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In order to investigate the feasibility of in vitro screening the antitumor activity of natural compounds by trypsin, porcine trypsin was used to for screening test, which is marked by inhibition of enzyme activity. Four compounds, namely daidzin, genistin, matrine and oxymatrine, were selected as test subjects. The natural antitumor drug camptothecin was used as the control. The inhibitory effect was detected by two experimental methods: direct detection of trypsin activity inhibition and hydrolysis of bovine serum albumin by trypsin. The results showed the inhibitory effects of the four natural compounds on trypsin, and the inhibition rates of the four natural compounds were significantly different. The enzyme activity assay showed that the inhibitory effect of matrine was better than that of oxymatrine, indicating that trypsin had a good screening resolution. The inhibitory effect was significantly increased with the increased ratio of sample to trypsin, suggesting the structure-activity correlation and dose-effect correlation of the screening methods. Altogether, the experimental method of screening antitumor activity of natural compounds by trypsin has good application values. Since porcine trypsin is similar to human trypsin in terms of molecular structure and performance, it is more applicable for screening of antitumor efficacy of natural pharmacodynamic compounds.

Protein complexes are involved in a variety of biological activities. Accurate and comprehensive characterization of the structures and interactions of protein complexes is crucial in determining their biological functions. Chemical cross-linking coupled with mass spectrometry (CXMS) is an emerging investigative technique for protein complexes. CXMS enables the sensitive high-throughput analysis of protein complexes without the requirements of molecular weight and purification. These attributes have spurred the increased use of CXMS for the structure and interaction characterization of purified protein complexes and complicated cell lysate samples. CXMS utilizes chemical cross-linking reagents to covalently connect two reactive amino acids in or between proteins that are spatially close to each other. Subsequently, the cross-linked proteins are digested into cross-linked peptides, followed by LC-MS/MS analysis, as well as database searching to provide cross-linking information for the composition, interaction, and structural site distance restrictions of protein complex identification. Therefore, identification of cross-linked sites has a decisive influence on the characterization of protein complexes. This identification is limited by the unsatisfactory quality of the cross-linked peptide spectrum. Insufficient b/y fragment ions and poor continuity of amino acid sequence matching lead to low coverage and accuracy of cross-linked site identification. Based on the complementary feature of mirror-cutting digestion, an orthogonal digestion strategy based on LysargiNase combined with trypsin was developed in this study. Trypsin is the most commonly utilized digestion enzyme in proteomics, with extremely high enzyme activity and specificity. Trypsin generates C-terminally charged peptides after lysine (K) and arginine (R). LysargiNase is a mirror protease complementary to trypsin that cleaves before the K and R residues. This generates peptides with an N-terminal positively-charged residue. Owing to the different physical and chemical micro-environments of the cross-linked peptides digested by LysargiNase and trypsin, the behavior of their detection ability in MS analysis is diverse. Using the orthogonal digestion strategy, both simple and complicated cross-linked samples were analyzed in this study. For the analysis of bovine serum albumin (BSA), 291 pairs of non-redundant cross-linked sites were obtained, of which 216 pairs of cross-linked sites were provided by trypsin digestion, whereas 75 pairs of cross-linked sites were exclusively supplied by LysargiNase digestion. Except for the 35% increase in the number of identified cross-linked sites, 32% of the spectra of the commonly identified cross-linked peptides have better quality with more b-type fragment ions and consecutive sequence matching. Furthermore, for the Escherichia coli sample, 726 pairs of cross-linked sites were obtained in total, among which, 624 and 274 pairs were identified from trypsin and LysargiNase digestion, respectively. LysargiNase digestion yielded 120 individual cross-linked sites, which resulted in a 16% increase in single trypsin digestion. Consistent with the BSA sample, the quality was improved in 35% of the spectra of commonly identified cross-linked peptides. Corresponding to the identified cross-linked peptides, 242 structural constraints with 607 pairs of intra-cross-linked sites and 29 sets of protein-protein interactions with 119 pairs of inter-cross-linked sites were obtained. The collective results demonstrated that, mirror cutting-assisted orthogonal digestion strategy could significantly increase the number of identified fragment ions and amino acid sequences matching the continuity of the spectra by contributing b-and y-type ions, respectively. This improved the accuracy and coverage of cross-linked peptide identification. The findings additionally demonstrate the superiority of our method in the accurate identification of the cross-linked peptide spectra and the increased number of identified cross-linked sites. In a word, this method is expected to provide new insights for the large-scale and highly accurate characterization of protein complexes.

