FIC 2017 Poster: Targeted Peptide Indentification for the ... Detection of Soy Proteins in ... two...
1
Target Peptide Identification for the Mass Spectrometric Detection of Soy Proteins in Food Matrices INTRODUCTION • Soybeans are one of the “Big eight” allergenic foods in the US, with soy allergies affecting approximately 0.4% of children. 1 • Because of their functional properties and high protein content (~38%), soybeans have been widely used in various food products such as baked foods (e.g. cookies, crackers), beverages, and meat products in the forms of soy flour, soy protein isolates, and soy protein concentrates. • Mass spectrometry (MS) is playing an increasingly important role in allergen detection and quantification since it has shown better performance in allergen quantification, in some cases, compared with other technologies such as ELISA. • The objective of the current study was to identify and evaluate target peptides from a variety of soy-derived ingredients for the parallel reaction monitoring (PRM) detection of soy protein in foods. OVERVIEW • Comparison of protein profiles of six commercial soy ingredients • Selection of target peptides for the soy ingredients • Comparison of abundance of the selected peptides across six soy ingredients MATERIALS AND METHODS Tryptic digestion and LC-MS/MS Extraction method Soy sample in buffer (1:20 w:v) (50 mM Tris-HCl, pH 8.6, with 6 M Urea, 20 mM DTT, and 1% PVPP) Heat at 60 °C for 30 min Sonicate at RT for 10 min Obtain supernatant Centrifuge at 13,000 x g for 10 min Quantify protein by Thermo Scientific™ Pierce™ 660 nm Protein Assay Reagent Soy ingredients information 5 μL of 20- fold diluted sample Alkylation Reduction Digestion C18 Spin Column clean-up Acronym Product type Processing method Composition NRSF Non-roasted soy flour Minimally heat processed RSF Roasted soy flour Fully heat treated SPI-A Soy protein isolate SPI-B Soy protein isolate Hydrolyzed SPC-A Soy protein concentrate SPC-B Soy protein concentrate RESULTS Figure 2. SDS-PAGE gel of six commercial soy ingredients with three extraction buffers Buffer P 1 2 3 4 5 6 7 50 mM Tris-HCl, 6 M Urea 250 150 100 75 50 37 25 20 15 10 5 kD a M W N R SF RSF SPI -B SPI -A SPC -A SPC -B 250 150 100 75 50 37 25 20 15 10 5 kD a Buffer M Buffer N 1 2 3 4 5 6 7 8 91 0 1 1 1 2 1 3 1 4 M W 50 mM Tris-HCl, 6 M Urea, 1% PVPP, 20 mM DTT 50 mM Tris-HCl, 6 M Urea, 1% PVPP N R SF RSF SPI -A SPI -B SPC -A SPC -B 250 150 100 75 50 37 25 20 15 10 5 kD a N R SF RSF SPI -A SPI -B SPC -A SPC -B M W Major protein (258) No “M” (228) Std<1.5 (124) Unique, conserved (57) High confidence (149) 6-25 aa (180) All detectable peptides (15332) Criteria for target peptides selection: (1) belongs to major seed storage proteins (2) does not contain methionine (3) 6-25 aa (4) high confidence (5) the standard deviation of peak area between all soy ingredients is less than 1.5 (6) unique amino acid sequence (7) conserved among protein isoforms (8) have good performance at low concentration Figure 3. Target peptide selection a. Example of target peptide selection b. Visualization of target peptide identification for soy protein, number of peptides is given in brackets Figure 1. SDS-PAGE comparison of protein extraction efficacy from unroasted soy flour 50 mM Ammonium Bicarbonate pH 8.0 Tris-HCl 1 M Gu-HCl or 6 M