in single MS/MS analysis. In particular, many membrane proteins identified in this study were detected by a new combination, but not by a traditional trypsin digestion method.     

Rapid protein identification using monolithic enzymatic microreactor and LC-ESI-MS/MS

A monolithic enzymatic microreactor was prepared in a fused-silica capillary by in situ polymerization of acrylamide, glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, followed by ammonia solution treatment, glutaraldehyde activation and trypsin modification. The choice of acrylamide as co-monomer was found useful to improve the efficiency of trypsin modification, thus, to increase the enzyme activity. The optimized microreactor offered very low back pressure, enabling the fast digestion of proteins flowing through the reactor. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at high flow rate. The digests were then characterized by CE and HPLC-MS/MS with the sequence coverage of 57.7%. The digestion efficiency was found over 230 times as high as that of the conventional method. In addition, for the first time, protein digestion carried out in a mixture of water and ACN was compared with the conventional aqueous reaction using MS/MS detection, and the former solution was found more compatible and more efficient for protein digestion.     

Hydrophilic polydopamine‐coated magnetic graphene nanocomposites for highly efficient tryptic immobilization

In this work, polydopamine‐coated magnetic graphene (MG@PDA) nanocomposites were synthesized by a facile method. Trypsin was then directly immobilized on the surface of the nanocomposites through simple PDA chemistry with no need for introducing any other coupling groups. The as‐made MG@PDA nanocomposites inherit not only the large surface area of graphene which makes them capable of immobilizing high amount of trypsin (up to 0.175 mg/mg), but also the good hydrophilicity of PDA which greatly improves their biocompatibility. Moreover, the strong magnetic responsibility makes them easy to be separated from the digested peptide solution when applying a magnetic field. The feasibility of the trypsin‐immobilized MG@PDA (MG@PDA‐trypsin) nanocomposites for protein digestion was investigated and the results indicated their high digestion efficiency in a short digestion time (10 min). In addition, the reusability and stability of the MG@PDA‐trypsin nanocomposites were also tested in our work. To further confirm the efficiency of MG@PDA‐trypsin nanocomposites for proteome analysis, they were applied to digest proteins extracted from skimmed milk, followed by nano RPLC‐ESI‐MS/MS analysis, and a total of 321 proteins were identified, much more than those obtained by 16‐h in‐solution digestion (264 proteins), indicating the great potential of MG@PDA‐trypsin nanocomposites as the supports for high‐throughput proteome study.     

Infrared-assisted tryptic proteolysis for peptide mapping

In this report, infrared (IR) radiation was employed to enhance the efficiency of tryptic proteolysis for peptide mapping. Protein solutions containing trypsin in sealed transparent Eppendorf tubes were allowed to digest under an IR lamp at 37 degrees C. The feasibility and performance of the novel proteolysis approach were demonstrated by the digestion of BSA and myoglobin (MYO) and the digestion time was significantly reduced to 5 min. The obtained digests were identified by MALDI-TOF MS with the sequence coverages of 69% (BSA) and 90% (MYO) that were much better than those obtained by conventional in-solution tryptic digestion. The present IR-assisted proteolysis strategy is simple and efficient, offering great promise for high-throughput protein identification.     

An Efficient In-gel Digestion Protocol for Mass Spectral Analysis by MALDI-TOF-MS and MS/MS and Its Use for Proteomic Analysis of Vigna mungo Leaves

An efficient protocol for in-gel digestion of Coomassie-stained protein spots has been established for mass analysis by matrix-assisted laser desorption/ionization-mass spectrometry (MS) and for tandem mass spectrometry (MS/MS). Identification of Vigna mungo leaf proteome from two-dimensional gel electrophoresis was done employing the protocol. About 300 proteins spots were consistently detected in three replicate gels. Optimization of the destaining process, digestion using 25 ng/μl trypsin in 20 μl trypsin buffer, and omission of peptide extraction step significantly increased the number of matched peptides and sequence coverage. Reliable characterization of 109 proteins by MS as well as tandem sequencing by MS/MS (PRIDE Accession no. 15318) suggests the potential application of the modified protocol for high throughput proteome analysis to unravel disputes in characterization of plant proteins in fundamental or applied research.     

