Gluten, comprised of glutenin and gliadin proteins, is a viscoelastic polymer which imparts the unique breadmaking characteristics to wheat flour. It is comprised of high-molecular and low-molecular (HMW-GS and LMW-GS) glutenin subunits which are linked via intramolecular disulfide bonds from amino acids at their N & C termi. Although mass spectrometry of wheat proteins is in its infancy, current investigation of Canadian and Australian wheat cultivars has revealed a series of HMW and LMW-GS not accounted for in any existing database. Varieties’ LMWGS- and HMWGS were analyzed by LC-MS (G2 Synapt Q-ToF or Orbitrap Fusion Lumos). The CNHR variety Lillian has a By variant at 75 868 Da, only 19 Da different from traditional By8, yet elutes over 2 min later. Australian varieties Yitpi, Suntop and Sunvale had the same HMW. Australian varieties Bonnie Rock, Cobra and Spitfire yielded another unknown at 75 924 Da while CWSWS AC Reed displays an uncharacterized, heavier By 8 at 75 887 Da. CWRW Flourish highlights a Uniprot unknown Ax2* HMW at 86 020 Da, 735 Da lighter than other Canadian varieties. Additionally, unique peptide cleavages have suggested the presence of caspase-like enzymes in the wheat kernel due to presence of unique HMW-GS co-variants detected for Bx7, Bx7OE and Dy10. These mass differences correspond to a hexapeptide (565 Da) at the C terminal end immediately following an aspartic acid residue. Australian varieties Bonnie Rock, Cobra and Spitfire revealed the same phenomena in their unreported Bx proteo-pairs. Analyses of durum samples has identified a previously unreported 120K Da protein present in the gluten extract. While post-translational modifications (PTMS) are not established in HMW-GS, MS analyses of Canadian wheat does suggest they are prevalent to varying extents in LMW-GS. A consistent set of protein-pairs, differing by 162+/-2Da and exhibiting consistently delayed retention times are discussed. One pair, CWRS variety Cardale, exhibited masses of 40 344 and 40 181 Da. Chymotrypsin digests for each differed by only a single peptide which Uniprot matched to the LMW-GS accession B2Y2Q7 with 99% sequence coverage and the mass difference between these peptides was 163.066 Da, matching tyrosine (163.063 Da). This peptide corresponded to the C-terminal peptide sequence GVGTGVGSY, with the mass difference explained by the loss of the C-terminal tyrosine. This conforms with the 257 unique wheat LMWGS sequences in Uniprot, where there are only 11 sequence variants for the C-terminal hexapeptide and all of them carry a C-terminal tyrosine.