The world leader in serving science

Michal Godula Ph.D. Thermo Fisher Scientific

Meat Authenticity Testing using Thermo Scientific Orbitrap HRAM MS Technology

2

Meat Substitution

A real scandal !!

3

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

4

Unit mass resolution MS • Is targeted (triple quadrupole)

• Selectivity 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)

No false positives!

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

610/

true

truemeas

m

mmzm

• Quadrupole MS

• Orbitrap MS

ppmzm 20010500

0.5001.500/ 6

ppmzm 21010314.500

10314.50010414.500/ 6

100 mDa error

1 mDa error

6

•Resolution

Mass Resolution FWHM

m

mR

m (FWHM)

• Quadrupole MS • Orbitrap (HR)MS

10004.0

400R 100000

004.0

400R

7

Resolution vs. m/z

0

50000

100000

150000

200000

250000

300000

350000

100 200 300 400 500 600 700 800 900 1000

Resolu

tion (

FW

HM

)

Q Exactive HF Q Exactive

Q-TOF

m/z

Much higher resolution

at low m/z range

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Thermo ScientificTM MS Portfolio

HR/AM

MS, MSn

Ap

plie

d

Mark

ets

Researc

h

Mark

ets

Non-targeted Analysis

Targeted Analysis

Quantitative Qualitative

• Biomarker Discovery

• Proteomics

• Metabolism

• Metabolomics

• Proteomics

• Bioanalysis

• Food Safety

• Environmental

• Clinical/Toxicology

• Metabolomics

• PTM Analysis

• Lipidomics

Transform Your Science

Ion Traps Triple Quads

Tribrid Orbitrap

MS

(Q) Exactive Series MS

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Principle Of OrbitrapTM Operation

12

2

R

Rmz 2

2

R

Rmzr

zm

kz

/

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

Characteristic frequencies: • Frequency of rotation ωφ

• Frequency of radial oscillations ωr

• Frequency of axial oscillations ωz

Inte

nsity

m/q

10

• Fundamental difference to other HRAM instruments

• Parameter measured is frequency, not time/voltage/current

• Resolution allows more accurate m/z determination

• Less prone to ambient conditions changes

• Usually stable within <2 ppm during several days

• No need for lock mass in “routine work”

• Small footprint

• Easy to setup

Orbitrap Mass Analyzer Features

11

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

of 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

0 5 10 15 20 25 30 35 40 45

Time (min)

Porcine TICSus 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

…

14

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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Targeted Analysis Of Myoglobin Proteotypic Peptides

Strategy

Survey MS Scan

Select ions

of 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

Extracted ion chromatograms with mn ± 5 ppm

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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

751.8378

752.3393

752.8404

753.3409

∆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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Identification Of Specific Product Ions – MS/MS

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Survey MS Scan

Select ions

of 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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766.8 1298.5681 (y13) 766.8 1395.6209 (y14)

751.8 1268.5576 (y13) 751.8 1365.6103 (y14)

744.8 1351.5947 (y14)

759.8 1381.6053 (y14)

Beef

Horse

Pork

Lamb

Very Specific Xics Allow Species Differentiation

759.8 1284.5525 (y13)

744.8 1254.5419 (y13)

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Q Exactive Analysis At 140 000 FWHM Resolution.

0 5 10 15 20 25 30 35 40 45

Time (min)

100%

50%

25%

10%

1%

Peak areas observed were

proportional with the relative

concentration

0 20 40 60 80 1000

1000000

2000000

3000000

% W/W

Pe

ak A

rea

R2 = 0.9676

Pork meat was mixed at weight % ratios of 1, 2, 10, 25, 50 and 100 with a mixture of 1:1:1

beef:horse:lamb meat

Digestion was performed in triplicate.

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Sam

ple

1

Sam

ple

2

Sam

ple

3

Sam

ple

40.0

5.0×106

1.0×107

1.5×107

2.0×107

2.5×107

Pe

ak A

rea

Assessment Of The Apparent Intra- And Inter Method Reproducibility

Sam

ple

1

Sam

ple

2

Sam

ple

3

Sam

ple

40.0

5.0×107

1.0×108

1.5×108

2.0×108

2.5×108

3.0×108

3.5×108

Pe

ak A

rea

Beef (Myoglobin peptide 120-134)

% CV < 20%

Sam

ple

1

Sam

ple

2

Sam

ple

3

Sam

ple

40.0

5.0×107

1.0×108

1.5×108

2.0×108

Pe

ak A

rea

Horse (Myoglobin peptide 120-134)

% CV < 16%

Pork (Myoglobin peptide 120-134)

% CV < 22%

Sam

ple

1

Sam

ple

2

Sam

ple

3

Sam

ple

40

1×107

2×107

3×107

4×107

Pe

ak A

rea

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)

Rela

tive a

bundance (

%)

Pure beef are in red

Pure beef are in blue Pure beef are in blue

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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 Practive

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Conclusions

Targeted bottom-up proteomics approach was applied for meat species

detection down to 0.1% w/w

Quick and simple workflow for any laboratory

High resolving power (140.000 FWHM) was needed to obtain sufficient

selectivity (not suitable for QQQ)

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