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 v5-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 v-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. Copyright: ß 2013 Lagrain et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: BL wishes to acknowledge the Research Foundation – Flanders (FWO, Brussels, Belgium; www.fwo.be) for his position as postdoctoral fellow. IR wishes to acknowledge the Special Research Fund (KU Leuven, Leuven, Belgium; www.kuleuven.be) for a position as postdoctoral fellow. IR and BL both wish to acknowledge the FWO for financial support during a research stay at the German Research Center for Food Chemistry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.