• 분석과학
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• 제30권2호
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• pp.96-101
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• 2017

This study investigated the effect of centrifugation on tryptic digestion. This was done by applying different centrifugation speeds (6,000, 8,000, 10,000, 20,000, and $30,000{\times}g$) over various durations (0, 10, 20, 30, 40, 50, and 60 min) to digest two model proteins - cytochrome c and myoglobin. The intact proteins and resulting peptides were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Centrifugation greatly improved the tryptic digestion efficiency of cytochrome c, where either an increase in centrifugation speed or in digestion duration significantly improved the digestion of cytochrome c. However, centrifugation did not noticeably improve the digestion of myoglobin; 16 h of centrifuge-assisted tryptic digestion at $30,000{\times}g$ barely removed the myoglobin protein peak. Similar results were also obtained when using conventional tryptic digestion with gentle mixing. When acetonitrile (ACN) was added to make 10% ACN buffer solutions, the myoglobin protein peak disappeared after 6 h of digestion using both centrifuge-assisted and conventional tryptic digestions.

• Bulletin of the Korean Chemical Society
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• 제35권10호
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• pp.3115-3118
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• 2014

• Bulletin of the Korean Chemical Society
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• 제18권4호
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• pp.428-432
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• 1997

The dephosphorylation specificity of protein phosphatase 2A (PP2A), calcineurin (PP2B) and protein phosphatase 2C (PP2C) were studied in vitro using myelin basic protein (MBP) as a model substrate which was fully phosphorylated at multiple sites by protein kinase C (PKC) or cyclic AMP-dependent protein kinase (PKA). In order to determine the site specificity of phosphates in myelin basic protein, the protein was digested with trypsin and the radioactive phosphopeptide fragments were isolated by high performance liquid chromatography (HPLC) on reversed-phase column. Subsequent analysis and/or sequential manual Edman degradation of the purified phosphopeptides revealed that Thr-65 and Ser-115 were most extensively phophorylated by PKA and Ser-55 by PKC. For the dephosphorylation kinetics, the phosphorylated MBP was treated with calcineurin or PP2C with various time intervals and the reaction was terminated by direct tryptic digest. Both Thr-65 and Ser-115 residues were dephosphorylated more rapidly than any other ones by phosphatases. However it can be differentiated further by first-order kinetics that the PP2B dephosphorylated both Thr-65 and Ser-115 with almost same manner, whereas PP2C dephosphorylated somewhat preferentially the Ser-115.

• Mass Spectrometry Letters
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• 제3권2호
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• pp.39-42
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• 2012

Phosphorylation and glycosylation are two of the most important and widespread post-translational modifications (PTMs) in an organism. Proteomics analysis of the PTMs has been challenged by low stoichiometry of the modified proteins and suppression effects by high abundance proteins, typically no-functional house-keeping proteins. In this study, a novel method was applied for not only isolating PTM peptides from intact peptides but also concurrently characterizing of glyco- and phosphoproteome using electrostatic repulsion hydrophilic interaction chromatography (ERLIC) packed with silica coated by crosslinked polyethyleneimine. For 2 mg tryptic digest of mouse proteome of epicardial adipose tissue with fat diet, 802 N-glycosylated peptides of 316 glycoproteins and 159 phosphorylated peptides of 75 phosphoproteins were identified using HPLC chip/quadrupole time-of-flight (Q-OF) tandem mass spectrometer.

• The Korean Journal of Physiology and Pharmacology
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• 제13권1호
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• pp.49-54
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• 2009

The mushroom Cordyceps militaris has been used for a long time in eastern Asia as a nutraceutical and in traditional Chinese medicine as a treatment for cancer patients. In the present study, a cytotoxic antifungal protease was purified from the dried fruiting bodies of C. militaris using anion-exchange chromatography on a DEAE-Sepharose column. Electrophoretic analyses indicated that this protein, designated C. militaris protein(CMP), has a molecular mass of 12 kDa and a pI of 5.1. The optimum conditions for protease activity were a temperature of $37^{\circ}C$ and pH of $7.0{\sim}9.0$. The enzyme activity was specifically inhibited by the serine protease inhibitor phenylmethylsulfonyl fluoride. Amino acid composition of intact CMP and amino acid sequences of three major peptides from a tryptic digest of CMP were determined. CMP exerted strong antifungal effect against the growth of the fungus Fusarium oxysporum, and exhibited cytotoxicity against human breast and bladder cancer cells. These results indicate that C. militaris represents a source of a novel protein that might be applied in diverse biological and medicinal applications.

• BMB Reports
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• 제33권3호
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• pp.234-241
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• 2000

A new type of the human TSA homologous gene was cloned from a HeLa cell cDNA and characterized. The gene product consists of 161 amino acids with a molecular mass of 16,900. The TSA homologous protein, as a new 6th member of the human TSA (hTSA VI), exerted a thioldependent peroxidase activity with the use of thioredoxin system as a physiological electron donor. The values of $V_{max}/K_m$ of hTSA VI for $H_2O_2$ and t-butyl hydroperoxide (t-BOOH) were calculated as $5.53{\times}10^{-2}$ and $3.70{\times}10^{-2}$, respectively. This implies that hTSA VI is a peroxidase, which reduces $H_2O_2$ and t-BOOH. The mutation of $Cys^{47}$ to serine resulted in a complete loss of the peroxidase activity. This suggests that $Cys^{47}$ acts as a primary site of catalysis. The analysis of the tryptic digest derived from hTSA VI revealed that the $Cys^{47}$ exists as a free thiol form. Taken together, these results suggest that the TSA homologous protein is a new type of the human family, which exerts thioredoxin-linked peroxidase activity toward $H_2O_2$ and alkyl hydroperoxide.

• Journal of Microbiology and Biotechnology
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• 제18권9호
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• pp.1555-1563
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• 2008

A novel $\alpha$-amylase ($\alpha$-1,4-$\alpha$-D-glucan glucanohydrolase, E.C. 3.2.1.1), ScAmy43, was found in the culture medium of the phytopathogenic fungus Sclerotinia sclerotiorum grown on oats flour. Purified to homogeneity, ScAmy43 appeared as a 43 kDa monomeric enzyme, as estimated by SDS-PAGE and Superdex 75 gel filtration. The MALDI peptide mass fingerprint of ScAmy43 tryptic digest as well as internal sequence analyses indicate that the enzyme has an original primary structure when compared with other fungal a-amylases. However, the sequence of the 12 N-terminal residues is homologous with those of Aspergillus awamori and Aspergillus kawachii amylases, suggesting that the new enzyme belongs to the same GH13 glycosyl hydrolase family. Assayed with soluble starch as substrate, this enzyme displayed optimal activity at pH 4 and $55^{\circ}C$ with an apparent $K_m$ value of 1.66 mg/ml and $V_{max}$ of 0.1${\mu}mol$glucose $min^{-1}$ $ml^{-1}$. ScAmy43 activity was strongly inhibited by $Cu^{2+}$, $Mn^{2+}$, and $Ba^{2+}$, moderately by $Fe^{2+}$, and was only weakly affected by $Ca^{2+}$ addition. However, since EDTA and EGTA did not inhibit ScAmy43 activity, this enzyme is probably not a metalloprotein. DTT and $\beta$-mercaptoethanol strongly increased the enzyme activity. Starting with soluble starch as substrate, the end products were mainly maltotriose, suggesting for this enzyme an endo action.