Meat Authenticity Testing by Orbitrap MS Technology Michal Godula, - - PowerPoint PPT Presentation

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Michal Godula, Ph.D. Thermo Fisher Scientific

Meat Authenticity Testing by Orbitrap MS Technology

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Meat Substitution A real scandal !!

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How testing is done?

• Two-dimensional polyacrylamide gel

electrophoresis and western-blot analysis

• Qualitative Real-Time PCR
• Enzyme-linked immunosorbent assay (ELISA)

Challenges

• These methods are mostly qualitative
• Molecular information obtained is limited
• Data can’t be revisited post-acquisition for data

mining

• They are not generic approaches and need to be

heavily customized

Motivation : $$$

• Addition of meat from undeclared species to a

specific meat product in order to lower production cost and increase profitability Cost per kg: Horse meat << Beef meat

An international issue

• It is economic fraud
• It represents health issues due to specific dietary

restrictions

• It is an ethical problem
• It is also an important cultural and religious

issues

Meat Substitution

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Unit mass resolution MS

• Is targeted (single or triple quadrupole)
• Selectivity is provided by tandem MS/MS (SRM transition needed)
• False positives are reality
• Need to setup instrument (SRM) before analysis
• Realistic breakpoint is 200-300 compounds in a run
• Time consuming data processing
• Can perform the same level of quantitation as MS/MS
• Selectivity obtained by accurate mass measurement (only m/z needed)
• Less false positives and negatives
• No need to setup instrument (SRM) before analysis
• Unlimited number of compounds in a run – perfect for screening
• Automated data processing

High Resolution Accurate Mass

What About Mass Spectrometry Options

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How Accurate Is Your Mass?

• Mass accuracy usually expressed as ppm or mDa error

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10 /    

true true meas

m m m z m

• Quadrupole MS
• Orbitrap MS

ppm z m 200 10 500 . 500 1 . 500 /

6 

    ppm z m 2 10 10314 . 500 10314 . 500 10414 . 500 /

6 

   

100 mDa error 1 mDa error

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• Resolution

Mass Resolution FWHM

m m R  

m (FWHM)

• Quadrupole MS
• Orbitrap (HR)MS

1000 4 . 400   R

100000 004 . 400   R

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Principle Of OrbitrapTM Operation

1 2

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        R Rm

z

  2

2

        R Rm

z r

 

z m k

z

/   Makarov A. Anal. Chem. 2000, 72, 1156-1162.

• Characteristic frequencies:
• Frequency of rotation ωφ
• Frequency of radial oscillations ωr
• Frequency of axial oscillations ωz

Intensity m/q

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Resolution vs. m/z

50000 100000 150000 200000 250000 300000 350000 100 200 300 400 500 600 700 800 900 1000

Resolution (FWHM) Q Exactive HF Q Exactive

Q-TOF

m/z

Much higher resolution at low m/z range

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Thermo ScientificTM Q ExactiveTM MS - a 3D View

Quadrupole Mass Filter HCD Cell Orbitrap Mass Analyzer C-Trap RF-Lens

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Why Bottom-up Proteomics Workflow Is An Interesting Option To Develop An MS Based Assay?

All life forms are related by common ancestry and descent. The construction

• f phylogenies provides explanations of

the diversity seen in the natural world. Today, phylogenies are usually constructed using DNA sequence data. Relationship between genes and species is central for meat speciation

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Traditional Peptide Fingerprinting Approach Using MS

5 10 15 20 25 30 35 40 45

Time (min) Porcine TIC Sus scrofa

Generate a mass list (MS and MS/MS data) Database search (MASCOT) MS and MS/MS matching Peptide fingerprinting

7 429 common unique peptides identified

Beef Horse Lamb Pork

17 specific peptides 9 specific peptides 14 specific peptides 23 specific peptides Using a relatively high abundance threshold …

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PMF

Bargen et al. (2013), Journal of Agricultural and Food Chemistry, 61:11986-11994

Peptide Mass Fingerprinting

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• It relies heavily on the quality of the MS and MS/MS data
• It strongly relies on bioinformatics and parameterizations
• It requires highly skilled scientists to obtain comprehensive

results Is this really appropriate for implementation in a routine food analysis laboratory ? Can we propose alternative strategies ?

What Are The Main Limitations Of This Analytical Approach?

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Targeted Bioinformatics Analysis Example: Myoglobin

Extraction of gene information In silico sequence alignment In silico protein translation and alignment Sequence analysis and In silico tryptic digestion Generate proteotypic mass lists

Proteotypic peptides can be identified (120-134)

YLEFISDA IIHVLHAKHP SDFGADAQAA MSKALELFR YLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFR YLEFISEA IIQVLQSKHP GDFGADAQGA MSKALELFR YLEFISDA IIHVLHAKHP SDFGADAQGA MSKALELFR

120 134

Beef Horse Pork Lamb

Myoglobin is the primary oxygen-carrying protein of muscle tissues It is a highly abundant protein

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How Bottom-up Proteomics Can Be Used For Meat Speciation

Phase 1 Proteome mapping Targeted Meat products Protein Extraction Proteolysis Peptide enrichment MS and MS/MS Protein / peptide identification

Genome annotation

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Bottom-up Proteomics Sample Preparation

