Climate change is impacting food production yields and quality. In addition, the constant global population growth, which demands more and better resources, is a greater concern for the future of the food supply. Thus, it is necessary to promote the production of safe and affordable alternative nutritious food sources of high-quality protein content [1], [2].

Amaranth (Amaranthus hypochondriacus L.) and quinoa (Chenopodium quinoa Will.) are phylogenetically close promising crops from the Amaranthaceae sensu lato family, belonging to the order Caryophyllales. They produce seeds of high nutritional quality and possess resistance to different types of abiotic stress, such as drought and salinity [3], [4], [5]. These species were cultivated in America since 3000–5000 BCE by the Aztecs, Mayans, and Incas. Although their cultivation was halted due to cultural adaptation between civilizations of the Old and New Worlds, they have now been established in North America, Europe, Africa, and Asia [4], [6], [7].

These crops have been described as “grains of the 21st century” due to the rediscovery of their nutritional characteristics in recent decades, as amaranth and quinoa seeds have a higher and better-quality protein content than most cereals, with a content of up to 21% and 18% of the dry mass, respectively, and with levels of essential amino acids close to those recommended by the FAO for human consumption, being particularly rich in cysteine, methionine, and lysine [8]. The proteins from these seeds are also characterized by their low prolamin content and low allergenicity, making them recommended for improving the quality of foods and beverages for people with celiac disease [9], [10].

Biochemical and molecular studies of seed storage proteins (SSPs) from amaranth and quinoa began in the late 1980s and early 1990s [11], [12], [13], [14]. Since then, work has continued focusing on the characterization of their techno-functional properties to promote their application in food technology by determining parameters such as their solubility under different pH and ionic strength conditions, assembly capacity, surface hydrophobicity, gel formation, and ability to form and stabilize emulsions, as well as obtaining amyloid nanofibrils for their application in nanomaterials [15], [16], [17], [18], [19].

One of the most widely used methods for extracting proteins from seeds is that established by Thomas B. Osborne [20]. Considered the first systematic classification of plant proteins, Osborne's approach defines seed proteins based on their solubility/extractability in different solvents: albumins, soluble in water; globulins, soluble in neutral or slightly alkaline saline solutions; prolamins, soluble in alcohol/water mixtures (60–70% v/v) but insoluble in water or saline solutions; and glutelins, which become soluble in dilute acidic or alkaline solutions [20], [21], [22]. As Osborne himself pointed out, this method of protein classification may yield inadequate or unsatisfactory results, as it introduces uncertainty. For example, protein extractability depends mainly on flour preparation conditions and the order in which fractionation is performed. In addition, many proteins exhibit intermediate solubility, so it is not always possible to clearly distinguish between the groups [20], [23], [24]. Even with these reservations, Osborne's method is useful for the initial characterization of seed proteins and for studies focused on their physicochemical and functional properties [21], [23].

In proteomic studies, the need to obtain such a large number of extracts per biological system can be strenuous, increasing analysis time and the likelihood of introducing experimental bias and human error from excessive sample handling. Therefore, proteomic analyses usually employ total-protein extraction methods to obtain the entire proteome represented in a single fraction [25]. This approach is successful for studying systems such as bacteria and cell lines, but obtaining satisfactory results is difficult in samples with a wide dynamic range of protein abundance [26]. In plants, for example, a few proteins can represent 50% or more of the total protein content, as is the case with RuBisCO in vegetative tissues and with storage proteins in seeds [20], [27].

When working with seeds, it is necessary to develop extraction methods that allow, on the one hand, reducing the number of fractions to be analyzed and, with this, decreasing the probability of making human errors, and, on the other hand, allow the removal or differential obtaining of protein subgroups to counteract the effect of a wide dynamic range. Although the study of seed proteins of promising emerging crops such as amaranth and quinoa has increased in recent years, it remains necessary to develop methods that enable deeper proteomic analysis through novel approaches. Thus, in the present work, we compare amaranth and quinoa seed proteins using the Osborne fractionation approach and the polarity-based method developed by our work group for the characterization of the amaranth seed proteome [28], [29], which has not been used for the profiling of quinoa seed proteins.