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BSE Control: Detection of gelatine-derived peptides in animal feed by mass spectrometry Analyst (2004) 129: 111-115
‘Applying mass spectrometry to the use of mankind’
Submitted by:Yasamin Badiei
Bovine spongiform encephalopathy (BSE):
discombobulatedbubu.blogspot.com
Mad cow disease
Neurodegenerative disease
www.sptimes.com/.../photos/wd-madcow-map.jpg
In 1986, BSE epidemic affected cattle in the UK.
PAP- processed animal proteinMBM- meat and bone meal
Infectious agent- PrionsMisfolded protein Induce normal proteins to fold into abnormal structures (amyloid)
Heat resistant
Effect of mad cow disease
www.mapleleafweb.com/files/cartoon/jan505c.jpg
Creutzfeldt-Jakob disease (vCJD)
www.niah.affrc.go.jp/.../images/prion_04_e.jpg
http://en.wikipedia.org/wiki/Prion
"holes" in prion-affected brain tissue (spongy shape.)
Spongiform encephalopathies (sick brain)
Reasons for concern
Long incubation period (4 years)No medical diagnosisSymptoms could be associated with neurological diseases e.g. Alzheimer’s disease
No treatment
Prevention- testing for MBM contaminationMass spectrometry- sensitive, speed, $$
$, specificityGelatine- partially hydrolyzed, water
soluble derivative of collagen which is found in connective tissue
http://en.wikipedia.org/wiki/Gelatin
Aim1. Gelatine acid hydrolysis with HCl or
CF3COOH: Determine optimum hydrolysis conditions for obtaining peptides
2. MS: Detect and quantify gelatine-derived peptides to identify contamination in feedstuffs
1- Gelatine hydrolysisHCl digestion methodAim: To determine effect of a) digestion time (0 to 180 min) and b) HCl concentrations (1M to 6M) on
yield of hydrolytic peptide products
Gelatine at a concentration of 100 mg ml-1 and an internal standard substance P was used.
Analyst (2004) 129: 113
a) Selected-ion monitoring chromatogram (SIM) for the optimization of gelatine digestion time using 3M HCl:
Analyst (2004) 129: 113 Signal intensity ratio of the gelatine peptide [M + H]+ 1044 plus [M + Na]+ 1066 against substance P [M + H]+ 1347 plus [M + Na]+ 1369 (dashed line).
Stronger marker ion signals observed between 10 to 30 minutes: optimum digestion time
b) Similarly gelatine was hydrolyzed for 40 min. using different concentration of HCl:
3M of HCl, the optimum HCl concentration, was the lowest concentration that reproducibly yielded marker peptides as monitored by MALDI-TOF.
2- Soft ionization MS techniquesFor producing gas-phase ions of nonvolatile
peptide mixtures:
i) Matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI TOF-MS)
ii) Liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS-MS)
2- Soft ionization MS techniques
Samples:1 g of cattle feed was contaminated
with gelatine at levels of 0.1 %, 0.3 %, 0.5 %, 1 % and 5 %
Control samples contained no gelatine
MALDI-TOF MSMALDI is based on the bombardment of sample
molecules with a laser light to bring about sample ionization. The matrix transforms the laser energy into excitation energy for the sample. In positive
ionisation mode the (M+H+) ions are usually the dominant species,
http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
MALDI-TOF MSUnder optimized conditions for HCl
hydrolysis (3M, 40 min.) the limit of detection for observing the gelatine marker peptides is 100 g ml-1
5% gelatine-spiked feeds of undiluted, 1+9 dilution and 1+99 dilutions were analyzed.
MALDI-TOF MSDue to complexity of feed matrix and the
suppression of marker peptide signals:
Analyst (2004) 129: 113 MALDI TOF spectra of 1 + 9 dilution of 5 % gelatine hydrolytic peptides (3M HCl, 40 min.)
Unfractionated digested peptides
Fractionated digested peptides
LC-ESI-MS-MSReverse phase HPLCComponents of a mixture are
partitioned between a nonpolar matrix and a polar solvent mobile phase
Compounds are separated based on their hydrophobic interaction between the analyte and the nonpolar matrix
http://www.ionsource.com/tutorial/chromatography/rphplc.htm
The reverse phase solvents are installed on the HPLC channels A and B.
Compounds stick to reverse phase HPLC columns in high aqueous mobile phase (water, 0.1% acid) and are eluted from RP HPLC columns with high organic mobile phase (50% ACN, 50%water, 0.1% acid).
HPLC: reverse phase-C18 column
aqueous organic
LC-ESI-MS-MSTandem MS/MS
http://en.wikipedia.org/wiki/Tandem_mass_spectrometry
LC-ESI-MS-MS: SRM vs. full MS-MS scanSelected reaction monitoring (SRM):The two analyzers are adjusted to
monitor a chosen precursor-product pair of analyte.
Higher sensitivity
Precursor Product Precursor Product
m/z 1044 m/z 471 m/z 556 m/z 278
m/z 915 m/z 697 m/z 397
m/z 810 m/z 425
m/z 342
m/z 567
m/z 681
Specific precursor ions are transmitted through MS 1 and specific fragments arising from these ions are measured by MS 2
For 0.1 % Gelatine feed Internal standard
Full MS-MS spectra scanThe MS spectra obtained 0.3 %
gelatine feed showed intense signals of gelatine peptide fragments
With full scan MS-MS extended information can be obtained from numerous MS-MS fragments confirming the presence of gelatine.
Quantitative analysisUse of leucine enkephalin as an
internal standardThe gelatine marker at m/z 1044
and the internal standard at m/z 556 elute closely from the LC
Using of LC-ESI-MS-MS in the detection of gelatine hydrolytic peptides with the ion trap and using the full MS-MS scan method
LC-ESI-MS-MS for 0.3 % gelatine-spiked feed
LC-ESI-MS-MS for blank feed
Analyst (2004) 129: 114
The signal area ratio of the gelatine-derived peptide at m/z 1044 against the internal standard at m/z 556 provides the amount of gelatine present in the sample:
Analyst (2004) 129: 115
ConclusionA sensitive and rapid analytical
technique was developed for the detection of peptide products from HCl hydrolysis of gelatine standards.
The manufacture of real animal feed samples with known quantities of MBM would be used to test the practicality of this analytical strategy.
ReferencesBSE Control: Detection of gelatine-derived
peptides in animal feed by mass spectrometry. Analyst (2004) 129: 111-115.
http://rarediseases.about.com/od/rarediseases1/a/vcjd.htm
http://learn.genetics.utah.edu/content/begin/dna/prions/
http://www.ionsource.com/tutorial/chromatography/rphplc.htm
Thank you !
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