Putting proteins together &
taking them apart
a review and critique of
current technologies for
protein
quantitation
Eric Eccleston
ric@aminoacids.com
R1
H N C OH
+ etc.
R2
R3
+ H N C OH + H N C OH
H2O
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
1
R1
amino acid :
-
H N
H2O
C OH
R1
residue :
N
C
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
+ H2O
R1
H N C OH
R2
R3
+ H N C OH + H N C OH
+ etc.
2
FOOD
the “great unwashed”
solubility
protein
intact
“digested”
tryptophan
280nm
CH
2
N
H
R1 R2 R3 R4
Warburg 1
approximation
R6 R7 R8
N C N C N C N C N C N C N C N C N
intact protein
3
Lowry
color
CEM SPRINT
DYE
conversion factor
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
Biuret
color
intact protein
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C N
Near Infra Red
NIR 800-2500 nm
SWNIR 800-1000 nm
conversion factor
intact protein
FOSS
FOODSCAN
NIPALS, PCA BP-ANN, ICA LS-SVM
chemometrics
4
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
Kjeldahl
Dumas
Combustion
H2SO4
Reduce NO x & etc
conversion factor
NH +
4
N
2
NH
2
N
H N
2
N
N
NH
2
Melamine
5
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
o
6 N HCl
R1
H N C OH
110 C 24 hrs
phew!
R2
R3
+ H N C OH + H N C OH
+ etc.
the “great unwashed”
6 N HCl
110 C 24 hrs
phew!
6
?
?
cysteine:
methionine:
SCH
3
(CH )
CH
22
2
R1
R3 R4 R5
R7 R8
SH
N C N C N C N C N C N C N C N C
7
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
o
performic acid 0 C 18 hr
o
6 N HCl 110 C 24 hr
O
O
cysteic acid:
S
O
double phew!
O
SCH 3
OH methionine sulfone: (CH )
2 2
CH 2
H N C OH
H N C OH
+ etc.
tryptophan
CH
2
N
H
R1 R2 R3 R4
R6 R7 R8
N C N C N C N C N C N C N C N C N
8
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C
o
4.2 N NaOH 110 C 24 hrs
phew!
CH
2
N
H
tryptophan + etc.
H N C OH
OH
H
H
H
OH
H
OH
H
OH
OH
H
OH
IEC
postReaction
column
9
OH
H
H
H
OH
H
OH
H
OH
OH
H
OH
Reaction
preRP
column
MS Confirm
Leather protein in Milk
nouvelle haute cuisine?
10
collagen
glycine
hydroxyproline
hydroxylysine
6 N HCl
110 C 24 hrs
the “great unwashed”
A
A=B?
6 N HCl
110 C 24 hrs
B
11
Intact protein
•Dye – SPRINT
calibration
•NIR – FoodScan
Digested protein
•Kjeldahl, Dumas - nonprotein N
•Hydrolysis
-composition
-protein as sum of Aas
-MS confirmation
Intact proteins
•CEM SPRINT
•NIR
calibrate
Digests
•Kjeldahl, Dumas
•Hydrolysis
-composition
-MS confirmation
12
$
$$
$ COST
$ $$
☺☺ ☺
☺ BENEFIT
☺ ☺☺
Graded Response
Triage
13
Food Protein Workshop: Developing a Toolbox of Analytical
Solutions to Address Adulteration
USP Headquarters, Rockville, Maryland
USP Meeting Center
Tuesday, June 16, 2009
4b. Breakout Session B
Status of nitrogen-based methods for protein
measurement
By
Jürgen Möller
FOSS Analytical, Sweden
Dedicated Analytical Solutions
Agenda
TOPICS/QUESTIONS FOR SPEAKER TO ANSWER:
• Status and overview of N-based methods and how they are being used for
protein measurement
• Advantages of these methods and why they are considered “gold standard
methods”
• Susceptibility to adulteration
• Need for reference standards
• N to protein conversion factors and accuracy, specificity/selectivity, reference
standards
• View on future research opportunities to advance protein measurement science
Dedicated Analytical Solutions
14
Kjeldahl - Reference Method still in use
• Kjeldahl from 1883
For Nitrogen / Protein
• Dumas method 1980’s
For Nitrogen / Protein
• NIR/NIT methods 1980’s
For Crude Protein
• Change of Kjeldahl
catalysts in the 1990’s
•
•
Mercury banned
CuSO4, TiO2 and Selenium as alternative
Dedicated Analytical Solutions
Kjeldahl / Protein standards in OMA
•
920.53 (Hg)
•
930.33 (Cu)
•
920.70 (Hg)
•
932.08 (Hg)
•
920.87 (Hg)
•
939.02 (Hg)
•
920.109 (Cu)
•
940.25 (Hg)
•
920.123 (Cu)
•
941.06 (Cu)
920.155 (Hg)
•
945.01 (Hg)
920.176 (Hg)
•
945.18 (Hg)
925.31 (Hg)
•
945.23 (Hg)
928.08 (Hg)
•
945.48 (Cu)
930.01 (Hg)
•
950.09 (Hg)
930.02 (Hg)
•
950.10 (Hg)
930.25 (Hg)
•
950.48 (Hg)
930.29 (Cu)
•
955.04 (Hg)
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
962.10 (Hg)
969.37 (Hg)
976.05 (Hg)
977.02 (Hg)
978.04 (Hg)
979.09 (Hg)
981.10 (Hg)
984.13 (Cu)
988.05 (Cu/Ti)
991.20 (Cu)
2001.11 (Cu)
Dedicated Analytical Solutions
15
Kjeltec™ from 1970’s
•
•
1970 introduction of block digestion by
FOSS Tecator
Since 1974 introduction of direct
steam distillation and other
improvements by FOSS Tecator
- Decreased use of chemicals
- Improved efficiency of the
digestion
- No sample transfer
- Alkali added in closed system
- Distillation into boric acid receiver,
reducing the distillation times and
avoiding back titration
Dedicated Analytical Solutions
AOAC 2001.11
•
A method based on block digestion/ steam distillation /boric acid
receiver solution, having a wide scope of applicability fullfilled definitely
a need of the international laboratory society.
Method for the determination of Crude Protein in
Animal Feed, Forage, Grain, and Oilseed using
Block Digestion, Copper Catalyst and Steam
Distillation into Boric Acid
Study director: Nancy J. Thiex, SD State University, Brookings, US
Study report: JAOAC, 85 (2), 2002, p 309 – 317
Summary: In Focus, 26 (2), 2002, p 10 - 12
Dedicated Analytical Solutions
16
AOAC Crude Protein study
•
12 participating labs from US, UK, DE, DK and SE using Foss-Tecator
Kjeltec equipment / applications
• Results:
- Excellent repeatability and reproducibility
(0.4 – 2.4 %)
- No statistical difference between 1% and 4%
boric acid as titrant
Dedicated Analytical Solutions
AOAC study
•
14 different samples
representing animal feed,
forage, grain and oilseed.
•
Nitrogen content from 1–15 %
( 7 – 80 % crude protein)
•
Recoveries of nitrogen from
tryptophan 98,8 % and
acetanilide 100,1 %
90,00
80,00
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00
Dedicated Analytical Solutions
17
AOAC study Protein
ID
Protein Block
Swine Pellets
Corn Silage
Grass Hay
Fish Meal
Dog Food
Chinchilla Feed
Albumin
Bird Seed
Meat & Bone Meal
Milk Replacer
Soybeans
Sunflower Seeds
Legume Hay
Mean
40.20
37.03
7.10
7.11
64.60
24.51
18.08
79.10
13.52
50.11
20.79
38.76
17.42
18.85
# Lab a (b)
11(1)
11(1)
12
12
12
12
10(2)
11(1)
11(1)
12
12
10(2)
12
12
Sr
0.17
0.17
0.11
0.13
0.46
0.2
0.15
0.31
0.12
0.92
0.28
0.22
0.40
0.27
% RSDr
0.43
0.46
1.57
1.88
0.72
0.83
0.83
0.40
0.88
1.84
1.33
0.57
2.28
1.42
SR
0.29
0.21
0.15
0.13
0.65
0.22
0.15
0.36
0.12
0.92
0.28
0.22
0.40
0.27
% RSDR
0.73
0.57
2.07
0.88
1.00
0.88
0.83
0.46
0.91
1.84
1.33
0.57
2.28
1.42
Dedicated Analytical Solutions
AOAC method 2001.11
– digestion in aluminium block at 420o C in 12
ml sulfuric acid with K2SO4 and a copper
catalyst. Digestion time 60 min.
