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The analysis of gluten proteins has a long and distinguished history of more than 250 years. Investigation of gluten by current methodologies is often challenging due to the limited genome information of cereals and their poor solubility in aqueous solvents. Enrichment and general isolation of gluten proteins can be achieved by extraction, precipitation and traditional column chromatography. For in-depth characterization of gluten proteins other techniques are required. For example, reversed-phase high-performance liquid chromatography (RP-HPLC) and gel-permeation (GP) HPLC with UV detection have been widely applied to gluten proteins and have proven to be highly efficient tools for the qualitative and quantitative investigation and isolation of gluten protein types and subunits. After suitable calibration, these techniques provide absolute concentrations of proteins. While these one-dimensional (1D) separation techniques are proficient for the analysis of gluten profiles from flours, they are inadequate for individual gluten proteins due to limited selectivity and sensitivity. Modern 2D proteomics approaches combine high-resolution separation (isoelectric focusing, SDS polyacrylamide gel electrophoresis or chromatography) with mass spectrometry (MS) using soft ionization such as matrix-assisted laser desorption/ionization (MALDI) or electrospray ionization (ESI) followed by time-of-flight (TOF), ion trap or triple quadrupole detection. For gluten proteins, most MS applications involve protein identification after proteolytic digestion and chromatographic separation prior to MS of individual ions from the peptide mixtures. The identification of gluten proteins or peptides after MS analysis requires extensive database searching with powerful tools to predict protein/peptide masses, isotopic patterns, enzymatic cleavage sites and possible modifications.