Proteomics is a fast-growing discipline that aims at systematic identification, quantification of proteins and their post-translational modifications in cells. Mass spectrometry-based shotgun proteomics technology is currently one of the mainstream methods for proteomics research. With this method, proteins need to be digested to peptides by site-specific proteases before they can be detected with mass spectrometry. Therefore, site-specific proteases played key roles in this process and so far, a variety of specific proteases have been developed and used in proteomics study. Particularly, the identification, characterization and development of proteases that cleave at the N-termini of corresponding amino acid residues, which are just mirrors to those of typical C-termini proteases, provide novel tools for proteomics analysis. In this review, we summarized the proprieties of LysargiNase, a most recently identified mirror trypsin, and its applications in proteomics research to promote its more widespread usage.

Proteomics is a powerful subject focusing on large-scale study of protein structures and functions. A complete enzymatic digestion of protein complexes is the key step in modern high-resolution and high-throughput mass spectrometry (MS)-based identification and quantification. To achieve MS analysis, both peptide sample pretreatment and data acquisition are prerequisite in proteomic studies. In this paper, we summarized both the enzymatic proprieties of three common proteolytic enzymes, Trypsin, Lys-C and Glu-C, the optimization of multi-enzyme combination and an advanced sample pretreatment in proteomics research.

Trypsin was treated by high pressure technology, and its spatial structure was changed, the relationship between structural changes and trypsin activity was investigated. The secondary structure change of trypsin after pressure treatment was observed by Fourier transform infrared spectroscopy(FTIR). Moreover its tertiary structure change was observed by fluorescence spectroscopy; and its activity was tested using Folin phenol method. The results showed that, compared with the untreated(0.1 MPa), trypsin activity change was significant(p<0. 05) under different pressure(100~600 MPa) treatment at 37 °C for 20 min. After treated with 300 MPa, its activity was 0. 386 times higher than the untreated. Secondary structure of trypsin was analysed using FTIR, and the peak area ratio of α-helix and β-tum in secondary structure was the maximum(2. 749); Endogenous fluorescence spectra intensity was the maximum (1 353) at excitation wavelength 295 nm, and was 4 262 at excitation wavelength 280 nm; exogenous fluorescent spectra intensity was 2 022 at excitation wavelength 228 nm, all these change was remarkable(p<0. 05) comparing with the untreated. Therefore, ultrahigh pressure processing influence on the spatial structure of trypsin and induce enzyme activity.change. Trypsin activity is relate to the peak area ratio of α-helix and β-turn and the exposure degree of Trp and other hydrophobic a mino acid residues and Tyr.

We applied immobilized enzyme reactors prepared with different sizes of magnetic particles into protein and proteome digestion. In addition, the influences of different sizes of the magnetic particles were studied on the reunion, enzyme efficiency and leakage sites. The experimental results showed that in comparison with the submicron magnetic particles, the amount of trypsin immobilized on the magnetic nanoparticles was 3. 5 times more than that of the submicron magnetic particles. However, the enzymatic efficiency was at the same level when the same amount of trypsin was used, and the reunion phenomenon was obviously improved when the size of the magnetic nanoparticles increased. Taking the immobilized enzyme reactor of 20 nm magnetic nanoparticles as an example, the digestion performance was further examined. The experimental results showed that rapid digestion could be achieved within 1 mm when the mass ratio of the trypsin and bovine serum albumin was 1:1. The peptide number of 0 missed cleavage site and the sequence coverage changed little after the protein was digested for 10 mm. It was concluded that the digestion efficiency of the immobilized enzyme reactor was much better than that of the in-solution digestion. When the immobilized enzyme reactors and the free trypsin were used for digestion, little differences of the leakage sites were found. Therefore, the immobilized enzyme reactors prepared with different sizes of magnetic particles can be applied in proteomic research for quick and efficient digestion.