– manual or automatic addition of water (or
steam, SAfE) to avoid destillation of solidified
digests and exothermal reaction.
– addition of NaOH and steam to liberate and
distill ammonia.
– trapping of ammonia in boric acid and titration
with standardized acid (photometric endpoint).
Dedicated Analytical Solutions
18
Quality control
•
•
•
•
Each run has to contain a quality control sample and standards to verify
the nitrogen recovery and to check the accuracy of the equipment and
procedure:
Nitrogen loss: Mixture of ammonium sulfate and sucrose is digested and
distilled under the same conditions as the samples. Recovery > 99%.
Distillation/Titration efficiency: Ammoniumsulfate is distilled directly.
Recovery > 99,5%.
Digestion efficiency: Acetanilide or Tryptophan are digested in a mixture
with sucrose. Recovery > 98%.
Dedicated Analytical Solutions
Global Proteinstandard on basis of AOAC 2001.11
EN ISO 5983-2:2005
Animal feeding stuffs — Determination of nitrogen
content and calculation of crude protein content
—
Part 2: Block digestion/steam distillation method
ICS 65.120
EN ISO method on basis of AOAC 200.11
Dedicated Analytical Solutions
19
Performance of EN ISO 5983-2 standard (AOAC 2001.11)
•
Validated range: 0,3 – 70 % protein
•
Validation samples:
- Sample 1: protein block
- Sample 2: swine pellets
- Sample 3: corn silage
- Sample 4: grass hay
- Sample 5: fish meal
- Sample 6: dog food
- Sample 7: chinchilla feed
- Sample 8: albumin
- Sample 9: bird seed
- Sample 10: meat and bone meal
- Sample 11: milk replacer
- Sample 12: soybeans
- Sample 13: sunflower seed
- Sample 14: legume hay
-
Sample 15: fish feed, small floating pellets
Sample 16: fish feed, large floating pellets
Sample 17: shrimp feed, crumble
Sample 18: shrimp feed, large sinking pellets
Sample 19: shrimp feed, small sinking pellets
Sample 20: larvae feed, flake
Sample 21: wheat grain
•Validated by 24-26 international laboratories
•Photometric end point determination
•Developed on basis of FOSS Kjeltec equipment
•Reference method also for Dumas
•Excellent repeatability and reproducibility
Dedicated Analytical Solutions
Good repeatability …
s dr vs % prote in ISO 5983-2
1
0,9
0,8
0,7
0,6
sdr
0,5
Linear
0,4
0,3
0,2
0,1
0
0
20
40
60
80
100
Dedicated Analytical Solutions
20
… and reproducibility
sdR and sdr vs % protein ISO 5983-2
1
0,9
0,8
0,7
sdr
0,6
sdR
0,5
Linear (sdR)
0,4
Linear (sdr)
0,3
0,2
0,1
0
0
20
40
60
80
100
Dedicated Analytical Solutions
EN ISO 20483:2006
• Cereals and pulses –
Determination of the nitrogen
content and calculation of the
crude protein content –
Kjeldahl method
• Block digestion with Cu/Ti
catalyst, steam distillation
into boric acid, automatic
titration with photometric (or
pH) end point determination
• 2 h digestion time, 20 ml acid
Dedicated Analytical Solutions
21
prEN ISO/DIS 20483:2006
Perform ance EN ISO 5983-2
Perform ance EN ISO 20483
1,6
1,6
1,4
1,4
1,2
1,2
y = 0,0129x + 0,061
sd %
1
1
sdr
sdr
sdR
0,8
sdR
0,8
Linear (sdR)
Linear (sdR)
Linear (sdr)
Linear (sdr)
0,6
0,6
y = 0,0065x + 0,1414
0,4
0,4
y = 0,0058x + 0,0248
0,2
0,2
y = 0,0047x + 0,16
0
0
20
40
60
80
0
100
0
Protein %
20
40
60
80
100
Pr o t ein %
Dedicated Analytical Solutions
ISO 8968–3:2004 and IDF 20-3 (2004)
• Milk -- Determination of
nitrogen content -- Part
3: Block-digestion
method (Semi-micro
rapid routine method)
• Joint development with
IDF and AOAC
Dedicated Analytical Solutions
22
Non-protein N and protein N
•
•
•
•
Nitrogen in milk
Total Nitrogen (AOAC 991.20 / ISO 8968-2/3 / IDF 20-2/3 )
Nonprotein Nitrogen (AOAC 991.21 / ISO 8968-4/ IDF 20-4)
Protein Nitrogen (AOAC 991.23/AOAC 991.24, ISO 8968-5, IDF
20-5 )
• TCA precipitation of proteins allows a separation of non-protein
Nitrogen (Urea, Ammonia) in the filtrate from protein nitrogen
(casein..) in the filter cake
Dedicated Analytical Solutions
EN ISO 16634-1:2008 and TS 16634-2
•
•
•
Food products - Determination of the
total nitrogen content by combustion
according to the Dumas principle and
calculation of the crude protein
content
Part 1: Oilseeds and animal feeding
stuffs
Part 2: Cereals, pulses and milled
cereal products (TS)
•
Using the same factors as Kjeldahl
•
AOAC methods:
-
992.15 (meat)
992.23 (cereal grains & oilseeds)
Dedicated Analytical Solutions
23
Protein = Protein ?
• Dumas determines total
Nitrogen, including inorganic
fractions like NO2/NO3.
• Kjeldahl determines organic
nitrogen plus ammonia.
• For many samples the
difference might be negligible
– but you have to check.
Dedicated Analytical Solutions
Can Dumas replace Kjeldahl ?
S. Seling et al., Max Rubner Institute, DE
(2005)
•
•
•
•
Wheat harvest 2000-2004:
Some 2% of ”Dumas protein” is
not determined by Kjeldahl
method
Kjeldahl protein = 0,959*Dumas
+ 0,258
Difference depends on growing
year, cultivar and growing
condition
Dedicated Analytical Solutions
24
Protein = Protein ?
Sample
French Bean
Summer Barley
Nitrate
8,9 / 6,9
0,1 / 150
Lettuce
33,2 / 9,0
Cucumber
Yam (dioscorea)
Cabbage
Spinach
Saw-dust
7,2 / 10,3
4,9 / 9,6
7,1 / 5,2
27,2 / 5,0
0,074/ 113%
Example:
• 33 000 mg/kg NO3 = 7,45 g
N/kg = 0,75 % Nitrogen
• 0,75 x 6,25 = 4,7 % Protein
Conclusion:
• Dumas is a routine method
• Applicability has to be
checked vs Kjeldahl
Dedicated Analytical Solutions
Trade conflicts ?
• Argentine supplier of soymeal claims protein content
of 47,2 %
• Malaysian importer of soymeal claims protein content
of 44,9 %
• Reason: Seller uses Dumas method, buyer applies
Kjeldahl method
Dedicated Analytical Solutions
25
Can Dumas replace Kjeldahl ?
