Authenticity testing described solutions

1. LC-MS authenticity testing
December 2018

2. Authenticity
Multiplex Protein LC-MS methods for animal species authenticity testing in
(finished) food products
Published method TrustGel (Food Chemistry 243 (2018): 461–467)
Collagen portfolio extends to all relevant species in food and pharma market
Collagen 1α2 GXY domain Euarchontoglires –  cDNA level
Other examples: milk
proteins, blood proteins
Example application in
pharma: Selective protein
quantification in humanized
models

3. Typical full scan chromatogram of milk from five different species.
RT: 0.00 - 18.01
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
6.08
390.75284
5.26
415.73145 7.18
374.69180
1.10
269.06256
7.90
317.69907
9.75
692.40533
4.18
345.68640
12.68
742.45215
3.21
323.66498 11.51
772.094362.77
309.67572 13.15
158.15367
14.12
154.99001
15.14
185.94099
16.23
185.94102
5.95
390.75250
7.06
374.688814.59
352.68661
7.83
317.69937
9.67
692.40430
12.33
654.36230
0.91
259.17084
3.58
287.18390 10.50
526.78278
2.50
276.16254
13.16
158.15387
14.12
154.99023
15.20
185.94101
16.26
185.94101
5.87
390.75241
0.98
247.65784
6.97
374.68851
3.53
287.18393
4.52
352.68634
7.70
317.69962
9.78
650.36902
12.13
654.36108 12.66
742.4513511.01
771.76410
2.46
285.67114
13.16
344.24347
14.91
185.94098
15.46
185.94098
16.55
185.94096
5.50
344.69022 6.02
350.71530 9.41
478.302371.29
269.06219
11.20
718.936834.76
249.67377
12.63
742.94739
8.53
502.59616
6.83
444.56012
1.00
323.20145 4.17
308.68652
2.63
322.50705
10.74
642.84113
12.88
1237.91772 14.15
154.99004
15.24
174.07608
16.77
174.07608
1.14
185.94089
6.13
390.75250
5.28
415.72995
6.38
419.26437
9.86
692.40448
3.90
416.19666
7.81
598.34576
12.70
742.45160
11.32
746.745791.43
185.94098 13.43
354.28525
14.98
185.94096
15.65
185.94099
16.60
185.94101
NL: 5.77E9
Base Peak F: FTMS
+ p ESI Full ms
[150.00-2000.00]
MS 180219_004
NL: 8.01E9
Base Peak F: FTMS
+ p ESI Full ms
[150.00-2000.00]
MS 180219_008
NL: 8.60E9
Base Peak F: FTMS
+ p ESI Full ms
[150.00-2000.00]
MS 180219_012
NL: 4.72E9
Base Peak F: FTMS
+ p ESI Full ms
[150.00-2000.00]
MS 180222_004
NL: 7.86E9
Base Peak F: FTMS
+ p ESI Full ms
[150.00-2000.00]
MS 180301_004

4. Example of sequence differences for one milk protein and five species.
Result: peptide 4 is specific for cow milk. Specific peptides for other species
were found in other milk proteins.
A2 B2 C2 D2 E2 F2 G2 H2 I2 J2
PEP2 n n n n
PEP3 n n n n
PEP4 n n
PEP6 n n n n n n n n
PEP7 n n n n n n n n
PEP8 n n n n n n n n
PEP9 n n n n n n n n
PEP10 n n n n n n n n n n
PEP11 n n n n
A2, B2
C2, D2 E2, F2 G2, H2
I2, J2

5. Detection of cow milk in milk of other species.
RT: 0.00 - 18.01
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (min)
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 9.90
BP: 979.56195
RT: 7.89
BP: 490.28622
RT: 2.14
BP: 490.28595
RT: 13.04
BP: 490.28238
RT: 9.81
BP: 979.56262
RT: 1.92
BP: 490.28531
RT: 13.74
BP: 490.28430
NL: 1.00E8
NL: 1.00E9
NL: 1.00E8
NL: 1.00E9
RT: 0.00 - 18.01
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (min)
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
0
20
40
60
80
100
RelativeAbundance
RT: 10.02
BP: 979.56018
RT: 1.92
BP: 490.28693
RT: 13.74
BP: 490.28458
RT: 10.06
BP: 979.56299
RT: 1.71
BP: 490.28671
NL: 1.00E8
NL: 1.00E9
NL: 1.00E8
NL: 1.00E9
+ 10 %+ 10 %
+ 10 %+ 10 %

6. Detection of protein isoforms. Spheres indicate amino acids and similar colors
indicate identical amino acids.
RT: 0.00 - 18.00
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
10.06
979.56299
1.70
490.28632
4.27
327.19342
10.30
490.28442
1.63
490.28607
8.79
490.28375
1.91
490.28528
13.75
490.28467
4.84
327.19284
10.63
1082.56641
7.69
541.78955
10.88
541.78717
4.34
361.52844
13.79
541.78760
3.70
541.78931
14.81
541.78644
16.65
1082.57214
10.71
1052.55640
10.91
526.78253
12.89
1052.55420
0.71
526.78259
10.45
526.78094
13.81
526.78259
15.18
526.77985
NL: 4.27E8
m/z=
327.19030-327.19358+
490.28180-490.28670+
979.55627-979.56607 F: FTMS
+ p ESI Full ms
[150.00-2000.00] MS
180301_006
NL: 8.94E8
m/z=
361.52540-361.52902+
541.78444-541.78986+
1082.56158-1082.57240 F:
FTMS + p ESI Full ms
[150.00-2000.00] MS
180301_006
NL: 8.36E8
m/z=
351.52193-351.52545+
526.77924-526.78450+
1052.55116-1052.56168 F:
FTMS + p ESI Full ms
[150.00-2000.00] MS
180301_006

7. Quick and specific detection of blood proteins from different species.
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
RAT SUS MACFA
MUS BOV RABIT
CANLF FELCA HUM
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
MACFA
HUM
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
SUS
BOV
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
CANLF
FELCA
2.5 3 3.5 4 4.5 5 5.5 6 6.5
Retention time (min)
MUS
RAT
RABIT