INTRODUCTION• Thinopyrum intermedium, commonly known as

intermediate wheatgrass (IWG) is a novel perennial crop,with both environmental and nutritional benefits (1 and 2).

• IWG is currently mainly used as a forage but shows greatpotential to be developed as a grain crop.

• IWG has higher protein, fiber and antioxidant contentsthan that of common wheat (3).

• IWG is mainly consisting of gliadins and low molecularweight glutenins (LMWG) and deficient in high molecularweight glutenins (HMWG) suggesting a poor gluten formingability (4 and 5).

• Therefore, the objective of this study was to betterunderstand the protein distribution of IWG lines and itsrelationship to bread-making quality parameters.

CONCLUSIONS• IWG samples have higher extractable albumins and globulins compared to

the wheat controls while wheat samples have higher unextractable HMWPPin contrast to IWG.

• Unextractable UP1 (HMWPP) of IWG has significant and positive correlationwith dough stability and extensibility.

• Extractable EP4 (gliadins) of IWG has significant and positive correlation withwater absorption.

• Despite the higher protein content, all 17 IWG varieties are deficient ofHMWG which is responsible for strength and elasticity of the dough.

• HMWG present in IWG are of lower molecular weight in size but confirmed asHMWG by LC-MS/MS.

• The molecular weight of intact proteins from all fractions of SE-HPLC will bedetermined by MALDI-TOF

METHODMaterials: 16 different IWG lines, grown by the Plant GeneticsDepartment at the University of Minnesota along with twowheat controls, Hard Red Wheat (HRW) and Arapahoe.

Extraction and Protein Fractionation: Flour proteins wereextracted by SDS buffer and extractable and unextractableprotein fractions were analyzed for molecular weightdistribution by size exclusion HPLC (SE-HPLC) (6).

Statistical Analysis: Correlation coefficients were calculatedusing the CORR procedure in SAS.

Mass Spectrometry Analysis: In gel chymotrypsin digestion was performed and followed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

Rheological Properties. Farinograph was done for optimumwater absorption, peak time and stability. Kieffer method wasused for dough extensibility.

Protein Quantification. Determined by a nitrogen analyzerfollowing the AOAC Dumas method.

ACKNOWLEDGEMENTSThis project was supported through research grants provided by the ForeverGreen Initiative, Minnesota Department of Agriculture and a gift fund by theLand Institute, Kansas.

REFERENCES 1. Wagoner, P. and Schauer, A. Intermediate wheatgrass as a perennial grain crop.

Advances in New Crops 1990; 143-145.2. Vogel, K. P., and Jensen, K. J. Adaptation of perennial triticeae to the eastern Central

Great Plains. J. Range Manage 2001; 54:674-697.3. Bunzel, M., Tyl, C. E. and Ismail, B. Chemical composition of intermediate wheatgrass.

(Abstr.) Cereal Foods World 2014; 59:A11.4. Marti, A., Qiu , X.,Schoenfuss, T. and Seetharaman, K. Characteristics of perennial

wheatgrass (Thinopyrum intermedium) and refined wheat flour blends: impact on rheological properties, Cereal Chem 2015; 92(5):434–440.

5. Becker, R., Wagoner, P., Hanners, G. D. and Saunders, R. M. Compositional, nutritional and functional evaluation of intermediate wheatgrass (Thinopyrum intermedium). J. Food Process Preserv 1991. 15:63-77.

6. Ohm, J., Hareland, G., Simsek, S., Seabourn, B., Maghirang, E. and Dowell, F.Molecular weight distribution of proteins in hard red spring wheat: relationship to quality parameters and intrasample uniformity. Cereal Chem (2010). 87(6):553–560.