• European Commission confirms the Kjeldahl method
as the community method for official controls
(Commission Regulation (EC) No 152/2009
Dedicated Analytical Solutions
Kjeldahl factors
Most common: 6,25 = 16% N
Established on basis of the respective amino acid profile
Dedicated Analytical Solutions
26
Tryptophan
204.225 g/mol - 13.72% N – factor 7.3
Dedicated Analytical Solutions
Protein factors
Amino acid
Formula
MW (g/mol)
%N
Factor
Alanine
C3H7NO2
89,09
15,71
6,37
Arginine
C6H14N4O2
174,2
32,15
3,11
Asparagine
C4H8N2O3
132,12
21,19
4,72
Aspartic acid
C4H7NO4
133,1
10,52
9,51
C3H7NO2S
121,16
11,55
8,66
C5H9NO4
147,13
9,52
10,50
Cysteine
Glutamic acid
C5H10N2O3
146,14
19,16
5,22
Glycine
Glutamine
C2H5NO2
75,07
18,65
5,36
Histidine
C6H9N3O2
155,15
27,07
3,69
Leucine
C6H13NO2
131,17
10,67
9,37
Lysine
C6H14N2O2
146,19
19,15
5,22
Methionine
C6H11NO2S
149,21
9,38
10,66
Phenylalanine
C9H11NO2
165,19
8,48
11,79
Proline
C5H9NO2
115,13
12,16
8,22
Serine
C3H7NO3
105,09
13,32
7,51
Threonine
C4H9NO3
119,12
11,75
8,51
C11H12N2O2
204,23
13,71
7,29
Tyrosine
C9H11NO3
181,19
7,73
12,94
Valine
C5H11NO2
117,15
Tryptophane
Dedicated Analytical Solutions
11,95
8,37
27
Recovery of Nitrogen
Lysine hydrochloride
16
100%
15
90-93%
% Protein
14
Hg
Se
13
Cu
Ti
12
11
10
10
20
30
40
50
60
70
digestion time (min)
Dedicated Analytical Solutions
Recovery in real samples
% recovery of protein
100
98
96
94
Se
Cu
92
Ti
90
Hg
88
86
84
Lysine
Dog food
Meat
Fishmeal
Wheat
Dedicated Analytical Solutions
28
EGN collaborative study
Method
% CP
ICC 105/2 (Kjeldahl, Cu)
12,46
ISO 5983-2 (Kjeldahl, Cu)
12,45
ISO 20483 (Kjeldahl, Cu/Ti)
12,39
ISO 16634 (Dumas/Combustion)
12,55
Dedicated Analytical Solutions
AAFCO proficiency testing scheme (2008)
Cu
Cu/TiO2
Dumas
Sample
% CP
sd %
% CP
sd %
% CP
sd %
AAFCO 0826
18,2
0,35
18,2
0,29
18,4
0,32
AAFCO 0827
12,9
0,36
12,8
0,46
13,0
0,46
AAFCO 0828
40,8
0,77
40,0
0,68
41,0
0,43
AAFCO 0829
23,6
0,42
23,0
0,58
23,5
0,33
AAFCO 0830
18,2
0,27
18,2
0,24
18,5
0,32
AAFCO 0831
27,5
0,40
27,3
0,64
27,8
0,46
23,5
0,43
23,2
0,48
23,7
0,39
Average
Dedicated Analytical Solutions
29
Comparison of ”Protein contents”
Sample
Type
Kjeldahl
Dumas
Amino Acid
AAFCO 200921
Chicken
17,29 (0,15)
17,64 (0,33)
14,22 (0,17)
AAFCO 200922
Pig starter
23,94 (0,33)
24,51 (0,39)
19,73 (1,18)
AAFCO 200923
Chow
12,3 (0,52)
12,51 (0,65)
7,16 (0,19)
•
•
Sum of 18 reported AA: Alanine, Arginine, Aspartic Acid, Cystein.
Glutamic acid, Glycine, Histidine, Iso-Leucine, Leucine, Methionine,
Phenylalanine, Proline, Serine, Threonine, Tryptophane, Tyrosine,
Valine
Significant differences, possible sources:
- Non-Protein Nitrogen (Ammonia, Urea…)
- Wrong factor ?
Dedicated Analytical Solutions
N-based methods
Method
cost/analysis
(USD)
thruput
(samples/day)
Accuracy
Applicability
Kjeldahl
1,50 - 2,50
100-200
+++
+++
Dumas
1-2
100-200
++
+++
0,1
400-500
+
+
NIR
•
•
•
•
•
•
Most widely used for (crude) protein analysis
Simple to use, with adequate accuracy and wide applicability
Probably > 50,000 installed units
Probably > 30 Mio analyses / year
NIR calibrations based on Kjeldahl or Dumas
Susceptible to adulterations
Dedicated Analytical Solutions
30
Adulterations
• Positive (allowed), e.g. urea, biuret …
• Negative (prohibited), e.g. melamin …
• At contamination levels (ppm, ppb)
• At ”fraud” levels ( > 0,2-0,4 % CP)
Dedicated Analytical Solutions
Melamine
66% Nitrogen
”Protein” content = >400%
Solubility in water: 3,2 g/l
Dedicated Analytical Solutions
31
Examples for N-fractionation schemes
•
•
•
Nitrogen fraction in wort and beer:
- Total nitrogen
- “Heat coagulable protein”
- HMW protein (MgSO4 precipitation)
Nitrogen in malts (ale, lager, distilling)
- Total Nitrogen
- Soluble Nitrogen (hot water extract)
Nitrogen in animal feed / forage
- Crude protein
- ADIP / ADIN
- NDIP / NDIN
- Urea
- Biuret
- Ammonia
Dedicated Analytical Solutions
.. more examples
•
•
•
•
Nitrogen in milk
- Total Nitrogen (AOAC 991.20 / ISO / IDF )
- Nonprotein Nitrogen (AOAC 991.21 / ISO / IDF )
- Protein Nitrogen (AOAC 991.23/AOAC 991.24, ISO , IDF )
(after TCA precipitation; Melamine will probably coprecipitate)
Nitrogen in eggs
- Nitrogen in eggs (AOAC 925.31)
- Water-soluble Nitrogen and Crude Albumin (AOAC 932.08)
(pI precipation at pH 4; salting out with NaCl / EtOH)
Nitrogen in soymeal
- Crude protein
- Protein dispersibility index
- NPN (after TCA precipitation)
General scheme possible?
- E.g. pI precipitation in 0,1 M acetic acid ?
Dedicated Analytical Solutions
32
.. more information
Joseph F. Zayes
Functionality of Protein in Food
Springer, 1997, ISBN 978-3-540-60252-1
(google book)
•
•
•
Data/ experience from ”normal” samples is needed
”unnormal” ratios of protein nitrogen to non-protein nitrogen might be detected
Possibly some preliminary results can be presented
Dedicated Analytical Solutions
Summary
•
Kjeldahl is still the an important
reference and routine method for
the determination of crude protein
/ protein fractions
•
New standards reflect the
instrumental and methodological
progress
made
•
Fractionation schemes to trace
adulterations should be
investigated
Thank you for your attention.
Questions?
Dedicated Analytical Solutions
33
A Kjeldahl nitrogen-based true protein
method that accounts for 12% TCA
soluble nitrogen.
David M. Barbano
Cornell University
Ithaca, NY 14853
dmb37@cornell.edu
USP Food Protein Workshop:
Developing a Toolbox of Analytical Solutions to Address Adulteration
June 16-17, 2009
Rockville, Maryland
Background: A Dairy Industry Perspective
Measurement of nitrogen fractions in milk and
milk products has been the basis of reference
analysis for protein determination in milk.
In 1938, Rowland published the well-recognized
Rowland nitrogen fractionation scheme for milk.
The Rowland fractionation method form the basis
for measurement of casein and non-protein
nitrogen in milk and milk products today.
The 3 most commonly measured nitrogen
fractions in milk are:
total nitrogen,
non-casein nitrogen, and
non-protein nitrogen.
