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INTRODUCTION
MATERIAL
REFERENCES
Immunochemical methods
Hofmann, Fischer, Mueller, and Babel (1999)
i) gelatin type
ii) gelatin quality
iii) concentration used
iv) led to false negative or positive readings (in some cases)
i) antibodies were found to be very sensitive to the alkaline or acidic process used for the gelatin
production
ii) not enough species-specific to allow a sensitive detection of mixture of low concentration of bovine
gelatin in porcine gelatin
Chromatographic methods
Spectroscopy method
Cao & Xu, 2008; Muyonga, Cole,& Duodu, 2004
Nemati, Oveisi, Abdollahi, and Sabzevari (2004)
Determination of gelation and intermolecular cross-linking of collagen and proteins: FTIR spectroscopy with attenuated total reflectance (ATR) or transmission accessories
Hashim et al. (2010)
Other potential method:
PCR-based methods
Tasara, Schumacher, and Stephan (2005)
Chemisorption method
Hunter, Nyburg,and Pritzker (1986)
Amorphous calcium phosphate (ACP)
promote by
collagen, gelatin, and agarose gels
Hydroxyapatite (HAP)
=HAP is a compound which is formed by chemisorption of gelatin
Hidaka and Liu (2003)
• Using in vitro formation of calcium phosphate precipitates.
• Study on the reaction of calcium phosphate precipitation
• Need further study to clarify these effects.
Mass spectrometric methods
Ocana et al. (2004)
Zhang et al. (2009)
OBJECTIVES
i) During MS/MS data processing, the threshold for specific peptide identification might be different from
one species to another - very high homology between the collagen sequences of mammals
ii) proline hydroxylation is another challenge making identification of peptide more difficult than most of the
proteins
Alternative
Fish gelatine
Other previous study
in developing method
of source detection on gelatin
To establish proteomics based alternative method that can be applicable to gelatine differentiation in food system
Fact : pig skin and bovine hide-derived gelatins constitute a great majority of yearly gelatin production scale around the world
to develop analytical methods intended to detect the species origin of gelatin
Issue:
Outbreak of BSE
(Bovine Spongiform Encephalopathy)
Religious beliefs and convictions
(Islam ; Judaism ; Hinduism)
on consumption or the use of porcine derived gelatin in food materials
on use of bovine gelatin for human consumption, cosmetic and pharmaceutical products.
(Venien & Levieux, 2005).
Gelatine & Issue related:
Gelatin produced nearly 326,000 tons a year
(46%) pig skin-derived
Challenge
(29.4%) bovine hides
i) Data dependent acquisition mode, a
precursor ion is selected and fragmented for
sequencing.
ii) Data independent acquisition mode, rather
than selecting a precursor, a nonoverlapping
m/z window is selected and all the precursor
ions within the window are co-fragmented
Fig. 4 shows fragment ion information for three of the peptides identified.
(23.1%) bones
(1.5%) other sources (Karim & Bhat, 2009).
Fig. 4C. is a good candidate for a specific bovine peptide was IGQPGAVGPAGIR because there were no post translational modifications observed and it was identified with high confidence.
Fig. 4B shows an example for proline side chain hydroxylation, where a unique porcine peptide with the sequence GFP*GSP*GNVGPAGK was detected with two hydroxylations.
Process that causes spectrum processing sequence determination more complex
i) 2 hydroxylation detected;
ii) Deamidation of asparagine in the peptide.
Therefore, it will be better to pick unmodified peptides, as was observed for the peptide in fig 4C
Fig. 4A is the MS/MS spectrum for one of the unique porcine peptides with the sequence GETGPAGPAGPVGPVGAR.
