Purification and Characterization of Calpastatin from Grass Prawn Muscle (<i>Penaeus monodon</i>)

Jiang, Shann‐Tzong; Wu, Jianbing; Su, Mei-Jui; Tzeng, Shinn-Shuenn

doi:10.1021/jf0001177

Cited by 4 publications

(6 citation statements)

References 39 publications

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“…Unfortunately, this work did not get the samples from patient, and only the urine sample was collected from an adult healthy volunteer as a biological sample to test practical application of the proposed encoding strategy. It did not show the peak of “Trypsin ID” at m / z 761.6, indicating there was no trypsin or trypsin-like protease in the sample, which was consistent with that measured with BAEE method. − To obtain the calibration curve, various concentrations of trypsin were spiked in 10-fold diluted urine samples. After incubating the spiked samples in the wells of peptide 1-encoded microplate and mixing with the internal standard, the MS spectra showed obvious peaks of “Trypsin ID” and the IS-peptide 1 (Figure A), indicating that the components of urine sample did not obvoiusly affect the hydrolysis and MS analysis.…”

Section: Resultssupporting

confidence: 62%

“…To assess the accuracy of the proposed method, the activities of trypsin and chymotrypsin were also detected with BAEE ( N -α-benzoyl- l -arginine-ethylester at 253 nm) and BTEE ( N -α-benzoyl- l -tyrosine-ethylester at 256 nm) methods, respectively. − Briefly, the assay was carried out in 0.1 M Tris-HCl buffer (pH 8.0) containing 6 mM CaCl 2 , 10 μM substrate (BAEE or BTEE), and trypsin or chymotrypsin at different concentrations, or 10× diluted samples. The absorbance was measured at 25 °C on a UV-3600 spectrophotometer (Shimadzu, Japan).…”

Section: Methodsmentioning

confidence: 99%

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Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Liu

2015

Anal. Chem.

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A peptide-encoded microplate was proposed for MALDI-TOF mass spectrometric (MS) analysis of protease activity. The peptide codes were designed to contain a coding region and the substrate of protease for enzymatic cleavage, respectively, and an internal standard method was proposed for the MS quantitation of the cleavage products of these peptide codes. Upon the cleavage reaction in the presence of target proteases, the coding regions were released from the microplate, which were directly quantitated by using corresponding peptides with one-amino acid difference as the internal standards. The coding region could be used as the unique "Protease ID" for the identification of corresponding protease, and the amount of the cleavage product was used for protease activity analysis. Using trypsin and chymotrypsin as the model proteases to verify the multiplex protease assay, the designed "Trypsin ID" and "Chymotrypsin ID" occurred at m/z 761.6 and 711.6. The logarithm value of the intensity ratio of "Protease ID" to internal standard was proportional to trypsin and chymotrypsin concentration in a range from 5.0 to 500 and 10 to 500 nM, respectively. The detection limits for trypsin and chymotrypsin were 2.3 and 5.2 nM, respectively. The peptide-encoded microplate showed good selectivity. This proposed method provided a powerful tool for convenient identification and activity analysis of multiplex proteases.

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Section: Resultssupporting

confidence: 62%

Section: Methodsmentioning

confidence: 99%

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Liu

2015

Anal. Chem.

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“…In shellfish, proteases from digestive tracts are considered to be the main causative agents for rapid postmortem degradation and deterioration of muscle tissue . Calpain and cathepsins are two other proteolytic systems present in muscle tissue, which have been linked to postmortem protein degradation ,, . Hence, some of these potential proteases were tested in the present study to establish their potential roles in the observed proteolytic changes in red swamp crayfish.…”

Section: Resultsmentioning

confidence: 99%

“…For freshwater prawn, muscle mushiness appeared to be due to the diffusion of proteolytic and collagenolytic enzymes from the hepatopancreas into the muscle tissue , . Calpain, an intracellular neutral endogenous protease, has also been implicated in textural changes in postmortem shellfish muscle , , although the exact role of this enzyme is controversial .…”