34
Background: A Dairy Industry Perspective
Non-casein nitrogen is the nitrogen soluble after
a milk sample has been treated with a
combination of acetic acid and sodium acetate
designed to precipitate casein. The nitrogen
remaining soluble is called non-casein nitrogen.
Total nitrogen minus non-casein nitrogen equals
casein.
Non-protein nitrogen is the nitrogen soluble after
a milk sample has been treated with
trichloroacetic acid to a final concentration of
12%. Total nitrogen minus non-protein nitrogen
equals true protein.
Background: A Dairy Industry Perspective
In the late 1980’s and early 1990’s it became
common to base part of the payment to dairy
farmers based on the protein content of milk.
Traditionally, milk protein content was expressed
on a total nitrogen basis (N x 6.38).
However, as value of protein in milk increased
the proportion of non-protein nitrogen naturally
present in milk became more of an issue because
it did not have the same value as protein and the
dairy industry did not want to send an economic
signal to dairy farmers to produce milk with
higher non-protein nitrogen content.
35
Background: A Dairy Industry Perspective
How much non-protein nitrogen is naturally
present in milk?
Typically about 5% of the total nitrogen is nonprotein nitrogen but it can vary from 2 to nearly
10% of the total nitrogen depending on dairy
cattle feeding.
What is the non-protein nitrogen in milk?
On average, non-protein nitrogen in milk is
about 50% urea and 50% other low molecular
weight metabolic nitrogen containing
compounds. The variable portion of natural
non-protein nitrogen in milk is urea.
Background: A Dairy Industry Perspective
The dairy industry uses to following terms to
distinguish the basis of milk protein
measurement.
1) Crude Protein or Total Nitrogen Basis (TN x
6.38)
2) True Protein Basis (TN minus NPN) x 6.38
36
Background: A Dairy Industry Perspective
Routine measurement of the protein content of
milk is done by infrared milk analysis but the
infrared milk analyzers are calibrated based on
Kjeldahl reference values.
In the US, it was proposed in the early 1990’s that
the basis of milk payment and calibration of
infrared milk analyzers be switched to a True
Protein basis. This would have doubled the
number of Kjeldahl nitrogen measurements to
make calibration samples for the routine method
and was considered too costly. There was a
need for a way to measure true protein nitrogen
directly by Kjeldahl without doubling the number
of Kjeldahl analyses.
Background: A Dairy Industry Perspective
As a result, a new method for directly measuring
the True Protein Nitrogen content of milk was
developed to replace the two test (i.e., TN and
NPN separately) analysis procedure. The Kjeldahl
method is unchanged the direct true protein
method is just a sample preparation procedure.
The Kjeldahl methods for measurement of total
nitrogen, nonprotein nitrogen, noncasein
nitrogen, direct true protein nitrogen, direct
casein nitrogen have all been collaboratively
studied and are official final action AOAC
methods for milk.
37
Status of Kjeldahl Methods for Milk
In the US and several other countries, true
protein is the basis for milk payment, feeding
management, and record keeping for genetic
selection.
In these countries the true protein Kjeldahl
reference is used as the basis of calibration
for milk protein determination by routine midinfrared milk analysis.
The USDA Federal Milk Markets adopted true
protein as the basis for protein payment in
the USA in 2000. The USDA laboratories use
the Kjeldahl direct true protein method as a
reference.
Status of Kjeldahl Methods for Milk Powder
For the purpose of international trade the basis
for the definition of protein content of nonfat dry
milk powders was left as a crude protein basis.
This leaves the official testing of milk powders
by Kjeldahl total nitrogen open to adulteration
with added non-protein nitrogen sources. This
analytical problem could be eliminated by
switching to a true protein basis as the standard
for milk powders. The Kjeldahl methodology is
already in place. The definitions of minimum true
protein level of milk powder would have to be
agreed upon by the IDF, ISO, Codex international
committees.
38
Method Precision and Accuracy
All of these Kjeldahl methods (for the milk matrix)
have well documented within and between lab
method performance statistics.
Method - Kjeldahl Total nitrogen in milk.
(Barbano, et al., JAOAC 1990. 73:849-859)
RSDr = 0.38% (within lab)
RSDR = 0.50% (between lab)
Method – Kjeldahl Direct True Protein in milk
(Barbano et al., JAOAC 1991. 74:281-288)
RSDr = 0.28% (within lab)
RSDR = 0.70% (between lab)
Method – Kjeldahl Non-protein nitrogen in milk
(Barbano et al., JAOAC 1991. 74:281-288)
RSDr = 2.81% (within lab)
RSDR = 5.70% (within lab)
Method Precision and Accuracy
All of these Kjeldahl methods have well
documented within and between labortory method
performance statistics based on collaborative
studies.
Method – Kjeldahl Total nitrogen in cheese.
(J. M. Lynch and D. M. Barbano. JAOAC 2002. 85:445-455)
RSDr = 0.38% (within lab)
RSDR = 0.50% (between lab)
Trouble Shooting Guide for Kjeldahl Analysis
J. M. Lynch and D. M. Barbano. 1999. Kjeldahl nitrogen
analysis as a reference method for protein
determination in dairy products. JAOAC. 82:1389-1398.
39
Kjeldahl True Protein Methods:
Susceptibility to Adulteration.
What influence does adulteration of milk with
Melamine have on the Kjeldahl true protein
analysis?
Melamine is low molecular weight, contains a
large amount of nitrogen, and is soluble partitions
into the 12% TCA soluble fraction. At low levels
of Melamine addition, the Kjeldahl true protein
method will correctly estimate true protein
content of milk.
At high levels of Melamine addition (e.g., 2 grams
per liter) additional steps to more completely
rinse the protein precipitate are necessary to
wash out the Melamine from the collected true
protein.
Mid- Infrared Milk Analysis True Protein
Method: Susceptibility to Adulteration.
What happens in the routine mid-IR method
whena milk sample is adulterated by with
Melamine?
Unlike with urea addition to milk, Melamine
does absorb infrared light at the sample
wavelength where protein is measured. This is
a major weakness of the routine mid- infrared
method.
40
Mid- Infrared Milk Analysis True Protein
Method: Susceptibility to Adulteration.
What happens in the mid-IR method when
a milk sample is adulterated by the addition
of Melamine?
Two different approaches are used for the core
protein method by mid-infrared.
Traditional fixed wavelength calibration
PLS full spectral calibration
The impact of adulteration of milk with
Melamine on the traditional fixed virtual filter
wavelength approach on an mid-FTIR will be
the same on all instruments using the same
wavelengths and instrument gain adjustments.
Mid- Infrared Milk Analysis True Protein
Method: Susceptibility to Adulteration.
True protein by IR (% )
Impact of Melamine on IR filter Protein Predciton - traditional fixed
filer wavelengths
4.00
3.90
3.80
3.70
3.60
3.50
3.40
3.30
3.20
3.10
3.00
0.00
0.20
0.40
y = 0.3502x + 3.1108
R2 = 0.9999
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
grams per Liter of Melamine
With spectral calibrations the sensitivity of the protein
estimate to Melamine addition can vary from one spectral
calibration model to another. Spectral calibrations will be
much less predictable in their susceptibility to adulteration
than traditional fixed filter models.
41
What can be learned from the dairy industry
experience with protein adulteration?
Reference methods (e.g., Kjeldahl) and
secondary routine methods for protein
measurement (e.g., mid-IR transmission
spectrophotometry) can be influenced
differently by different adulterants.
Just because the reference method is not
influenced by the adulterant, it does not mean
the secondary method will not be influenced.
Different secondary methods for protein
measurement may be influenced differently by
different protein adulterants.
Protein Analysis
Colorimetric Method
Sam KC Chang, Ph.D.