urea Gu-HCl Urea Gu-HCl 1% (w:v) PVPP + + - - + + - - + + 20 mM DTT + - + - + - + - + - Buffer A B C D E F G H I J 50 mM Tris-HCl (pH 8.6) PBS 1 M Gu-HCl or 6 M urea Gu-HCl Urea 1% (w:v) PVPP - - + + - - 20 mM DTT + - + - + - Buffer K L M N O P Q A B C D E F G H I J K L M N O P Q MW MW MW MW 250 150 100 75 50 37 25 20 15 10 5 kD a 250 150 100 75 50 37 25 20 15 10 5 kD a 250 150 100 75 50 37 25 20 15 10 5 kD a 250 150 100 75 50 37 25 20 15 10 5 kD a Protein Sequence Contain Cysteine? Contain Methionine? Soy- specific peptide? number of soy ingredients can detect the peptide Identified as a potential target peptide? 2S albumin ELINLATMCR Yes Yes Yes 6 2S albumin KQLQGVNLTPCEK Yes Yes 6 Yes Basic 7S globulin LMVFDLAR Yes Yes 6 Glycinin G2 (A2B1a) GKQQEEENEGSNILSGFAPEFLK Yes 5 β-conglycinin alpha chain LITLAIPVNKPGR Yes 6 Yes a b CONCLUSIONS Buffers M, N, P (50 mM Tris - HCl with 6 M Urea) have shown the optimal extraction effect among all of the buffers. The protein profiles of most soy ingredients are very similar to each other except for the SPI - B (soy protein isolate - B ), which contains mostly low molecular weight peptides. 57 peptides that satisfy a set of pre - determined criteria were selected for future targeted MS analysis. FUTURE STUDIES Optimize extraction and digestion methods for the food matrices test. Evaluate quantitative performance of the selected target peptides. Develop a quantitative Parallel Reaction Monitoring (PRM) method using isotopically labeled internal standards. Reference: 1. Savage, J. H., Kaeding, A. J., Matsui, E. C. and Wood, R. A. The natural history of soy allergy. J. Allergy Clin. Immunol. 2010, 125, 683–686. ACKNOWLEDGEMENTS This research is part of a collaboration between FARRP/UNL and Thermo Fisher Scientific. Mass spectrometry was conducted on a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole - Orbitrap mass spectrometer placed at UNL. Figure 5. Peptide performance at each level of on-column total peptide content (sample: NRSF, two soy digests with duplicate injections) Figure 4. Comparison of abundance of selected peptides across six soy ingredients QLQ KQL SVA VGF CLD NLQ RPS VFD GKQ KPQ LSA LSA NLQ NNN QQE SQS VFD NNN IST KQI QGQ QIV FNE HFL QFG DTV NSW TSN VLI VSD FES LIT LQE NIL NIL SRD TIS VPS AIV DIE FES LQE QQQ SQS VLF AIL DLD ESY IPA KTI NFL NPI QEE QKQ QQE SPQ SRN TIS -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Log2(Peak area ratio) Y axis indicates Log 2 (Peak area of the peptide in the specified soy ingredient / peak area of the same peptide in unroasted soy flour), x axis indicates the first three amino acid of each peptide and the protein it belongs to. Peptide TISSEDEPFNLR 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 1 10 2 10 3 10 4 10 5 10 6 10 7 10 8 On-column total peptide content (ng) Peak Area LSAQYGSLR 10 -2 10 -1 10 0 10 1 10 2 10 3 10 3 10 4 10 5 10 6 10 7 10 8 On-column total peptide content (ng) Peak Area LC-MS/MS (Thermo Scientific™ Q Exactive™ MS) Shimin Chen 1 , Charles Yang 2 , and Melanie Downs 1 1 Food Allergy Research & Resource Program, Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA 2 Thermo Fisher Scientific, San Jose, CA RSF SPI-A SPI-B SPC-A SPC-B PO72461-EN 0717S
Transcript of FIC 2017 Poster: Targeted Peptide Indentification for the ... Detection of Soy Proteins in ... two...