Discrimination of in vitro and in vivo digestion products of meat proteins from pork,beef, chicken,and fish

In vitro digestion products of proteins were compared among beef, pork, chicken, and fish. Gastric and jejunal contents from the rats fed these meat proteins were also compared. Cooked pork, beef, chicken, and fish were homogenized and incubated with pepsin alone or followed by trypsin. The digestion products with molecular weights of less than 3000 Da were identified with MALDI‐TOF‐MS and nano‐LC‐MS/MS. Gastric and jejunal contents obtained from the rats fed the four meat proteins for 7 days were also analyzed. After pepsin digestion, pork, and beef samples had a greater number of fragments in similarity than chicken and fish samples, but the in vitro digestibility was the greatest (p < 0.05) for pork and the smallest for beef samples. After trypsin digestion, the species differences were less pronounced (p > 0.05). A total of 822 and 659 peptides were identified from the in vitro and in vivo digestion products, respectively. Our results could interpret for the differences in physiological functions after the ingestion of different species of meat.     

A proteomic study of sodium/D-glucose cotransporter 1 (SGLT1): topology of loop 13 and coverage of other functionally important domains

In order to obtain further information about the structure and function of human sodium/D-glucose cotransporter 1 (hSGLT1), the recombinant protein was subjected, either after reconstitution into liposomes or in its free form, to proteolysis followed by nanoscale microcapillary liquid chromatography electrospray ionization tandem mass spectrometry (LC-MS/MS). The peptides released from SGLT1 proteoliposomes by trypsin bead digestion represented the early N-terminal, loop 7, and loop 9, supporting topology models that place these domains on the extracellular side of the protein. Trypsin bead digestion generated, however, also a number of peptides derived from loop 13 whose topology with regard to the membrane is hitherto a point of debate. Sequence coverage was provided from amino acids 559 to 644, suggesting that loop 13 is almost completely accessible at the extravesicular face of the proteoliposomes. These results support the notion that major parts of loop 13, essential for the interaction with transport inhibitors in vivo, are located extracellularly in intact cells. In-gel trypsin, chymotrypsin, and in particular trypsin/chymotrypsin digestion of recombinant SGLT1 in combination with LC-MS/MS provide extensive sequence coverage of the protein, including domains involved in sugar and inhibitor binding and potential phosphorylation sites. These studies demonstrate that proteomic analysis combined with mass spectrometry is a useful tool to characterize regions of SGLT1 that are important for its function and regulation.     

Open Tubular Lab-On-Column/Mass Spectrometry for Targeted Proteomics of Nanogram Sample Amounts

A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ∼1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.     

Utilization of newly developed immobilized enzyme reactors for preparation and study of immunoglobulin G fragments

The newly developed immobilized enzyme reactors (IMERs) with proteolytic enzymes chymotrypsin, trypsin or papain were used for specific fragmentation of high molecular-mass and heterogeneous glycoproteins immunoglobulin G (IgG) and crystallizable fragment of IgG (Fc). The efficiency of splitting or digestion were controlled by RP-HPLC. The specificity of digestion by trypsin reactor was controlled by MS. IMERs (trypsin immobilized on magnetic microparticles focused in a channel of magnetically active microfluidic device) was used for digestion of the whole IgG molecule. The sufficient conditions for IgG digestion in microfluidic device (flow rate, ratio S:E, pH, temperature) were optimized. It was confirmed that the combination of IMERs with microfluidic device enables efficient digestion of highly heterogeneous glycoproteins such as IgG in extremely short time and minimal reaction volume.     

Ionic-strength-dependent changes in the structure of the major protein of the human erythrocyte membrane.

The effect of ionic strength on the proteolysis by trypsin of the major membrane-penetrating protein (polypeptide 3) in the erythrocyte membrane was studied. Both the intracellular and extracellular regions of the protein are susceptible to trypsin proteolysis under hypo-osmotic conditions, whereas under iso-osmotic conditions the extracellular region of the protein is resistant to trypsin, and the intracellular region yields only two cleavage products with trypsin. Studies of the fragments obtained from polypeptide 3 by trypsin digestion under iso-osmotic conditions of 'ghosts' radioiodinated with lactoperoxidase confirmed our earlier conclusions that the polypeptide chain of polypeptide 3 traverses the membrane twice. Ionic-strength-dependent changes were also observed in the incorporation of iodine by lactoperoxidase into the individual extracellular tyrosine sites of the protein. These results show that polypeptide 3 undergoes ionic-strength-dependent changes in structure.