Professor, IFT Fellow
84
Department of Cereal and Food Sciences
North Dakota State University
42
OUTLINES
Status and overview of these methods
y
How they are being used for protein measurement
Comparison for each method in terms of
Principle, selectivity,
Accuracy, precision,
y Simplicity/analysis time,
y Interferences,
y Cost
y
y
Susceptibility to adulteration
Need for reference standards
Food matrix issues
View on future research opportunities for advancing protein
measurement science
85
HOW ARE THEY USED
Measure proteins in situations where proteins could be
solubilized except in the case of dye binding in that
proteins form complex precipitate with remaining dye in
the solution.
During extraction, purification and characterization of
proteins.
y
y
y
y
y
y
Animal proteins
Plant seed storage proteins
Microbial proteins
Enzymes
Inhibitors
Toxins
86
43
GENERALLY, CAN ESTIMATE
True protein/peptide content.
Non-protein nitrogen (including melamine): By
differences between colorimetric and total crude
proteins by Kjeldhal or Dumas methods.
87
GENERAL ADVANTAGES AND DISADVANTAGES
OF COLORIMETRIC METHODS
Advantages
Relative simple, rapid
y Inexpensive
y Reproducible and accurate
y No corrosive reagents
y
Disadvantages
Most work for proteins that can be solubilized in the
liquid systems.
y Need to establish a standard curve for specific type of
food using a reference protein, or correlate with an
official total nitrogen methods.
y
88
44
BIURET METHOD
Measure peptide bonds (2 or more).
Cu+2 reagent complexes with peptide bonds.
Develop violet color (540 nm).
O C
Advantages:
Simplest method.
y Color deviation not too great.
y Few substances interfere.
y Does not measure non-protein N.
y
H
N
R
C
H
N
H
C
C
R
C
N
H
N
C
O
Cu
89
BIURET METHOD-- DISADVANTAGES
Disadvantages:
y
y
y
y
y
y
Not very sensitive (1-10 mg).
Bile pigment interferes.
Ammonium salts.
Color deviation, gelatin gives pinkish-purple color.
Large amount of lipid or CHO gives opalescence, not
clear, turbidity.
Not an absolute method, requires standardization
against known protein (bovine serum albumin).
90
45
BIRUET METHOD- APPLICATIONS
Protein isolation, purification and characterization.
Cereal proteins
Meat proteins
Soy proteins
Animal feed (AOAC 935.11)
91
LOWRY METHOD
Cu+2 reagent complexes with peptide bonds, plus
reduction of phosphomolybdic-phosphotungstic
reagent (Folin reagent) by tyrosine and tryptophan.
H2
Give blue color (750 nm).
C
H2
C
Advantages:
OH
N
H
Sensitive, 50-100 times more sensitive than biuret method.
Micrograms range (10 to 100 μg)
y Specific, less affected by turbidity.
y Rapid (45 min).
y
92
46
LOWRY METHOD (CONTINUED)
Disadvantages:
Need standardization with known protein.
Color varies with protein.
y Color not strictly proportional to concentrations.
y
y
Modified method (Hartree, 1972)
y
Interfered by sugars, ammonium sulfate, sulfhydryl
compounds such as mercaptoethanol.
93
LOWRY METHOD- APPLICATIONS
Used primarily in the extracts of foods,
Protein isolation, purification and characterization
94
47
ANIONIC DYE BINDING
SO3Na
NaO 3 S
N N
HO
N N
NaO 3S
Orange G, 470-5 nm
Acid Orange 12, 480 nm
NH2 OH
O2N
N N
NaO3S
N N
SO3Na
Amido Black 10B, 620 nm
95
DYE-PROTEIN REACTION
MECHANISMS
Bind cationic groups of the basic amino acid
residues
imidazole of histidine,
y guanidine of arginine and
y ε-amino group of lysine) and
y the free amino terminal group of the protein.
y
96
48
PRINCIPLE OF ANALYSIS
Protein + dye (in excess, known amount) →
protein-dye precipitate + dye (remained, free).
Measure the differences between known amount
and free in the remained solution to calculate the
amount of protein binding.
97
AUTOMATED DYE-BINDING MACHINE
CEM SPRINT RAPID PROTEIN
ANALYZER
Slide provided by Mr. John Urh
98
49
PLACE SAMPLE IN A CUP, FILL WITH DYE
SOLUTION, HOMOGENIZE, AND FILTER
BEFORE READING
Slide provided by Mr. John Urh
99
ADVANTAGES AND DISADVANTAGES
OF ANIONIC DYE-BINDING METHOD
Advantages:
y
y
y
y
y
Rapid.
Can detect lysine change during processing.
No corrosive reagents.
Measures Protein N.
Precision better than Kjeldhal.
Disadvantages:
Need milligrams of protein.
y Proteins have different binding capacities.
y Interfered by starch, Ca, and phosphate salts.
y
100
50
ANIONIC DYE-BINDING:
APPLICATIONS
Milk:
AOAC 967.12 using Acid Orange 12
y AOAC Method 975.17 using Amido Black (10B)
y
Meat
Soy and wheat flour:
y
AACC Method 46-14B using Acid Orange 12
Beer and wort
Other cereals: Oat groat, corn, rice
Rapeseed
Dairy foods: Ice cream and frozen desserts
Other legumes
Potato
101
BRADFORD METHOD USING
COOMASSIE BLUE DYE-BINDING
Coomassie blue G-250 dye (CBG, Max. 465 nm)
+ acid + protein
↓
Protein-CBG complex (595 nm)
CH3
H2
C N
O3S
H3C
H
C
C2H5
N
H2
C
C2H5
SO3
NH
CH2
CH2OH
102
51
BRADFORD METHOD
Advantages:
Sensitive, 1-100 μg.
Very quick, 2 min.
y Less interfered by K, Na salts, CHO than Lowry.
y
y
Disadvantages:
y
Interfered by large amounts of detergents such as
sodium dodecyl sulfate, triton X-100.
103
BRADFORD METHOD: APPLICATIONS
Protein isolation, purification and characterization
Wort and Beer
104
52
BICINCHONINIC ACID (BCA METHOD)
Principle:
y
Protein + Cu+2 (under basic condition) → Cu+1
Peptide bond, cystine/cysteine, tryptophan and tyrosine
y
Cu+1 + BCA apple green color → BCA-Cu+1 (Purple, 562 nm)
OOC
N
N
COO
Cu+1
OOC
N
N
COO
105
BCA METHOD (CONTINUED)
Advantages as compared to Lowry and Bradford
methods:
Sensitive: 0.5-100 μg.
y Less affected by sucrose, lipid, non-ionic detergent such
as Triton X-100, ammonium sulfate.
y Short analysis time: 15 min.
y More stable solution, easy to use.
y
Disadvantages:
1. Color changes with incubation time.
2. Color is not strictly proportional to conc.
3. High concentrations of reducing sugar, EDTA, poly- phenolic
and sulfhydryl compounds interfere.
106
53
MELAMINE
NH2
N
H2N
N
N
NH2
107
INFRARED OR NEAR INFRARED
METHOD
Proteins contain peptide bonds, which absorb
radiations at various wavelengths:
Mid-infrared bands: 6.47 μm
y Near infrared bands: 3.3-3.5 μm, 2.08-2.22 μm, 1.561.67 μm.
y We can measure reflectance, or transmittance.
y More proteins, less reflected or transmitted.
y
Applications:
Milk: Mid-infrared, AOAC 975.18, 972.16
y Grains, meat, dairy products: Near infrared
y
Advantages: Rapid.
Disadvantages: Expensive equipment, need
calibration.
108
54
SUSCEPTIBILITY TO
ADULTERATION
No research reports with respect to adulteration by
melamine
According to the principles of reactions, melamine
may not be detected by colorimetric methods.
