Limits...
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-DE spots that yielded different MS/MS identifications in polymer fractions than in a total protein extract. 2-DE gel pattern of UPP peak 1 proteins (pink) was overlaid with gel pattern of UPP peak 2 proteins (green). Spots labeled with red numbers were identified as LMW-GS in the UPP peak 1 fraction but either gliadins, triticin or different LMW-GS in a total protein fraction. In at least one of the four fractions, spots labeled with underscored numbers contained chain-terminating gliadins as the predominant proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: 2-DE spots that yielded different MS/MS identifications in polymer fractions than in a total protein extract. 2-DE gel pattern of UPP peak 1 proteins (pink) was overlaid with gel pattern of UPP peak 2 proteins (green). Spots labeled with red numbers were identified as LMW-GS in the UPP peak 1 fraction but either gliadins, triticin or different LMW-GS in a total protein fraction. In at least one of the four fractions, spots labeled with underscored numbers contained chain-terminating gliadins as the predominant proteins.

Mentions: Proteins in 69 of the most abundant 2-DE spots from the UPP peak 1 fraction were identified by MS/MS (Additional file1: Table S1). Multiple proteins were identified in many spots. However, in most cases, the majority of spectra could be assigned to one protein sequence that was deemed to be the predominant protein in the spot. The predominant proteins identified in 25 2-DE spots differed between a total protein extract examined in Dupont et al.[2] and the UPP peak 1 fraction (Table 1). Eleven spots contained gliadins as the predominant proteins in the total protein fraction, but LMW-GS in the UPP peak 1 fraction (124, 163, 166, 169, 320, 323, 324, 325, 330, 467, 468) (Figure 5). In three spots, different types of LMW-GS (i-type, s-type or m-type) were identified as the predominant proteins in the total protein fraction and the UPP peak 1 fraction (125, 315, 319) (Figure 5). In two spots where the predominant protein was a traditional alpha or a gamma gliadin in the total protein fraction, the predominant protein in the UPP peak 1 fraction was a chain-terminating alpha gliadin containing seven cysteines instead of the usual six (346, 546) (Figure 5). One spot identified as triticin in the total protein fraction was identified as a LMW-GS in the UPP peak 1 fraction (348) and one spot identified as a gamma gliadin in the total protein fraction was identified as a triticin in the UPP peak 1 fraction (134).


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-DE spots that yielded different MS/MS identifications in polymer fractions than in a total protein extract. 2-DE gel pattern of UPP peak 1 proteins (pink) was overlaid with gel pattern of UPP peak 2 proteins (green). Spots labeled with red numbers were identified as LMW-GS in the UPP peak 1 fraction but either gliadins, triticin or different LMW-GS in a total protein fraction. In at least one of the four fractions, spots labeled with underscored numbers contained chain-terminating gliadins as the predominant proteins.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: 2-DE spots that yielded different MS/MS identifications in polymer fractions than in a total protein extract. 2-DE gel pattern of UPP peak 1 proteins (pink) was overlaid with gel pattern of UPP peak 2 proteins (green). Spots labeled with red numbers were identified as LMW-GS in the UPP peak 1 fraction but either gliadins, triticin or different LMW-GS in a total protein fraction. In at least one of the four fractions, spots labeled with underscored numbers contained chain-terminating gliadins as the predominant proteins.
Mentions: Proteins in 69 of the most abundant 2-DE spots from the UPP peak 1 fraction were identified by MS/MS (Additional file1: Table S1). Multiple proteins were identified in many spots. However, in most cases, the majority of spectra could be assigned to one protein sequence that was deemed to be the predominant protein in the spot. The predominant proteins identified in 25 2-DE spots differed between a total protein extract examined in Dupont et al.[2] and the UPP peak 1 fraction (Table 1). Eleven spots contained gliadins as the predominant proteins in the total protein fraction, but LMW-GS in the UPP peak 1 fraction (124, 163, 166, 169, 320, 323, 324, 325, 330, 467, 468) (Figure 5). In three spots, different types of LMW-GS (i-type, s-type or m-type) were identified as the predominant proteins in the total protein fraction and the UPP peak 1 fraction (125, 315, 319) (Figure 5). In two spots where the predominant protein was a traditional alpha or a gamma gliadin in the total protein fraction, the predominant protein in the UPP peak 1 fraction was a chain-terminating alpha gliadin containing seven cysteines instead of the usual six (346, 546) (Figure 5). One spot identified as triticin in the total protein fraction was identified as a LMW-GS in the UPP peak 1 fraction (348) and one spot identified as a gamma gliadin in the total protein fraction was identified as a triticin in the UPP peak 1 fraction (134).

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