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

Meat Authenticity Testing by Orbitrap MS Technology Michal Godula, Ph.D. Thermo Fisher Scientific The world leader in serving science

A real scandal !! Meat Substitution 2

Meat Substitution Motivation : $$$ How testing is done? • Addition of meat from undeclared species to a • Two-dimensional polyacrylamide gel specific meat product in order to lower production electrophoresis and western-blot analysis cost and increase profitability • Qualitative Real-Time PCR Cost per kg: Horse meat << Beef meat • Enzyme-linked immunosorbent assay (ELISA) Challenges An international issue • These methods are mostly qualitative • It is economic fraud • Molecular information obtained is limited • It represents health issues due to specific dietary restrictions • Data can’t be revisited post-acquisition for data mining • It is an ethical problem • They are not generic approaches and need to be • It is also an important cultural and religious heavily customized issues 3

What About Mass Spectrometry Options 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 High Resolution Accurate Mass  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 4

How Accurate Is Your Mass? • Mass accuracy usually expressed as ppm or mDa error  m m    6 meas true m / z 10 m true  6  500 . 1 500 . 0    • Quadrupole MS m / z 10 200 ppm 100 mDa error 500  6  500 . 10414 500 . 10314    • Orbitrap MS m / z 10 2 ppm 1 mDa error 500 . 10314 5

Mass Resolution FWHM • Resolution m  R  m (FWHM)  m • Orbitrap (HR)MS • Quadrupole MS 400  400   R 1000  R 100000 0 . 4 0 . 004 6

Principle Of Orbitrap TM Operation  Characteristic frequencies: Frequency of rotation ω φ • Frequency of radial oscillations ω r • Frequency of axial oscillations ω z • Intensity k    2 2     R m R m             z 1 2 z / m z r z     R R 2 m/q Makarov A. Anal. Chem. 2000, 72 , 1156-1162. 7

Resolution vs. m/z Q Exactive HF Q Exactive 350000 300000 Much higher resolution 250000 at low m/z range Resolution (FWHM) 200000 150000 100000 Q-TOF 50000 0 100 200 300 400 500 600 700 800 900 1000 m/z 8

Thermo Scientific TM Q Exactive TM MS - a 3D View Quadrupole Mass Filter RF-Lens C-Trap HCD Cell Orbitrap Mass Analyzer 9

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 10

Traditional Peptide Fingerprinting Approach Using MS Generate a mass list (MS and MS/MS Porcine TIC data) Sus scrofa Database search (MASCOT) MS and MS/MS matching 0 5 10 15 20 25 30 35 40 45 Time (min) Peptide fingerprinting Beef Horse 17 specific peptides 23 specific peptides Using a relatively high 7 429 common abundance threshold unique peptides … identified Lamb Pork 9 specific peptides 14 specific peptides 11

Peptide Mass Fingerprinting PMF 12 Bargen et al. (2013), Journal of Agricultural and Food Chemistry, 61:11986-11994

What Are The Main Limitations Of This Analytical Approach? • 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 ? 13

Targeted Bioinformatics Analysis Example: Myoglobin Myoglobin is the primary oxygen-carrying protein of muscle tissues Extraction of gene It is a highly abundant protein information In silico sequence alignment In silico protein Proteotypic peptides can be translation and identified alignment (120-134) 120 134 Sequence analysis and In silico tryptic Beef YLEFISDA IIHVLHAKHP SDFGADAQAA MSKALELFR Horse YLEFISDA IIHVLHSKHP GDFGADAQGA MTKALELFR digestion Pork YLEFISEA IIQVLQSKHP GDFGADAQGA MSKALELFR YLEFISDA IIHVLHAKHP SDFGADAQGA MSKALELFR Lamb Generate proteotypic mass lists 14

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 Genome annotation Protein / peptide identification 15

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 Proteomic grade trypsin is added and the reaction is performed at 40ºC for 24h. 7. Trypsin cleavage occurs after basic amino acids : Lys (K) & Arg (R) III. IV. I. II. + H 2 O 16

Each Targeted Peptides Can Be Detected And Extracted From Tics 751.8378 ∆ m is - 0.7 ppm 752.3393 752.8404 753.3409 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 Very complex TIC’s Over 7000s of unique peptides can be found. Need for very high resolution data (>100 000). 17

Data Dependent MS/MS Used For Targeted Work Beef Horse Pork Lamb 766.8435 751.8383 744.8304 759.8357 Survey MS Scan (z=2) (z=2) (z=2) (z=2) 140 000 FWHM m/z 500-2000 Select ions of interest Isolation width = 1.5 AMU Beef Horse Pork Lamb 1298.5681 1268.5576 1254.5419 1284.5525 Acquired MS/MS (y 13 ) (y 13 ) (y 13 ) (y 13 ) spectra 1395.6209 1365.6103 1351.5947 1381.6053 (y 14 ) (y 14 ) (y 14 ) (y 14 ) 17 500 FWHM Generation of post-analysis XICs for selected proteotyping peptides Extracted ion chromatogram at y 14 or y 13 ± 5 ppm 18

Assessment Of The Apparent Intra- And Inter Method Reproducibility Beef (Myoglobin peptide 120-134) Horse (Myoglobin peptide 120-134) % CV < 20% 3.5×10 8 2.0×10 8 % CV < 16% 3.0×10 8 1.5×10 8 2.5×10 8 Peak Area Peak Area 2.0×10 8 1.0×10 8 1.5×10 8 1.0×10 8 5.0×10 7 5.0×10 7 0.0 Sample 1 Sample 2 Sample 3 Sample 4 0.0 1 2 3 4 e e e e l l l l p p p p m m m m a a a a S S S S Pork (Myoglobin peptide 120-134) Lamb (Myoglobin peptide 120-134) 2.5×10 7 % CV < 22% % CV < 19% 4×10 7 2.0×10 7 Peak Area 3×10 7 1.5×10 7 Peak Area 2×10 7 1.0×10 7 5.0×10 6 1×10 7 0.0 0 1 2 3 4 e e e e 1 2 3 4 l l l l p p p p e e e e m m m m l l l l p p p p a a a a m m m m S S S S a a a a S S S S 19

Tryptic Digestion Optimization After 1h, peptides can be detected . At 4h, we observed 30-40% of the maximum abundance. 20

Chromatograms From Beef Meat Sample Spike With 1 % Pork Meat MS 1 Relative abundance (%) XIC 744.8304 ± 5 ppm Pure beef are in red 7 8 9 10 Time (min) Pure beef are in blue Pure beef are in blue 21

What about Gelatine What is gelatine ? About 50% of production Gelatine is an hydrolyzed form of collagen • Mainly, Type I, Type III and Type II collagen • Porcine During the preparation of gelatin, triple helical tropocollagen is denatured by heat and chemical hydrolysis forming 3 Gelatine collagen specific polypeptidique chains. Beef Fish Chicken 22

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 23

Routine Practice Protein We can use labeled peptides Proteotypic peptides as internal standards ! MS/MS spectra Quality LC-MS Targeted MS Control ! 1. Select precursor ion Introduction of internal standards ( 13 C, 15 N) MS Digestion Fractionation 2. Precursor fragmentation This is a must for methods to MS/MS be used for routine analysis in Homogenization/Lysis multiple sites 3. Use Precursor-Fragment pairs for identification 24

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 25

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 26