A possible case is the dye-binding method. Since
melamine has 3 amino groups, and therefore, may
bind dyes (and form precipitate??).
y
CEM-Sprint: 5% does not interfere, but higher may
interfere.
109
NEED FOR REFERENCE STANDARDS
Need protein references for standard curves
Standardize with Kjeldhal or Dumas for each type
of foods
y
correlation/regression analysis
110
55
FOOD MATRIX ISSUES
Solubility of protein
Processing effect: heat, acid
Protein-CHO interactions
y Protein-lipid interactions: emulsion
y
y
Insoluble particulates
y
Carbohydrates
Sugars
Starch
Celluloses
Hemicelluloses
Lignins
y Lipids
y
Other solutes: sugar, salts
111
FUTURE RESEARCH OPPORTUNITIES FOR
ADVANCING PROTEIN MEASUREMENT
SCIENCE
Some opportunities
Specific type of foods.
y Standardization by official method such as Kjeldhal.
y Overcoming processing effect on the food matrix:
Improving solubilization.
y Interferences with the colorimetric methods by
adulterants.
y
112
56
113
Food Protein: What role for biosensors?
Harvey E Indyk
Fonterra Dairy Group
New Zealand
[insert document information here]
57
Why Quality Measurements Are Needed
Compliance with Customer Specifications
Compliance with International Regulatory Guidelines
Maintain Production Process Control
Conduct International Trade
Support Fundamental Research:
Milk composition, Effect of processing,
Seasonality, breed, lactation, feed etc
BUT
Bottom Line in Competitive Global Food Market:
Food Safety
Brand Protection
Profitability
[insert document information here]
Current Routine “Protein” Methods in Dairy
Industry
eg Kjeldhal, Dumas, AAA, IR, Colorimetric,
Spectrophotometric, IA (ELISA, RID), HPAC,
PAGE (Proteins)
Biosensor assays…a promising alternative?
[insert document information here]
58
The Biosensor: Principle Elements
Biorecognition
Transducer
Element
Analyte
Signal
Detector
R
A
RA
SelectivitySpecificity
Sensitivity
[insert document information here]
The Biosensor Family
SPR Instrument Companies
Biacore, Affinity Sensors, Windsor,
Nippon, Texas, GWC, Jandratek, IBIS,
Applied Biosystems, Autolab,
Plasmonic, Reichert, BioSuplar,
Virtech…….
Optical-Electronic
Evanescent Wave
SPR
Light Addressable
Potentiometric (LAPS)
Electrochemiluminescence
2007: ~1180 papers based
on optical biosensors
Electrochemical
Potentiometric
Amperometric
Conductimetric
Optical
Y
Bioaffinity
sensor
Absorbance
Fluorescence
Luminescence
TIRF
Acoustic
Quartz crystal
Piezoelectric
Surface Acoustic Wave
Surface Transverse Wave
[insert document information here]
59
SPR: Information Rich
Ab screening for concentration IA (ELISA,
SPR) development
Qualitative
Quantitative
Mapping
(5-10%)
Important:
Measures concentration of “biologically active” analyte
that binds with immobilised interaction partner vs
physicochemical protein assays measure “total” amounts.
[insert document information here]
Types of molecular interactions
[insert document information here]
60
An SPR experiment: overview
[insert document information here]
SPR-Biosensor Characteristics
Attributes
Regenerable sensor surface
High throughput and automation
RealReal-time, label free measurements
Low nonnon-specific binding surface
Various immobilisation chemistries and assay formats
Simplified or elimininated sample preparation
Precision
Exchangable chip flexibilityflexibility-but limited multianalyte ability
Considerations
Cost
Availability of antibody or binding protein
Differentiation of specific and nonnon-specific interactions
Ligand stability to repeated regeneration
RIRI- Extreme temperature sensitivity
[insert document information here]
61
Biacore Systems: 4 Integrated Components
[insert document information here]
SPR - Surface Plasmon Resonance
Detector
array
Lightsource
Polarized
light
Prism
Optical detection
unit
Reflected
light
Sensor chip
Flow system
Flow channel
Intensity
Resonance
signal
Time
Angle
Sensorgram
[insert document information here]
62
[insert document information here]
SPR - Surface Plasmon Resonance
Detector
array
Lightsource
Polarized
light
Prism
Optical detection
unit
Reflected
light
Sensor chip
Flow system
Flow channel
Intensity
Resonance
signal
Time
Angle
Sensorgram
[insert document information here]
63
[insert document information here]
The Flow Cell
[insert document information here]
64
The Sensor Surface
CM-dextran
Thiol-alkane
linker
(~2%)
Gold
(~50 nM)
Glass
• Biocompatible
• Low non-specific binding
• Typically > 100 runs in one flow cell
[insert document information here]
Immobilisation Chemistry
2007: >90% via
amine coupling
[insert document information here]
65
[insert document information here]
Biacore Q: Designed for Concentration
Analysis
Dedicated system for concentration analysis
Designed for assay development and routine testing
Fully automated
Wizard driven functionality
No. flow cells:
Injection volumes:
Flow rate:
Sample capacity
Time per sample
Flowcell volume
4
5 - 325µ
325µl
5 - 100µ
100µl/min.
2 x 96 samples
5 - 15min
~60nl
[insert document information here]
66
Method Development Tools
Concentration Immunoassay Development Tools:
Detecting molecule for direct assay
Detecting molecule for indirect assay
Non-Specific binding
Regeneration scouting
Specificity/Cross reactivity
Enhancement molecule
Assay stability
Matrix interference
[insert document information here]
Concentration Assay Formats
Direct
analyte
analyte
ligand
ligand
Direct binding assay
1
2
Direct binding assay
with enhancement
competing
analyte
detecting
molecule
analyte
Indirect
enhancement
molecule
ligand
Inhibition assay
(solution competition)
analyte
ligand
Surface competition assay
[insert document information here]
67
General Concentration Assay Procedure
Surface Preparation
Sample Injection
Regeneration
Evaluation
[insert document information here]
Food Applications of SPR biosensors
Fraudulent
Protein
concentrationprotein
(eg majoradulterants...eg
milk proteins, CNs, αmelamine???
-lac, β-lac)
Protein species adulteration (eg milk)
Protein conformation (native vs denatured)
Heat treatment (eg milk FBP and α-lactalbumin)
Bioactive components (eg minor milk proteins Ig’
Ig’s, Lf, FBP, LPO, GFs)
Steroid hormones
Veterinary residues (chloramphenicol, tylosin, nicarbazin, fenicol,
fenicol, β-lactams,
aminoglycosides, streptomycin, sulphonamides, β-agonists)
CanBbiosensors,
or any
VitaminsBUT:
(folate, biotin,
12, B5, B2)
Allergens
measurement method detect low-level
Pathogens (eg E. Coli, Staph SEB toxin, Salmonella)
fraudulent adulteration of food protein
Toxins (eg mycotoxins, aflatoxins, bacterial, marine)
without
prior knowledge of its identity?
Pesticide residues
General tool for antibody screening for ELISA, SPR method development
development
[insert document information here]
68
Detection of Adulterants in Milk Products
Detection of plant proteins in milk:
Direct assay
Milk species detection:
Inhibition assay
Haasnoot et al., J. Agric. Food Chem., 49, 5201-6, 2001
[insert document information here]
Haasnoot et al., J. Dairy Res., 71, 322-9, 2004
Why Measure IgG?