1. Meat sample mixed with water (1:5) is homogenized and the mixture is sonicated 2. Proteins in the suspension are precipitated with acetone (1:1) 3. Acetone is discarded and the generated protein pellet is dryed to remove all traces of acetone. 4. Protein pellets are dissolved in ammonium bicarbonate (pH 8.5). 5. Proteins are denatured by heating at 120˚C 6. Reduced with Dithiothreitol (DTT) and alkylated with Iodoacetamide IAA 7. Proteomic grade trypsin is added and the reaction is performed at 40ºC for 24h. Trypsin cleavage occurs after basic amino acids : Lys (K) & Arg (R)

+ H2O

I. II. III. IV.

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749.0 749.5 750.0 750.5 751.0 751.5 752.0 752.5 753.0 753.5 754.0 754.5 755.0

m/z

∆m is - 0.7 ppm

Very complex TIC’s Over 7000s of unique peptides can be found.

Each Targeted Peptides Can Be Detected And Extracted From Tics

Need for very high resolution data (>100 000).

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Survey MS Scan Select ions

• f interest

Acquired MS/MS spectra Generation of post-analysis XICs for selected proteotyping peptides

Beef Horse Pork Lamb 766.8435 (z=2) 751.8383 (z=2) 744.8304 (z=2) 759.8357 (z=2)

140 000 FWHM m/z 500-2000 17 500 FWHM

Isolation width = 1.5 AMU

Beef Horse Pork Lamb 1298.5681 (y13) 1268.5576 (y13) 1254.5419 (y13) 1284.5525 (y13) 1395.6209 (y14) 1365.6103 (y14) 1351.5947 (y14) 1381.6053 (y14)

Extracted ion chromatogram at y14 or y13 ± 5 ppm

Data Dependent MS/MS Used For Targeted Work

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S a m p l e 1 S a m p l e 2 S a m p l e 3 S a m p l e 4 0.0 5.0×106 1.0×107 1.5×107 2.0×107 2.5×107

Peak Area

Assessment Of The Apparent Intra- And Inter Method Reproducibility

Sample 1 Sample 2 Sample 3 Sample 4 0.0 5.0×107 1.0×108 1.5×108 2.0×108 2.5×108 3.0×108 3.5×108

Peak Area

Beef (Myoglobin peptide 120-134) % CV < 20%

S a m p l e 1 S a m p l e 2 S a m p l e 3 S a m p l e 4 0.0 5.0×107 1.0×108 1.5×108 2.0×108

Peak Area

Horse (Myoglobin peptide 120-134) % CV < 16% Pork (Myoglobin peptide 120-134) % CV < 22%

S a m p l e 1 S a m p l e 2 S a m p l e 3 S a m p l e 4 1×107 2×107 3×107 4×107

Peak Area

Lamb (Myoglobin peptide 120-134) % CV < 19%

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Tryptic Digestion Optimization

After 1h, peptides can be detected. At 4h, we observed 30-40% of the maximum abundance.

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Chromatograms From Beef Meat Sample Spike With 1 % Pork Meat

MS1 XIC 744.8304 ± 5 ppm

7 8 9 10 Time (min)

Relative abundance (%)

Pure beef are in red Pure beef are in blue Pure beef are in blue

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What about Gelatine Gelatine

Porcine Beef Chicken Fish

About 50% of production

What is gelatine ?

• Gelatine is an hydrolyzed form of collagen
• Mainly, Type I, Type III and Type II collagen

During the preparation of gelatin, triple helical tropocollagen is denatured by heat and chemical hydrolysis forming 3 collagen specific polypeptidique chains.

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Gelatine Adulteration Detection

Complementary Type I collagen tryptic peptides [831-846] / [847-879] Specific to pork gelatin

Data Courtesy F. Beaudry, University of Montreal, Canada

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Protein Proteotypic peptides MS/MS spectra

Fractionation Digestion LC-MS Homogenization/Lysis Targeted MS

We can use labeled peptides as internal standards !

• 1. Select precursor ion

MS

• 2. Precursor fragmentation

MS/MS

• 3. Use Precursor-Fragment

pairs for identification

Quality Control !

Introduction of internal standards ( 13C, 15N)

This is a must for methods to be used for routine analysis in multiple sites

Routine Practice

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Conclusions

• Targeted bottom-up proteomics approach is applied for meat species detection down to 0.1%

w/w both in meat and gelatine

• Quick and simple workflow for any laboratory
• High resolving power (140.000 FWHM) was needed to obtain sufficient selectivity
• Isotopically labelled peptides recommended for routine control
• Tested in routine – applying HPLC-Q Exactive

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Acknowledgments

Special Thank you to dr. Francis Beaudry and dr. Alberto Ruiz from the Université de Montréal, Canada for providing the data DOI: 10.1080/19440049.2015.1064173

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Webinar: Thermo Fisher Scientific Meat Adulteration Resource

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• Current IC/LC-MS/MS portfolio allows determination of polar pesticides in both food and environmental

samples well below regulatory limits

• Simple sample preparation for IC separation – no FMOC needed!
• Good separation efficiency of IC makes it a suitable method for most polar pesticides
• TSQ Quantiva is the recommended MS/MS for water analysis @ ppt levels
• IC-TSQ Endura is suitable for food sample analysis @ ppb levels (LC-TSQ Quantiva an option if more

sensitivity or more difficult matrix is analyzed)

• Full application support is provided for the methods by EU Sales Support Team

Conclusions