Target Peptide Identification for the Mass Spectrometric Detection of Soy Proteins in
Food Matrices
INTRODUCTION• Soybeans are one of the “Big eight” allergenic foods in the US, with soy allergies affecting approximately 0.4% of children.1• Because of their functional properties and high protein content (~38%), soybeans have been widely used in various food products such as baked foods
(e.g. cookies, crackers), beverages, and meat products in the forms of soy flour, soy protein isolates, and soy protein concentrates.• Mass spectrometry (MS) is playing an increasingly important role in allergen detection and quantification since it has shown better performance in allergen
quantification, in some cases, compared with other technologies such as ELISA. • The objective of the current study was to identify and evaluate target peptides from a variety of soy-derived ingredients for the parallel reaction monitoring
(PRM) detection of soy protein in foods.
OVERVIEW• Comparison of protein profiles of six commercial
soy ingredients• Selection of target peptides for the soy ingredients• Comparison of abundance of the selected peptides
across six soy ingredients
MATERIALS AND METHODS
Tryptic digestion and LC-MS/MS
Extraction method
Soy sample in buffer(1:20 w:v)
(50 mM Tris-HCl, pH 8.6, with 6 M Urea, 20 mM DTT, and 1% PVPP)
Heat at 60 °C for 30 min
Sonicate at RT for 10 min
Obtain supernatant
Centrifuge at 13,000 x g for
10 min
Quantify protein by Thermo Scientific™
Pierce™ 660 nm Protein Assay
Reagent
Soy ingredients information
5 μL of 20-fold diluted
sample
AlkylationReduction Digestion C18 Spin Column clean-up
Acronym Product type Processing method Composition
NRSF Non-roasted soy flour Minimally heat processed
RSF Roasted soy flour Fully heat treated SPI-A Soy protein isolateSPI-B Soy protein isolate HydrolyzedSPC-A Soy protein concentrate SPC-B Soy protein concentrate
RESULTS
Figure 2. SDS-PAGE gel of six commercial soy ingredients with three extraction buffers
Buffer P
1 2 3 4 5 6 7
50 mM Tris-HCl, 6 M Urea
250
150
100 75
50
37
25 20
15 10 5
kDa
MW
NR SF
RSF SPI-B
SPI-A
SPC-A
SPC-B
250
150 100 75
50 37
25 20
15 10 5
kDa
Buffer M Buffer N
1 2 3 4 5 6 7 8 9 10
11
12
13
14
MW
50 mM Tris-HCl, 6 M Urea, 1%
PVPP, 20 mM DTT
50 mM Tris-HCl, 6 M Urea,
1% PVPP
NR SF
RSF SPI-A
SPI-B
SPC-A
SPC-B
250
150
100 75
50 37
25 20
15 10 5
kDa
NR SF
RSF SPI-A
SPI-B
SPC-A
SPC-B
MW
Major protein (258)
No “M” (228)
Std<1.5 (124)
Unique, conserved
(57)
High confidence (149)
6-25 aa (180)
All detectable peptides(15332)
Criteria for target peptides selection:(1) belongs to major seed
storage proteins(2) does not contain
methionine (3) 6-25 aa(4) high confidence (5) the standard deviation
of peak area between all soy ingredients is less than 1.5
(6) unique amino acid sequence
(7) conserved among protein isoforms
(8) have good performance at low concentration
Figure 3. Target peptide selection a. Example of target peptide selection b. Visualization of target peptide identification for soy protein, number of peptides is given in brackets
Figure 1. SDS-PAGE comparison of protein extraction efficacy from unroasted soy flour
50 mM Ammonium Bicarbonate pH 8.0 Tris-HCl1 M Gu-HClor 6 M urea Gu-HCl Urea Gu-HCl
1% (w:v)PVPP + + - - + + - - + +
20 mM DTT + - + - + - + - + -Buffer A B C D E F G H I J
50 mM Tris-HCl (pH 8.6)
PBS
1 M Gu-HCl or 6 M urea Gu-HCl Urea1% (w:v)
PVPP - - + + - -
20 mM DTT + - + - + -Buffer K L M N O P Q
A B C D E F G H I J K L M N O P Q MWMWMWMW
250
150
100 75
50
37
25 20
15 10 5
kDa 250
150
100 75
50
37
25 20
15 10 5
kDa250
150
100
75
50
37
25 20
15
10 5
kDa250
150
100 75
50
37
25 20
15
10 5
kDa
Protein Sequence Contain Cysteine?
