TrustGel, a validated LC-MS (liquid chromatography - mass spectrometry) method for the animal species specific quantification of gelatin

1. Animal species specific quantification of gelatin with TrustGel
Anne Kleinnijenhuis
anne.kleinnijenhuis@triskelion.nl
November 2017

2. Contents
Introduction
TrustGelTM method
Theoretical target selection
Experimental target selection
Internal standardization strategy
Conclusions & Future

3. Introduction (1)
Gelatin is a valuable ingredient in food & pharma applications
It is produced by partial hydrolysis of collagen
Acid
Base
Hot water extraction
Predominantly collagen 1 (1α1 and 1α2)

4. Introduction (2)
Commercial gelatin is often produced from bone and skin
Bone mainly contains collagen 1α1 and 1α2
Skin mainly contains collagen 1α1, 1α2 and 3α1
Qualitative assessment of collagen types
in Bos taurus skin using analogous target
peptides (which have the same position
in the aligned sequence).

5. Introduction (3)
Fibrillar collagens almost entirely consist of GXY amino acid repeats
G = glycine, X and Y are often (hydroxy)proline and alanine
One fifth of the amino acids is proline
Often prolines are posttranslationally modified to hydroxyproline
GXY repeats result in the typical triple helix structure
Collagen crosslinks are partially cleaved by acid/base treatment
In alkaline-treated gelatin: N/Q deamidation

6. Introduction (4)
The gelatin industry will benefit from a sensitive and reliable species
determination method.
Important to manufacturers:
Label claim
Regulatory requirements
Also important to consumers:
Know what you eat
Halal foods

7. Introduction (5)
Currently available gelatin analytical methods are DNA- or protein-
based
DNA-based e.g. PCR (polymerase chain reaction)
Protein-based e.g. ELISA (enzyme-linked immunosorbent assay)
Issues reported regarding (false) negatives, false positives,
inconsistencies, low reactivity (Food Chemistry 190 (2016) 276–284)

8. Introduction (6)
Bottom up LC-MS offers the following advantages:
Triskelion developed and validated
The development of the TrustGelTM method has been initiated
and supported by Rousselot, the global gelatin market leader
Reliable technique. Particular targets remain intact during
processing. Easy implementation of quantitative targets.
Multiple target detection:
Confirm presence of animal species
Monitor other collagen types
Monitor collagen from other animal species
Production conditions
Biological variation in hydroxylation

9. (1)
TrustGel has unique advantages.
Diverse validation set (variables: tissue source, continent of origin,
processing conditions, bloom value, animal species)
Bovine in porcine and Porcine in bovine
Developed according to the Triskelion general workflow (Link)
Utilizes combined internal standardization approach
CALs/QCs prepared with reference gelatin => analyte processing
Implementation of SIL IS (stable isotope labeled internal
standard)

10. Acceptable and accountable method performance on the single
sample level
Validated under GLP
Traceability
Data integrity
Validated according to bioanalytical guidelines
Scientifically sound
Completeness
LLOQ (lower limit of quantification): 0.05%
(2)

11. Proteogenomic analysis
Differences between Bos taurus and Sus scrofa collagen were
studied on the nucleotide, codon group, amino acid and target
peptide level
The findings are presented in Food Chemistry 243: 461-467 (2018)
Example comparison Homo sapiens / Mus musculus (Link)

12. Theoretical target selection
The theoretical target selection was related to:
Peptide length
Peptide structure unambiguity
Peptide stability
Target uniqueness

13. Experimental target selection
Performed on Dionex Ultimate 3000 – Q Exactive Orbitrap
(Thermo Scientific)
Peptide sequence confirmation using Orbitrap MS/MS data
The experimental target selection was related to:
Sensitivity
Specificity
Peak shape / chromatography

14. SIL IS synthesis
Equivalent to analyte targets
Containing 15N/13C labeled amino acid
E.g. porcine SIL IS:
GIpGEFGLpGPAGPR(-13C6
15N4)
Including Hyp (p) residues for accurate peak matching in the
complex chromatograms

15. Experimental & Results
The method consists of 4 parts:
Details are described in the Food Chemistry reference
1. Solubilization
2. Analyte processing
3. UPLC-MS/MS (QqQ, Waters)
4. SIL IS synthesis and implementation

16. Absolute recovery
calculation
According to:
Bioanalysis (2016) 8,
891–904

17. Conclusions
Reliable detection of porcine and bovine gelatin, LLOQ 0.05%
Validation study scientifically sound, complete and traceable
Combined internal standardization strategy
Diversity in the validation set
Experimental basis and theoretical justification

18. Future
The TrustGelTM method will be expanded in the near future …
Other quantitative / qualitative animal species?
Other collagen types?
Expanded ranges?
Triskelion is also open to your requests

19. SlideShare series Anne Kleinnijenhuis
Animal species specific quantification of gelatin with TrustGel
(November 2017)
Domain-specific analysis of collagen code (May 2017)
Exploring LC-MS peptide dynamic range (December 2016)
Strategies for bioanalysis of proteins using LC-MS (May 2016)
Proposal for the absolute quantification of modular molecules
(October 2015)