Protect commercial milk from colostrum
Immunotherapeutic Foods and Supplements
[insert document information here]
69
Bovine IgG: Methods for Quantitation
Methods include:
HPLC (Ion-Exchange, Size Exclusion, Reversed-phase, LC-MS, AAA)
Electrophoresis (PAGE, CE)
Affinity LC
MS
Immunoassay (RID, ELISA)
SPR-based Immunoassay:
Analyte:
Analyte:
Bovine IgG standards: 156 -10,000ng/ml
Samples: Milk
(1:1,000)
Colostrum (1:10,000)
Ligand:
Ligand:
Immobilization onto CM5 chip:
Goat α-bovine IgG
Run Conditions:
Injection time: 3min
Buffer: HBS-EP
Regeneration: ~ 1min 10mM H3PO4
Indyk & Filonzi J. AOAC Int., 86, 2, 386386-393, 2003
[insert document information here]
Immobilisation
RU
50000
Coupling
45000
Block
40000
35000
Response
Regeneration
30000
25000
Amount Immobilized
Activation
16KRU
20000
15000
10000
0
300
600
900
1200
1500
Time
1800
2100
2400
2700
[insert document information here]
3000
s
70
Bovine IgG over 4 Ligand Surfaces
RU
Rabbit α-IgG Fc1
1800
1600
10ug/ml IgG Std
Protein G Fc2
1400
1200
Goat α-IgG Fc3
Response
1000
800
600
400
Chicken α-IgG Fc 4
200
0
-200
-50
0
50
100
150
Time
200
250
[insert document information here]
s
IgG Calibration over Goat α-IgG
RU
676
576
Response
476
376
276
176
76
-24
0
1000
2000
3000
4000
5000
Concentration
6000
7000
8000
9000
10000
ng/ml
[insert document information here]
71
IgG Assay Specificity Summary
RU
300
NSB of colostrum to reference surface
250
1:1000
200
150
Response
• 1:1,000 yes
• 1:50,000 no
100
1:50,000
50
0
-50
-100
-50
0
50
100
150
200
250
s
Time
Sample matrix effect
Goat-all runs
1.0
0.8
0.6
RU norm
• Normalised responses stds and samples
0.4
0.2
0.0
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
IgG norm
Competition over Protein G surface
• Colostrum (1:25,000) + Prot G (200ug/ml)
800
700
600
500
400
Response
• Complete inhibition
RU
300
200
100
0
-100
-200
50
100
150
200
250
300
Time
[insert document information here]
350
s
Lactation IgG levels-Single Cow
IgG (mg/m L)
70
60
HPLC (mean)
50
RID
40
SPR-IA (mean)
30
20
10
0
Day postpartum
[insert document information here]
72
Production Effects on IgG
Host physiological activity depends on conformational integrity
$$$... 1% increase IgG yield = US$1250/MT
AbAb-based SPR measures intact, native, undenatured IgG
IgG Denaturation at High Pressure
110
1.0
Effects of heat and HPP on IgG (%)
100
Control
90
Residual IgG (%)
Undenatured IgG (mg/mL)
0.8
IgG (pure)
0.6
IgG (Colostrum)
0.4
80
600 MPa/ 1 min
70
600 MPa/3 min
60
Heat control
50
40
30
20
10
0
0.2
Control
600 MPa/ 1
min
600 MPa/3 min
Heat control
Treatment detail
0.0
0
10
20
30
Time (min)
40
50
Prototype colostrum product, Heat: 90°
90°C, 90 s
60
Under HPP, unidentified colostral components stabilise IgG
[insert document information here]
Potential advantages of HPP vs Thermal processing
Process Denaturation of IgG
Host immune function depends on IgG domain (Fc
(Fc and Fab)
Fab) stability
Exploit specificity of immobilised polyclonal α-IgG vs Protein G
anti-IgG
RU
Heated IgG (isolate and colostrum) detected
by immobilised α-IgG vs Protein G
750
700
650
600
550
500
Ligand: anti-IgG
450
350
Enhancement: HBS
300
66°C
(a)
68°C
Enhancement: anti-IgG
250
R e s id u a l Ig G ( % )
Response
400
200
150
100
50
0
-50
-100
0
50
100
150
200
250
300
350
400
450
500
s
Time
Protein G
RU
750
100
80
60
40
20
0
70°C
0
700
650
1000
600
2000
3000
sec
4000
550
(b)
500
Response
400
350
Enhancement: HBS
300
Enhancement: Protein G
250
66°C
Ligand: Protein G
450
68°C
100
70°C
80
200
150
60
100
50
40
0
-50
20
-100
0
50
100
150
200
250
Time
300
350
400
450
500
s
0
0
1000
2000
sec
3000
4000
Denaturation rate Fc ~ Fab in isolate
[insert document information here]
Denaturation rate Fc ≠ Fab in colostrum
73
The Final Word
[insert document information here]
Proteomics in Food Protein Analysis
Kevin J. Shefcheck
Center for Food Safety and Applied Nutrition
U.S. Food and Drug Administration
74
Protein allergens and toxins in food
Food safety and food defense
Protein allergens (safety)
Peanut, egg, milk, wheat
Accidental adulteration
ppm
Protein toxins (safety and defense)
Staphylococcal enterotoxin, shigatoxin, ricin
Deliberate adulteration
ppb
Purpose
Routine surveillance
Enforcement-labeling rules
Food defense- deliberate adulteration
Allergen Detection in Food
Unknown or suspected
hazard
Antibody-based
Screening
(ELISA, Bioplex, SPR)
Confirmation
(instrument based
method, LC/MS,
LC/MS/MS)
1. Rapid (minutes-hours)
2. Sensitive (sub-ppm) and can be
quantitative
3. Inexpensive
4. Specific test for each target
5. Cross reactivity/false positives
1. Specific and inherently multianalyte
2. Quantitative
3. Sensitive (can be matrix dependent)
4. Expensive infrastructure; low cost
per sample
5. Slower (hours)
75
Food Allergies
Food allergy is an immunological
response to a protein in food.
Prevalence of food allergies is on the
rise.
About 8% of children under 3 and 12% of general population have a food
allergy.
Thresholds are difficult to establish
because of lack of clinical data.
Food Allergens
The major food allergens are
glycoproteins, 10-70 kDa in size that
are abundant in allergenic food.
Food allergens are generally water
soluble (except gluten) and some
have resistance to acid and
proteolysis.
76
Food Processing
Allergens can change with heating
Sensitivity can increase due heat
modified protein
Matrix may play a large role in
processing
Allergen Analysis Scheme
Identification of markers for the food
of interest
Use markers for identifying and
quantifying the offending food in
adulterated samples
77
Picking a marker
Abundant protein from food of interest
Easily digestable protein
Look at LC-MS chromatogram to determine
the most abundant peptides
Easily fragmented peptides with LC-MS/MS
Prolines in moderation are a plus because they
give good fragment peptide signals
Peptide Identification
4
1
MKLLILTCLVAVALARPKHPIKHQGLPQE
VLNENLLRFFVAPFPEVFGKEKVNELSKD
IGSESTEDQAMEDIKQMEAESISSSEEIV
PNSVEQKHIQKEDVPSERYLGYLEQLLRL
KKYKVPQLEIVPNSAEERLHSMKEGIHAQ
QKEPMIGVNQELAYFYPELFRQFYQLDA
YPSGAWYYVPLGTQYTDAPSFSDIPNPI
GSENSEKTTMPLW
FFVAPFPEVFGK
Proteolytic
digestion
2
3
HPLC chromatogram
of digested protein
Identification
of protein
Theoretical digest
and fragmentation
MS spectrum of peak 4
E
VAPFPEVF
Peptide sequence
V
F
P F
P
A
V
MS/MS spectrum of the peptide
78
Protein Extraction
Extraction
Non-denaturing solvent extraction
PBS, Tris, etc.
Denaturing solvent extraction
8M Urea, high concentration of detergents, reducing
agents, etc.
Extraction/Digestion
Trypsin added to non-denaturing solvent
Cleanup
2-D or SDS-Page
Solid phase extraction
Reverse phase, ion exchange or mixed mode
Immunoaffinity extraction
Why Immunoaffinity Sample
Preparation?