As a kind of recognition molecule, aptamer has been studied and applied widely in numerous science fields in recent years. Immobilized enzymatic reactor has drawn much attention because of its striking advantages, such as high digestion efficiency and ease in coupling with the separation and detection systems. In this study, a novel microfluidic enzymatic chip, which immobilized trypsin based on aptamer, was prepared and proposed. An online analysis platform, which consisted of an aptamer-based chip and high performance liquid chromatography tandem mass spectrometry, was established by using a 6-port valve and applied to protein analysis. The enzymatic capacity and stability performance of chip reactor were characterized by using mixed protein sample, which consisted of bovine serum albumin (BSA), myoglobin (Mb) and cytochrome c (Cyt. c). The sample digestion time of the chip reactor was about 5.76 s while 1 microL/min of flow rate was adopted; and moreover, 5 ng of Mb was identified successfully with the sequence coverage of 37%. Furthermore, the sequence coverages and the relative standard deviations were 44.3% and 6.5% for BSA, 65.0% and 2.7% for Mb, 62.0% and 5.6% for Cyt. c respectively when 500 ng digest of mixed proteins were analyzed in three runs. According to experimental results, the online analysis platform possesses the ability of high sensitivity and good stability, which can provide a promising tool for rapid and high-throughput proteomics study in the near future.

Currently, the shotgun based strategy has been widely applied in proteomic research. In this strategy, protein identification relied on the identification of the corresponding proteolytic peptides. Therefore, rapid and efficient protein digestion is crucial for accurate protein identification and characterization. Even though traditional free protein digestion in solution has been widely adopted, it had a few inherent disadvantages including long incubation time, incomplete digestion and non-reusability of the protease. In this work, we developed a new type of trypsin immobilized on squamous polymer modified silica bead (SPMSB) for ultra fast and highly efficient protein digestion. The squamous polymer coated silica beads were prepared by surface initiated atom transfer radical polymerization (SI-ATRP), which leaded to surface confined growth of non-crosslinked polymer chains on the sufface of the silica beads for trypsin immobilization. The digestion efficiency of the obtained SPMSB-trypsin was evaluated using both standard proteins and complex protein extracts obtained from E. coil. Highly efficient digestion was achieved in only 1 - 2 mm digestion. Furthermore, the SPMSB-Trypsin exhibited both good stability and excellent recovery, therefore can be applied in proteomic research in the future.

The cDNA Djtry, encoding a planarian trypsin, was identified from the cDNA library of Dugesia japonica. Multiple alignment analysis showed that the Tryps&#95;SPc domain contained the incompletely conserved catalytic triad in which the first amino acid His was substituted by Lys. Phylogenetic analysis indicateed that Djtry protein falls at the base of other animal trypsins. The Djtry cDNA was cloned into a bacterial vector pET-28a and was transferred into E. coli BL21. The His-tagged Djtry fusion protein expression was induced by IPTG. SDS-PAGE analysis revealed that the Djtry was expressed as inclusion bodies in E. coli BL21 with the estimated molecular weight of approximately 26 kDa. Western blotting with His-tag antibody showed that the antibody was reacted with the fusion protein after refolding. Compared to bovine trypsin using BAEE as special substrate of trypsin, the enzyme activity of Djtry was measured. These results indicate that Djtry represents the archetype of animal trypsins, and this type of mutational trypsin Djtry still performs the trypsin nature with slightly weaker activity.

Structure of a recombinant chimeric anti-CD20 IgG1 monoclonal antibody was verified by the application of high-performance liquid chromatography-mass spectrometry (HPLC-MS)and N-terminal sequencer. Molecular masses, N-terminal sequences and peptide maps of the antibody treated with different reagents and enzymes were measured. Results indicate that the amino acid sequences of light and heavy chains and 10 disulfide bonds were consistent with theoretical structure. By comparison of molecular masses and peptide maps for the fully glycosylated and deglycosylated samples, the N-linked glycosylation site was identified. The method is simple, rapid, precise, and could be referred to the quality control and structure determination of other IgG1 products.