Contain Methionine?
Soy-specific peptide?
number of soy ingredients
can detect the peptide
Identified as a potential
target peptide?
2S albumin ELINLATMCR Yes Yes Yes 62S albumin KQLQGVNLTPCEK Yes Yes 6 Yes
Basic 7S globulin LMVFDLAR Yes Yes 6Glycinin G2 (A2B1a) GKQQEEENEGSNILSGFAPEFLK Yes 5
β-conglycinin alpha chain LITLAIPVNKPGR Yes 6 Yes
a
b
CONCLUSIONS Buffers M, N, P (50 mM Tris-HCl with 6 M Urea) have shown the optimal extraction effect
among all of the buffers.
The protein profiles of most soy ingredients are very similar to each other except for the SPI-B (soy protein isolate-B), which contains mostly low molecular weight peptides.
57 peptides that satisfy a set of pre-determined criteria were selected for future targeted MS analysis.
FUTURE STUDIES Optimize extraction and digestion methods for the food matrices test.
Evaluate quantitative performance of the selected target peptides.
Develop a quantitative Parallel Reaction Monitoring (PRM) method using isotopically
labeled internal standards.Reference:1. Savage, J. H., Kaeding, A. J., Matsui, E. C. and Wood, R. A. The natural history of soy allergy. J. Allergy Clin. Immunol. 2010, 125, 683–686.
ACKNOWLEDGEMENTSThis research is part of a collaboration between FARRP/UNL and Thermo Fisher Scientific. Mass spectrometry was conducted on a Thermo Scientific™ Q Exactive™ Hybrid Quadrupole-Orbitrap mass spectrometer placed at UNL.
Figure 5. Peptide performance at each level of on-column total peptide content (sample: NRSF, two soy digests with duplicate injections)
Figure 4. Comparison of abundance of selected peptides across six soy ingredients
QLQ
KQ
LSV
AVG
FC
LDN
LQ RPS VF
DG
KQ
KPQ LS
ALS
AN
LQN
NN
ESQ
SVF
DN
NN
IST
KQ
IQ
GQ
QIV
FNE
HFL
QFG DTV
NSW TS
NVL
IVS
DFE
SLI
TLQ
EN
ILN
ILSR
DTI
SVP
SA
IVD
IEFE
SLQ
EQ
SQS
VLF
AIL
DLD ES
YIP
AK
TIN
FL NPI
QEE
QK
QE
SPQ
SRN
TIS
-4.5-4.0-3.5-3.0-2.5-2.0-1.5-1.0-0.50.00.51.01.52.02.5
Log2
(Pea
k ar
ea r
atio
)
Y axis indicates Log2 (Peak area of the peptide in the specified soy ingredient / peak area of the same peptide in unroasted soy flour), x axis indicates the first three amino acid of each peptide and the protein it belongs to.
Peptide
TISSEDEPFNLR
10-3
10-2
10-1
100
101
102
103
101
102
103
104
105
106
107
108
On-column total peptide content (ng)
Peak
Are
a
LSAQYGSLR
10-2
10-1
100
101
102
103
103
104
105
106
107
108
On-column total peptide content (ng)
Peak
Are
a
LC-MS/MS (Thermo
Scientific™ Q Exactive™ MS)
Shimin Chen1, Charles Yang2, and Melanie Downs11Food Allergy Research & Resource Program, Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
2Thermo Fisher Scientific, San Jose, CA
RSFSPI-ASPI-BSPC-ASPC-B
PO72461-EN 0717S