Molecular Weight Distribution of Flour Proteins in Intermediate Wheatgrass (Thinopyrum Intermedium): Impact on End-Use Quality Parameters QR

CodeContact Information: C . Gajadeera at cgajadee@umn.edu

C. GAJADEERA1, J. OHM2, K. WHITNEY3, C. RAHARDJO1, S. SIMSEK3, B. ISMAIL1

1Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 551082USDA-ARS Cereal Crops Research Unit, North Dakota State University, Fargo, ND 581023Department of Plant Sciences, North Dakota State University, Fargo ND 58102

Figure 4. SDS-PAGE visualization ofgluten protein profileLane 1: Molecular weight marker Lane 2: Hard red wheat Lane 3: ArapahoeLane 4: Bulk IWG Lane 5: IWG L4-160Lane 6: IWG L4-157Lane 7: IWG L4-1Lane 8: IWG L4-32Lane 9: IWG L4-85

202

114

73

47

34

17

kDa

27

621

4

3

5

67

13121110

202

11473

34

47

27176

kDa1 2 3 4 5 6 7 8 9

HMWGω-gliadinsLMWG

LMWG & α,β and γ-gliadins

RESULTS

89

Marker HRW Bulk-IWG

Proteinband ID Allele (species) a

Assigned Protein

No. of unique peptides Coverageb

1 T. intermediuam gi311360246 α-gliadin 3 8%

2 T. aestivum gi.385845947 LMWG 4 9%

3 T. aestivum gi. 401787278 α-gliadin 1 6%

4 T. intermedium gi.31136023 α-gliadin 2 6%

5 T. aestivum gi.513130029 LMWG 2 15%

6 T. intermedium gi.572923925 HMWG 3 17%

7 T. intermedium gi.572923923 HMWG 9 22%

8 T. intermedium gi.572923923 HMWG 11 20%

9 T. Intermedium gi.572923919 HMWG 4 16%

10 T. Intermedium gi.572923917 HMWG 4 25%

11 T. Intermedium gi.572923917 HMWG 4 19%

12 T. Intermedium gi.572923931 HMWG 8 13%

13 T. Intermedium gi.572923931 HMWG 16 22%

Figure 5. SDS-PAGE visualization ofgluten proteins selected formolecular identificationThe chymotrypsin in-gel digestionof proteins for the analysis of LC-MS/MS

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

3 4 5 6 7 8

r va

lue

Retention time (min)

SDS Extractable

Chromatogram

r value

P=0.05

P=0.01

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

3 4 5 6 7 8

r va

lue

Retention time (min)

SDS Unextractable

Chromatogram

r value

P=0.05

P=0.01

Farinograph Kieffer

Protein FractionWaterAbsorption Stability Extensibility Protein %

SDS Extractable

EP1 0.46 (ns)0.04 (ns) -0.38 (ns) 0.58*

EP2 0.27 (ns) -0.07 (ns) -0.40 (ns) 0.45 (ns)

EP3 0.28 (ns)-0.03 (ns) -0.40 (ns) 0.61**

EP4 0.51* -0.43 (ns) -0.42 (ns) 0.69**

EP5 0.20 (ns) 0.02 (ns) -0.33 (ns) 0.130 (ns)

SDS Unextractable

UP1 0.41 (ns) 0.81*** 0.69** 0.52*

UP2 0.52* 0.27 (ns) 0.62** 0.69**

UP3 0.44 (ns) 0.05 (ns) 0.51* 0.66**

UP4 0.40 (ns) -0.21(ns) 0.32 (ns) 0.61**

UP5 0.34 (ns) -0.32 (ns) 0.26 (ns) 0.32 (ns)

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

3 4 5 6 7 8

r va

lue

Retention time (min)

SDS Unextractable

Chromatogram

r value

P=0.05

P=0.01

Extractable Protein Percentage (% of Total Protein) Unextractable Protein Percentage (% of Total Protein)