Previous research reported that:
Standard gelatin of bovine/porcine
Standard gelatin from bovine and porcine was added to food systems
Extracted (based on the modified method above)
ESI-qTOF-MSE
(electrospray ionization quadrupole time-of-flight)
Gelatin peptides
Analysis
The number of peptides identified in these samples was lower
But still multiple unique peptide Identified for porcine and bovine
For example:
All of sample added with porcine gelatin was identified to had 1 of porcine specific peptides, which have
Gelatin
Journal paper:
RESULT
&
DISCUSSION
Gelatin is a protein based product
Proteomics analysis based alternative methods that can be applicable to gelatin differentiation in real food systems.
i. MSE method had advantages on complex mixture analysis
because it based on parallel collision-induced-dissociation of
peptides and provides masses of the precursor and fragment
ions in parallel (Purvine, Eppel, Yi, & Goodlett, 2003).
ii Gives a unique opportunity to the researchers to selectively
and accurately study the shape of molecules, which are
beyond the reach of traditional techniques like NMR, electron
microscopy, and X-ray crystallography.
iii Compared to any other high resolution mass spectrometer,it
can be used to identify and quantify sample analytes at
lower concentrations by ion mobility separations.
X-Y is generally
Glycine - X - Y
GELATIN
Fig. 3 shows the peptides identified from standard porcine and bovine digests with different m/z values.
Figure 2 shows the total ion chromatograms for bovine and porcine standard gelatins tryptic digests acquired over 50-1600m/z
- the peptides were separated over 90 min before analyzed in the mass spectrometer
- high mass resolution (>10,000) achieved to increased mass accuracy of the peptide analysis, which, in turn, provided
high confidence in deducing peptide sequence information.
- distinct differences in the elution profiles along with many similar peptide elutions.
- identification of the lower abundant peptides which otherwise might be masked by the abundant peptides in the mixture.
In the IdentityE approach, an alternate scanning mode was applied, by which
- peptide information was collected at low collision energy
- peptide sequence information was collected at ramped higher collision energy.
In this way, more fragment ions per peptide and more peptides per protein were detected
A novel method to differentiate bovine and porcine gelatins in food products: NanoUPLC-ESI-Q-TOF-MSE based data independent acquisition technique to detect marker peptides in gelatin
MAISARAH SHAMSUDIN
90
Bovine
10
50
Standard gelatin samples were mixed ratios
(analyzed in triplicate)
10
50
90
Porcine
• Although there is a large homology between the two gelatin sources,
nano liquid chromatography was able to separate the unique peptides
which were identified using the IdentityE method.
• Even when bovine gelatin standard was 9-fold in excess, it was still
possible to identify porcine peptides in the mixture.
• Therefore, it is reasonable to speculate that, with some calibration to
the method, it might be possible to detect much lower contamination
of porcine gelatin in bovine or vice versa.
METHOD
It was cooled rapidly to 65oC
Cow milk was heated up to 90oC for 15 second in a boiler
Gelatin solution (either porcine or bovine) was added to the milk at the level of 0.1% (w/v)
Homogenized by stirring and temperature was reduced to 45oC
Raw data for peptide sequences
1. The internal standard amino acid sequence (yeast alcohol dehydrogenase, Uniprot accession # P00330) was included in
the FASTA file of the database.
2. The peptide sequences were acquired from the uniprot database and from an earlier published work (Zhang et al., 2009).
3. The Apex3D data preparation parameters were set to
4. Database search query was set to
5. Variable modification were set for
6. Absolute quantification of the peptides was calculated with the Hi3 functionality of the IDENTITYE system using the spiked
known amount of the internal standard.
7. The false positive rate (FPR) against the random database was set to 4% (according to ProteinLynxGlobal (PLGS) server
workflow).
8. Baseline for peptide selection for protein identification was based on many peptides, 3 fragments per peptide and 7
fragments per protein
9. Protein identifications were manually checked for almost complete consecutive fragments for sequence accuracy
Mixture was inoculated with 2% commercial yoghurt culture of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus
Then, it was stored at 3oC to cooled down and to terminate acid development.
Mixture was incubated at 43oC until pH decreased to about 4.5
2 mg of gelatin standard and the gelatin extracted (yoghurt, cheese or ice cream) were mixed with 1 mL of 50 mM NH4HCO3
Sample was sonicated with an ultrasonic homogenizer (10 s on, 10 s off, 3 cycles).
picric acid
The mixture was centrifuged at 15,000 rpm and filtered through a 0.22 µm syringe filter.