Section: Introductionmentioning

confidence: 99%

Protease Activity in Post-Mortem Red Swamp Crayfish (Procambarus clarkii) Muscle Stored in Modified Atmosphere Packaging

Chen

Guttmann

Xiong

et al. 2008

J. Agric. Food Chem.

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Protease activity during storage is thought to be an important contributor to decreased shelf life of fresh seafood. To examine this, three batches of red swamp crayfish ( Procambarus clarkii) tails, placed on trays, were packed with a polyvinyl chloride film (aerobic packaging or AP), under vacuum (vacuum packaging or VP), or under a modified atmosphere (MAP: 80% CO 2/10% O 2/10% N 2), and proteolytic activity was measured on days 0, 1, 3, 6, and 10 during storage at 2 degrees C. The crude extract from the crayfish digestive system (gut) did not have an apparent role in muscle proteolysis as negligible proteolytic activity was detected. However, the loss of calpastatin (the endogenous calpain inhibitor) was identified in MAP-stored muscle samples on day 10, suggestive of high m-calpain activity. Tail samples stored in AP showed no appreciable proteolysis, but those stored in MAP and VP showed significant decreases in the levels of 53, 66, 71, and 110 kDa polypeptides during storage. The observed proteolytic activity and myofibrillar protein degradation did not correspond to muscle textural properties as the MAP samples had an increased toughness ( P < 0.05) after storage for 10 days. These findings suggest that other physicochemical mechanisms are involved in postmortem alteration in the crayfish muscle structure under the packaging systems investigated.

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“…), which demonstrated the specificity of the proposed method in the analysis of complex samples and indicated that interference from co‐eluted ions with quantitation results was negligible. The measurement accuracy for trypsin and chymotrypsin in the same samples was also examined by comparing the results with those obtained from BAEE and BTEE methods, which were also reliant on the substrate cleavage to assay protease activities and showed relative errors less than 5.2%. Thus, the proposed peptide‐encoding strategy could be potentially applicated in clinical diagnosis and expanded to protease activity assays in blood samples, while necessary dilution and desalting protocols might be needed to obtain sacrifatory results.…”

Section: Resultsmentioning

confidence: 99%

Peptide codes for multiple protease activity assay via high‐resolution mass spectrometric quantitation

Liu

Feng

et al. 2016

Rapid Comm Mass Spectrometry

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RATIONALE: Proteases, which are involved in a number of biological processes, play important roles in human health. Alterations in protease activities have been associated with many diseases. Thus, effective methods for multiple protease assay are very essential and may help to discover more about their functions in different physiological processes. Here, we proposed a strategy of peptide codes for label-free analysis of multiple protease activities using high-resolution mass spectrometry (HRMS). METHODS:The peptide codes were designed to contain a coding region and the substrate of the protease, respectively. Upon the cleavage of target proteases, the coding regions could be rapidly identified and directly quantitated by accurate m/z extractions without internal standard. Therefore, the coding region could be used as the unique "Protease ID" for the identification of the corresponding protease, and the amount of the cleavage product was used for protease activity analysis. RESULTS: The method was validated by using trypsin and chymotrypsin as model proteases, with the designed "Trypsin ID" and "Chymotrypsin ID" occurring at m/z 761.270 and 711.243, respectively. The peak area of "Protease ID" was proportional to trypsin and chymotrypsin concentration in the range from 0.01 to 10 nM and 10 to 2000 nM, respectively. The proposed method could also be used for screening of protease inhibitors, which might be of great importance for drug discovery. CONCLUSIONS: This method provided a label-free and accurate quantitative platform for convenient identification and activity analysis of multiple proteases, which could potentially be applied in clinical diagnosis.

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Purification and Characterization of Calpastatin from Grass Prawn Muscle (Penaeus monodon)

Cited by 4 publications

References 39 publications

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Peptide Code-on-a-Microplate for Protease Activity Analysis via MALDI-TOF Mass Spectrometric Quantitation

Protease Activity in Post-Mortem Red Swamp Crayfish (Procambarus clarkii) Muscle Stored in Modified Atmosphere Packaging

Peptide codes for multiple protease activity assay via high‐resolution mass spectrometric quantitation

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