Simplify
Expedite
Multiplex
79
Milk Allergens
Several allergenic milk proteins are known,
with the most abundant being α-S1-casein
and β-casein
Casein encompasses almost 80% of the
total protein in milk, and α-S1-casein and
β-casein are about 31% and 28% of the
total Casein protein respectively.
Peptides FFVAPFPEVFGK (m/z 634.4) and
YLGYLEQLLR (m/z 692.9) are the largest
intensity peptides from α-S1 casein.
Peptide Markers for Casein in
Chocolate
634.36
692.87
100
100
%
%%
MS
100
0
04 0 0
4 00
450
450
500
500
550
550
600
600
650
650
7 00
7 00
75 0
75 0
800
800
850
850
900
900
950
950
1 00 0
1 00 0
1 05 0
1 05 0
1100
1100
1150
1150
m /z
m /z
0
400
450
500
550
600
650
700
750
800
136.08
900
950
1000
1050
1100
1150
m/z
920.47
120.08
295.15
100
850
100
249.16
MS/MS
991.55
%
%
277.16
0100
221.11
200
371.21
300
400
500
600
742.46
700
836.23
800
676.38
991.52
1090.60
351.21
934.55
900
394.21
1267.70
771.48
658.40
469.25529.36
552.84
1000
1104.65
1100
1203.79
1200
1323.69
1300
1455.55
1400
m/z
0100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
m/z
80
De Novo Sequencing
136.08
100
920.47
120.08
295.15
100
249.16
MSMS of
634.36
991.55
%
MSMS of
692.87
%
277.16
221.11
0100
200
371.21
300
400
469.25529.36
552.84
500
742.46
600
700
836.23
800
991.52
1090.60
676.38
351.21
934.55
900
394.21
1267.70
771.48
658.40
1000
1104.65
1203.79
1100
1200
1323.69
1300
1455.55
1400
0100
m/z
200
300
400
500
600
700
800
900
Waters MaxEnt 3
& PepSeq
R
100
136.08
a1
Y
L
L
Q
E
L
Y
G
L
Y
K
yMax
200
G
F
267.16
a2
100
249.17
b4 2+
a2
1200
1300
1400
m/z
V
EP
F
P
920.50
y8
A
V
F
F
yMax
295.15
b2
991.56
y8
0
100
1100
Waters MaxEnt 3
& PepSeq
%
175.13
y1
1000
394.22
b3
1267.71
277.17
b2
334.19
b3
300
%
400
469.26 529.36
y4
a4
500
771.48
658.40
y6
y5
867.46 934.55
835.45
y7
653.33
709.50
b7
600
700
800
900
992.68
1104.66
y9
1000
1100
1249.74
1200
227.11
1289.72
1300
1400
M/z
1500
YLGYLEQLLR
0
100
326.18
200
570.31 641.36 717.38
300
400
500
991.53
y9
676.38
y6
465.26
b4
199.12
600
700
823.45
y7
800
985.40
900
1090.61
y10
1083.59 1092.58
1000
1100
1384.75
1237.68
y11
1200
1385.70
1300
1400
M/z
1500
FFVAPFPEVFGK
Immunoaffinity Methodology
Extract protein in TBS +
0.1% Tween-20 pH 8
for 2 hours at 60 ºC
One gram of dark chocolate
Add 100 µL of anti-casein magnetic
beads to the supernatant
30 min.
at 10 ºC
2 hr. at 60 ºC
Centrifuge extract at 40000 xg
1 hr. at RT
Wash beads twice with TBS +
0.1% Tween-20 pH 8
Wash beads twice with
100 mM Tris pH 8
Add 5 µg of Trypsin
Collect beads in 50 µL of
100 mM Tris-0.1% Rapigest pH 8
Overnight at 37 ºC
UPLC-4000 QTrap
81
Immunoaffinity extraction of α-S1 casein from
dark chocolate spiked with milk solids
m/z 692.9
m/z 634.4
100
5.26
100
100 ppm
10 ppm
50
0.5
1.0
1.5
2.0
2.5
Time, min
3.0
3.5
0
4.0
991.4
1.7e6
50
1 ppm
4.25
0
Intensity
Intensity
MRM
4.6
5.0
5.5
6.5
7.0
920.3
3.0e5
1.5e6
6.0
Time, min
676.2
2.6e5
1.3e6
2.2e5
1.1e6
7.0e5
Intensity, cps
Intensity, cps
EPI
9.0e5
771.3
529.2
658.2
5.0e5
3.0e5
1.4e5
1.0e5
991.4
450.1
1090.5
6.0e4
934.3
2.0e4
1.0e5
400
1.8e5
500
600
700 m/z, amu
800
900
1000
1100
400
500
600
700
800
m/z, amu
900
1000
1100
Quantitation: Matrix Effects
Extractability
Trypsin digestion
Retention time and ionization
Internal standards are the best
method for remedying these
problems
82
Internal Standards
FFVAPFPEVFGK
Target peptide: high sensitivity, good MS/MS
FFAAPFPEVFGK
synthetic surrogate (elution time?)
FFVAPFPEVFGKEKVNEL
digest standard, control for digestion (elution time?)
FFVVPFPEAFGK (RS- Residue Swapping)
co-elutes, $50/ mg: fragments are shifted 42 Da;
can only be used for MS/MS
FFVAPFPEVFGK (15N,13C- AQUA)
co-elutes, $400/µg, fragments are shifted 10 Da
FFVAPFPEVFGK (18O)
co-elutes, $50-100/mg: fragments are shifted 4 Da
FFVAPFPEVFGK (15N)
stable labeled protein co-elutes; control for extraction, digestion; use
for MS or MSMS mode
Future considerations
Continue working on protein
extraction and sample cleanup for
different allergen proteins and
different sample matrices
Examine N15 labeled casein as a
potential internal standard with
immunoaffinity extraction
83
Acknowledgements
Steve Musser and John Callahan
Carmen Westphal and Lauren Jackson
Jinxi Li
What Proteomics
has to say about
protein quantitation,
nutrition & adulteration
in foods
Eric Eccleston
ric@aminoacids.com
84
PROTEOMICS
extract total protein from ...
organism
separate total protein
2D Gel
identify, quantitate,
compare
FOOD
unintended
consequence
peanut plant
food for growing embryo
- high abundance of a small
number of components
- lower complexity
85
the “great unwashed”
6 N HCl
1.
2.
3.
110 C 24 hrs
prepackaged preparation
kits
prepackaged separation
protocols with
prepackaged components
the “great unwashed”
2D size > 6.5 kD
specific protocols
86
flexible 2D kits
- pre-cast immobilized pH gradient IEF strips
3
pI
10
100
kD
- pre-cast SDS gels
orthogonal
40
3
pI
10
100
protein is a poly-acid
pI is the 0 charge pH
kD
only charged species migrate
40
87
3
pI
10
100
kD
tofu
40
s
3
pI
10
shredded
wheat
100
kD
40
w
88
3
pI
10
100
rye
bread
kD
40
r
3
pI
10
100
beer
kD
40
b
89
3
pI
10
100
oatmeal
kD
40
o
3
pI
10
100
kD
40
90
R1 R2 R3 R4 R5 R6 R7 R8
N C N C N C N C N C N C N C N C N
Near Infra Red
NIR 800-2500 nm
SWNIR 800-1000 nm
PATTERN
RECOGNITION
NIPALS, PCA BP-ANN, ICA LS-SVM
chemometrics
PATTERN
RECOGNITION
91
an example
CH C N
2
H N CH COOH
2
Cyanoalanine
PATTERN
RECOGNITION
92
CH C N
2
H N CH COOH
2
Pre-packaged cheap, robust methodology
well within the technological range and
budget of industry laboratories
Fingerprint ID of food “proteome”
Database - elements already in literature
93
$
$$
$ COST
$ $$
☺☺ ☺
☺ BENEFIT
☺ ☺☺
Graded Response
Triage
94