presentation2009

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 !

Any Questions?