研究目的
To analyze the protein-bound and unbound ZnPP formed in aqueous meat extracts during incubation at room temperature for up to 72 hours to gain insights into the pathway of ZnPP formation post mortem.
研究成果
The study confirmed that myoglobin is the primary protein for ZnPP formation, with ZnPP transitioning from protein-bound to free states during incubation. An unknown fluorescence peak suggests possible ZnPP occurrence in higher molecular weight proteins. The findings provide new insights into the post-mortem ZnPP pathway, indicating a need for further research on ZnPP release mechanisms and characterization of unknown fluorophores.
研究不足
The study was conducted using meat extracts rather than intact meat tissue, which may not fully replicate in vivo conditions. Only water-soluble proteins were investigated, potentially missing insoluble proteins. The unknown fluorescence peak in high molecular weight range requires further characterization. The release mechanism of ZnPP from myoglobin is not fully understood and needs more investigation.
1：Experimental Design and Method Selection:
The study used a SEC-HPLC-UV-fluorescence setup to separate and detect proteins in aqueous meat extracts, with SDS-PAGE and MALDI-TOF-MS for protein identification.
2：Sample Selection and Data Sources:
Pork meat (longissimus lumborum) was purchased from a local butcher, homogenized, and extracted with phosphate buffer.
3：List of Experimental Equipment and Materials:
Equipment includes a hand mixer (Braun), Ultra Turrax, centrifuge, Shimadzu Nexera HPLC with SPD M20A and RF 20A detectors, TosoHaas TSKgel column, Bio-Rad SDS-PAGE equipment, and Bruker AUTOFLEX-III LRF200-CID MALDI-TOF-MS. Materials include phosphate buffer, sodium azide, filters, and various chemicals from suppliers like Carl Roth and Sigma Aldrich.
4：Experimental Procedures and Operational Workflow:
Meat extracts were prepared, incubated at 30°C for 0-72 hours, centrifuged, filtered, and analyzed by SEC-HPLC. Fractions were collected and analyzed by SDS-PAGE and MALDI-TOF-MS.
5：Data Analysis Methods:
Fluorescence intensities were measured, and data were analyzed using statistical methods in R with packages nlme and lsmeans, including post hoc tests with Bonferroni correction.
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HPLC device
Nexera
Shimadzu
Separating and detecting proteins using SEC-HPLC
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detector
SPD M20A
Shimadzu
UV-VIS detection
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detector
RF 20A
Shimadzu
Fluorescence detection
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fraction collector
FRC-10a
Shimadzu
Collecting fractions from HPLC
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MALDI-TOF-MS
AUTOFLEX-III LRF200-CID
Bruker Daltonik
Mass spectrometry for protein identification
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laser
Smartbeam-Laser 200
Bruker Daltonik
Ionization in mass spectrometry
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software
FlexAnalysis version 3.3
Bruker Daltonik
Data analysis for mass spectrometry
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software
BioTools version 3.2
Bruker Daltonik
Sequence database search for protein identification
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hand mixer
Braun
Homogenizing meat samples
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Ultra Turrax
Extracting proteins from meat homogenate
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centrifuge
Centrifuging meat-suspension and extracts
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column
TSKgel G4000WXL
TosoHaas
Size exclusion chromatography for protein separation
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SDS-PAGE equipment
Mini-PROTEAN
Bio-Rad
Electrophoresis for protein separation
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software
Quantity One 1-D Analysis Software version 4.5.2
Bio-Rad
Analyzing SDS-PAGE results
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calibration kit
LMW Calibration Kit
Amersham
Calibrating molecular weights in SDS-PAGE
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filter
0.2 μm cellulose acetate filter
Carl Roth
Filtering samples for HPLC analysis
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