Identification of Intact High Molecular Weight Glutenin Subunits from the Wheat Proteome Using Combined Liquid Chromatography-Electrospray Ionization Mass Spectrometry

Abstract

The present paper describes a method for the identification of intact high molecular weight glutenin subunits (HMW-GS), the quality determining proteins from the wheat storage proteome. The method includes isolation of HMW-GS from wheat flour, further separation of HMW-GS by reversed-phase high-performance liquid chromatography (RP-HPLC), and their subsequent molecular identification with electrospray ionization mass spectrometry using a quadrupole-time-of-flight mass analyzer. For HMW-GS isolation, wheat proteins were reduced and extracted from flour with 50% 1-propanol containing 1% dithiothreitol. HMW-GS were then selectively precipitated from the protein mixture by adjusting the 1-propanol concentration to 60%. The composition of the precipitated proteins was first evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining and RP-HPLC with ultraviolet detection. Besides HMW-GS (≥65%), the isolated proteins mainly contained ω5-gliadins. Secondly, the isolated protein fraction was analyzed by liquid chromatography-mass spectrometry. Optimal chromatographic separation of HMW-GS from the other proteins in the isolated fraction was obtained when the mobile phase contained 0.1% trifluoroacetic acid as ion-pairing agent. Individual HMW-GS were then identified by determining their molecular masses from the high-resolution mass spectra and comparing these with theoretical masses calculated from amino acid sequences. Using formic acid instead of trifluoroacetic acid in the mobile phase increased protein peak intensities in the base peak mass chromatogram. This allowed the detection of even traces of other wheat proteins than HMW-GS in the isolated fraction, but the chromatographic separation was inferior with a major overlap between the elution ranges of HMW-GS and ω-gliadins. Overall, the described method allows a rapid assessment of wheat quality through the direct determination of the HMW-GS composition and offers a basis for further top-down proteomics of individual HMW-GS and the entire wheat glutenin fraction.

Characterization of Trypsin-Like Protease of Lactobacillus plantarum FNCC 0270

“…According to Lagrain et al (2013) with formic acid as the eluent, the sensitivity of the MS detector clearly increased. This was also underlined by improved mass intensities of the detected proteins in the Base Peak Chromatogram (BPC).…”

Portable gliadin-immunochip for contamination control on the food production chain

“…The extracts were also analyzed by reverse-phase HPLC/ESI-MS employing a method from previously published reports [44][45][46] with a Thermo (Thermo Electron, USA) Deca XP Max ion trap mass spectrometer using electrospray ionization in the positive ion mode (ESIþ ). The HPLC instrument was a Thermo (Thermo Electron, USA) MS Pump equipped with a quaternary solvent delivery system, autosampler, and column heater and variable wavelength detector.…”

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“…HMW glutenin subunits were detected by MALDI TOF MS and RP-HPLC/nESI-MS/MS in durum wheat (Muccilli et al, 2011;Lagrain et al, 2013). A heterotetrameric α-amylase inhibitor (ETI) was detected by MALDI-TOF MS in hulled emmer wheat as an assembly of proteins highly similar to that found in durum wheat (Capocchi et al, 2013).…”