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Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry.

Vensel WH, Tanaka CK, Altenbach SB - Proteome Sci (2014)

Bottom Line: Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins.All three types were found in the largest proportions in the SDS-extractable polymer fraction.These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution.

View Article: PubMed Central - HTML - PubMed

Affiliation: USDA-ARS, Western Regional Research Center, 800 Buchanan St, Albany, CA 94710, USA. william.vensel@ars.usda.gov.

ABSTRACT

Background: Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated.

Results: Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction.

Conclusions: This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.

No MeSH data available.


Related in: MedlinePlus

2-D gels showing the proteins present in extractable polymeric protein (EPP) and unextractable polymeric protein (UPP) fractions. A) EPP peak 1; B) UPP peak 1; C) EPP peak 2; D) UPP peak 2; E) EPP peak 3; F) UPP peak 3.
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Figure 4: 2-D gels showing the proteins present in extractable polymeric protein (EPP) and unextractable polymeric protein (UPP) fractions. A) EPP peak 1; B) UPP peak 1; C) EPP peak 2; D) UPP peak 2; E) EPP peak 3; F) UPP peak 3.

Mentions: EPP and the UPP extracts were further separated by SEC (Figure 3). The glutenin polymer as well as the monomeric gliadins are soluble in 50% aqueous acetonitrile in the presence of 0.1% TFA and elute in order of size. The superimposed chromatograms can easily be divided into an early emerging peak (peak 1) and a later emerging peak (peak 2). A third fraction, referred to as peak 3, corresponds to a shoulder on the back of peak 2. The elution profile is similar to that obtained by others[3,11,15] with the polymeric components emerging in the first peak and the monomeric components in the latter half of the chromatogram. Each fraction was analyzed by 2-DE and the results in Figure 4 show that the principal proteins of peak 1 from the EPP (Figure 4A) and the UPP (Figure 4B) fractions after reduction are the HMW-GS and LMW-GS. Serpins are also visible in peak 1, particularly in the EPP fraction. In addition, peak 1 of the EPP fraction contains several proteins identified in previous studies as triticin that are not apparent in any of the other fractions. In comparison, the omega, alpha and gamma gliadins are the predominant proteins in peak 2 from the EPP (Figure 4C) and the UPP (Figure 4D) fractions. Some proteins in the LMW-GS region are also visible in both fractions. Peak 3 from both the EPP (Figure 4E) and UPP (Figure 4F) fractions contained proteins identified previously as alpha amylase/trypsin inhibitors and purinins[2]. Their presence in this fraction suggests that these proteins are not cross-linked into the polymer. Some alpha and gamma gliadins were also detected in peak 3. This is not surprising since peak 3 was collected from the tail of peak 2.


Protein composition of wheat gluten polymer fractions determined by quantitative two-dimensional gel electrophoresis and tandem mass spectrometry.

Vensel WH, Tanaka CK, Altenbach SB - Proteome Sci (2014)

2-D gels showing the proteins present in extractable polymeric protein (EPP) and unextractable polymeric protein (UPP) fractions. A) EPP peak 1; B) UPP peak 1; C) EPP peak 2; D) UPP peak 2; E) EPP peak 3; F) UPP peak 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4016294&req=5

Figure 4: 2-D gels showing the proteins present in extractable polymeric protein (EPP) and unextractable polymeric protein (UPP) fractions. A) EPP peak 1; B) UPP peak 1; C) EPP peak 2; D) UPP peak 2; E) EPP peak 3; F) UPP peak 3.
Mentions: EPP and the UPP extracts were further separated by SEC (Figure 3). The glutenin polymer as well as the monomeric gliadins are soluble in 50% aqueous acetonitrile in the presence of 0.1% TFA and elute in order of size. The superimposed chromatograms can easily be divided into an early emerging peak (peak 1) and a later emerging peak (peak 2). A third fraction, referred to as peak 3, corresponds to a shoulder on the back of peak 2. The elution profile is similar to that obtained by others[3,11,15] with the polymeric components emerging in the first peak and the monomeric components in the latter half of the chromatogram. Each fraction was analyzed by 2-DE and the results in Figure 4 show that the principal proteins of peak 1 from the EPP (Figure 4A) and the UPP (Figure 4B) fractions after reduction are the HMW-GS and LMW-GS. Serpins are also visible in peak 1, particularly in the EPP fraction. In addition, peak 1 of the EPP fraction contains several proteins identified in previous studies as triticin that are not apparent in any of the other fractions. In comparison, the omega, alpha and gamma gliadins are the predominant proteins in peak 2 from the EPP (Figure 4C) and the UPP (Figure 4D) fractions. Some proteins in the LMW-GS region are also visible in both fractions. Peak 3 from both the EPP (Figure 4E) and UPP (Figure 4F) fractions contained proteins identified previously as alpha amylase/trypsin inhibitors and purinins[2]. Their presence in this fraction suggests that these proteins are not cross-linked into the polymer. Some alpha and gamma gliadins were also detected in peak 3. This is not surprising since peak 3 was collected from the tail of peak 2.

Bottom Line: Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins.All three types were found in the largest proportions in the SDS-extractable polymer fraction.These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution.

View Article: PubMed Central - HTML - PubMed

Affiliation: USDA-ARS, Western Regional Research Center, 800 Buchanan St, Albany, CA 94710, USA. william.vensel@ars.usda.gov.

ABSTRACT

Background: Certain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated.

Results: Flour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction.

Conclusions: This is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.

No MeSH data available.


Related in: MedlinePlus