Sample ID HMWPP LMWPP ω-gliadinα,β and γ

gliadinAlbumin and

GlobulinHMWPP LMWPP

ω-gliadin

α,β and γgliadin

Albumin and Globulin

HRW 13.6 4.44 5.00 31.7 18.1 12.83 3.90 2.13 4.85 3.33Arapahoe 14.4 4.67 5.10 32.8 18.9 10.92 3.59 2.01 4.43 3.07Bulk IWG 16.1 7.42 5.71 33.8 21.1 7.73 1.78 1.05 2.80 2.34IWG L4-1 15.7 8.20 5.89 37.5 21.4 3.77 1.24 0.83 2.90 2.40IWG L4-3 14.9 7.79 5.67 34.4 19.5 5.45 1.93 1.34 5.11 3.72IWG L4-29 15.0 6.66 5.08 35.4 18.9 5.01 1.92 1.36 6.49 4.00IWG L4-32 17.4 7.88 5.80 35.6 22.5 2.94 1.04 0.77 2.94 3.00IWG L4-57 17.9 8.21 6.28 37.5 19.0 3.36 1.18 0.86 3.17 2.46IWG L4-72 17.1 7.94 5.39 33.4 20.5 5.31 1.73 1.13 4.13 3.26IWG L4-84 14.5 6.77 5.24 36.3 17.6 6.40 2.17 1.45 5.85 3.60IWG L4-85 14.9 6.75 5.08 35.9 18.9 5.36 1.95 1.34 5.86 3.81IWG L4-103 16.7 7.87 5.82 33.4 21.9 3.48 1.43 1.09 4.49 3.71IWG L4-105 16.0 7.22 5.46 36.6 21.6 4.27 1.36 0.92 3.49 2.93IWG L4-139 15.8 7.53 5.31 35.5 19.2 4.87 1.91 1.28 5.01 3.46IWG L4-154 15.8 7.98 5.99 37.1 21.4 4.33 1.32 0.87 2.72 2.29IWG L4-157 14.8 7.03 5.57 36.3 21.4 4.16 1.54 1.12 4.66 3.30IWG L4-159 15.5 7.22 5.42 33.4 17.9 5.22 2.20 1.62 7.19 4.13IWG L4-160 16.3 7.63 5.36 36.0 21.7 4.23 1.41 0.93 3.20 2.78IWG L4-172 15.2 7.95 5.76 35.3 21.7 4.28 1.49 1.07 3.77 3.36

Figure 3: Correlation coefficients (r) of SE-HPLC absorbance area values of SDS-extractable proteins withfarinograph water absorption (A), SE-HPLC absorbance area values of SDS-unextractable proteins withfarinograph peak time (B) and stability (C).

Table 1. Protein components from extractable and unextractable fractions

EP, SDS-extractable proteins; UP, SDS-unextractableproteins; 1, HMWPP; 2, LMWPP; 3, ω-gliadins; 4, α, βand γ-gliadins; 5, albumin and globulin** and ***,significance at P < 0.01 and P < 0.001 respectively. ns,not significant at P < 0.05

Table 2. Correlation coefficients between quality parameters and protein fractions

Table 3: Molecular identification of extracted gluten proteins by LC-MS/MS

aGI accession number of the best matched sequence in NCBIbThe percentage of amino acid sequence coverage from uniquepeptides matched to the full-length protein sequences.

-50

0

50

100

150

200

250

300

350

400

0 1 2 3 4 5 6 7 8 9 10

Abso

rban

ce a

t 214

nm

Retention Time (min)

SDS-Extractable Proteins

Average_Bulk IWGAverage_HRWW

-50

-40

-30

-20

-10

0

10

20

30

40

0 1 2 3 4 5 6 7 8 9 10

Abso

rban

ce a

t 214

nm

Retention Time (min)

SDS-Unextractable Proteins

Average_Bulk IWGAverage_HRWW

Figure 1: SE-HPLC chromatograms of IWG and wheat. SDS-extractableproteins (A); SDS-unextractable proteins (B)

A B

-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

3 4 5 6 7 8

r va

lue

Retention time (min)

SDS Unextractable

Chromatogram

r value

P=0.05

P=0.01

-1.0-0.8-0.6-0.4-0.20.00.20.40.60.81.0

3 4 5 6 7 8

r va

lue

Retention time (min)

SDS Unextractable

Chromatogram

r value

P=0.05

P=0.01

Figure 2: Correlation coefficients (r) of SE-HPLCabsorbance area values of SDS-unextractableproteins with dough extensibility (A) and flourprotein content (B)

A

B

A B CF1, 3.5-5.5 min: HMWPP (High Molecular Weight Polymeric Protein)F2, 5.5-5.9 min: LMWPP (Low Molecular Weight Polymeric Protein)F3, 5.9-6.2 min: ω-gliadinsF4, 6.2-6.9 min: α,β and γ gliadinsF5, 6.9-8.0 min: albumins and globulins

Slide Number 1