Food product (gelatin present)
gelatin-picric acid precipitation
10 µL of the filtered homogenate was mixed with 40 µL of 50mM NH4HCO3.
5 mM DTT was added to the mixture and it was incubated at 60oC for 15 min.
10 mM IAA was added to the mixture and then, incubated in the dark at room temperature for 30 min.
picric acid
1) Prior to the injection:
2) 2µl volume of sample (containing 200 ng of tryptic peptide mixture) was loaded on the nanoUPLC-
ESI-qTOF-MSE system;
3) Data independent acquisition mode (MSE) was carried out by
- 6V low energy
- 15-40 V high energy collision
- over 1.5 s intervals
4) The internal mass calibrant Glu-fibrinopeptide was infused at every 45 s through the nanolockspray
ion source at 300 nl/min flow rate.
5) Peptide signal data were collected between 50–1600 m/z values.
Food product (gelatin absent)
picric acid precipitation
50 µL of 0.4% Rapigest in 50 mM NH4HCO3 and 1 µg of proteomics grade trypsin (50 µl, 20 ng/µl) in 50 mM NH4HCO3 was added to the mixture and it was incubated at 37oC for 16 h.
3 µL of TFA and 2 µL ACN was added to the mixture and was incubated at 60oC with constant shaking at 600 rpm for 2 hour
cooled down to 65oC
cooled down to 40oC
Raw milk was pasteurized at 85oC for 5 min
Gelatin solution (either porcine or bovine) was added at the level 0.1% (w/v)
During Rapigest removal, standard ADH tryptic digest internal calibrant was added to a final concentration of 50 fmol/µL.
The mixture was made up to 200 µL volume by addition of appropriate amounts of 50 mM NH4- HCO3 buffer.
The resulting mixtures had 100 ng/µLtryptic peptide concentration.
0.02% (w/v) of CaCl2 was added to the mixture
After Rapigest cleavage, the mixture was centrifuged at 15,000 rpm for 15 min and an aliquot was taken into a LC vial for analysis and the rest of the tryptic peptides were stored at -86 oC.
Rennet of microbial origin (Mucormiehei) was added & kept at 32oC for 90 min to complete coagulation.
The coagulum was cut into 1–3 cm3 cubes and the curds rested for 5–10 min.
Finally, the cheese blocks were placed in plastic containers filled with brine (12% w/w NaCl) and stored at 5 oC.
It was pressed, molded and salted in brine (12% (w/w) NaCl) at 15oC for 8 h.
Drained for 25 min
6) Label-free proteomics methodology was successfully used for complex mixture analysis.
Fig. 1 shows the experimental flow chart for the analysis applied in this study.
Mercury (Hg) dissolved in twice its weight of nitric acid and this solution was diluted 25 times to its volume with water
20 g of sample was added to 20 mL of acid Hg(NO3)2 solution to precipitate all proteins except for gelatin.
The mixture was shaken and added with 40 mL distilled water
Ingredients of:
The mixture was shaken again
The ingredients (except for emulsifier and gelatin) were mixed and pasteurized at 85oC for 15 min with constant stirring.
Cooled down rapidly to 50oC
Then, the mixture was let stand for 5 min and filtered into a test tube.
An equal volume of saturated aqueous picric acid solution was added to the portion of filtrate in the test tube. The presence of gelatin was confirmed by formation of yellow precipitate.
Homogenized for 3 min
7) After the gelatin extraction from the food materials, carbamidomethyl-cysteine modification was applied and the peptides were
generated by overnight trypsinization.
8) Tryptic peptides generated were then separated prior to mass spectrometry analysis.
-to separate most of the peptides
-detect the different among all the similarity.
Test tube was centrifuged for 15 min at 1200g (Nuve, NF400/R model, Turkey) and drained.
The ice cream mix was then stored at 4oC for 24 h for ageing
Gelatin precipitate adhered tenaciously to the bottom and walls of the tube was collected using a spatula and transferred into vials after washed with distilled water.
The emulsifier and gelatin solution were then added and the mixture was beaten in an ice-cream maker at -20oC for 15 min.
It was then cooled down to - 20 oC in a freezer and stored under this condition until analysis.
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.conclusion..