We have performed a detailed characterization and identification of wheat gluten proteins obtained from the Teal variety of Canadian hard red spring wheat. RP-HPLC separation of the sample into 35 fractions has reduced the spectral complexity; this was followed by MALDI mass spectrometry (MS), which showed the presence of six or fewer resolved protein components above 20 kDa in each RP-HPLC fraction, giving a total of 93 MS resolved peaks. These included 17 peaks in the omega-gliadin fractions (F1-4), 12 in the high molecular weight (HMW) glutenin subunit fractions (F5-8), 59 in the alpha- and beta-gliadins and low molecular weight (LMW) glutenin subunit fractions (F9-31) and 5 peaks in the gamma-gliadin fractions (F32-35). Peptide maps of tryptic digests of HPLC fractions were obtained from a tandem quadrupole time-of-flight mass spectrometer (MALDI QqTOF MS) and were submitted to the ProFound search engine. HMW glutenin subunits including Ax2*, Dx5, Bx7, and Dy10 (consistent with the known profile of Teal), and LMW glutenin subunits including six from group 3 type II and 1 from group 2 type I, were identified with reasonable sequence coverage from HPLC fraction 5, 7, 17, and 18. The identities of the peptides attributed to selected gluten proteins were confirmed using MS/MS with BioMultiView to match the predicted and measured partial amino acid sequences. Because of incomplete wheat DNA databases, many wheat gluten proteins could not be identified. These results suggest that the combination of RP-HPLC with MS and MS/MS techniques is a promising approach for the characterization of wheat gluten proteins.