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

The routine analysis of phosphoproteomics needs effective and specific enrichment methods. The strong cation exchange chromatography (SCX) separation conditions, including the buffer solution and the separation gradient, were studied to enrich the phosphopeptides in a short time. Firstly, the tryptic peptides of standard protein mixtures (bovine alpha-casein and beta-casein, chicken egg ovalbumin) were used to optimize the SCX strategy. Then, this method was applied to the complicated samples, the tryptic peptides from yeast. The experimental results showed that the phosphopeptides were eluted before 30 min in the optimized SCX systems with less interference from nonphosphopeptides. Phosphopeptides were successfully separated in a short time, and the ion signals corresponding to phosphopeptides were accordingly increased during mass spectrometric analysis. It was indicated that the optimized SCX separation system could be used as a simple enrichment method for the separation of phosphopeptides, especially for phosphopeptides in the complex real samples, in a short time, and to increase the relative abundance of phosphopeptides in the detection fraction. This study provides the pragmatic technique for phosphoproteomics analysis on a large-scale.

The existing of intermediate in the process of protein unfolding is necessary for studying the kinetic folding and unfolding pathway of protein. With trypsin as a model protein, the conformational changes of trypsin in the presence of different GuHCl concentrations were investigated by using fluorescence spectra (FR), and compared with the activity recovery of denatured trypsin. It was found that the maximum wavelength of FR of trypsin increases with the concentration of GuHCl increasing, and the maximum wavelength of FR of trypsin reaches the maximum when the concentration of GuHCl is 2 mol x L(-1), and then, the maximum wavelength of FR of trypsin decreases with the concentration of GuHCl increasing. There exists a intermediate state in the presence of low concentration of GuHCl. The conformation of the intermediate is different from the native and unfolding states. The maximum wavelength of FR of the intermediate is the maximum, and fluorescence intensity is the highest, while the bioactivity recovery is the lowest. The reasons for these were studied with the knowledge of the molecular structure.

Tryptic hydrolysis of whole casein was investigated by high performance size exclusion chromatography (HPSEC) in combination with the degree of hydrolysis (DH). In terms of HPSEC chromatograms obtained at different DH values, the complex process of enzymatic reaction and the relative molecular mass distribution of multiple hydrolysates were quantitatively characterized. Based on the information of casein micelle structure, the possible reaction mechanism was deduced from a series of chromatograms. Being taken into account the primary structure of whole casein and the target amino acid of trypsin, the distribution of theoretical peptides were accurately calculated by determining the split sites of complete enzymatic hydrolysis. According to the relationship between retention time and relative molecular mass, the corresponding HPSEC absorption peaks of active peptides in hydrolysates were identified, and caseinophosphopeptides sequences were also characterized.

High-performance ion exchange chromatography (HPIEC) is extensively used in the separation of peptides and proteins, especially in the biotechnology process. The principle of separation of proteins is based on the changes of pH and salt concentration in the mobile phase for the chromatographic model. A new synthetic method with the help of a catalyst for the bounding of diethylaminoethyl group on a home-made macro-pore silica sphere (the trade mark is Sinopak-s, with sphere size of 5 microm and pore diameter of 100 nm) was developed in our laboratory for the application of the scale-up separation of biotechnological target products in China. The Sinopak-s-DEAE weak anion ion exchange matrix for HPLC was prepared and characterized with various proteins. The pH value and reaction time were discussed for the reaction efficiency of ligand to the silica sphere. The coverage of the DEAE ligand on the silica surface were among 1.6 to 2.1 micromol/m2 for six batches of packings. The influences of the pH value and the salt concentration in mobile phase upon the retention of proteins on the DEAE column were also discussed. A bio-activity recovery up to 98% for trypsin was arrived after purification with the DEAE column under the chosen chromatographic conditions. The capacity of matrix for BSA was 80 mg/g. The column was successfully applied to separate a mixture of several standard proteins in a linear gradient elution condition from 0 to 0.4 mol/L of NaCl in a 50 mmol/L of Tris/HCl buffer (pH 7.0) at 1.0 mL/min flow rate and detected at 280 nm wavelength.