Bringing New Methods to the Seed Proteomics Platform: Challenges and Perspectives

Abstract

For centuries, crop plants have represented the basis of the daily human diet. Among them, cereals and legumes, accumulating oils, proteins, and carbohydrates in their seeds, distinctly dominate modern agriculture, thus play an essential role in food industry and fuel production. Therefore, seeds of crop plants are intensively studied by food chemists, biologists, biochemists, and nutritional physiologists. Accordingly, seed development and germination as well as age- and stress-related alterations in seed vigor, longevity, nutritional value, and safety can be addressed by a broad panel of analytical, biochemical, and physiological methods. Currently, functional genomics is one of the most powerful tools, giving direct access to characteristic metabolic changes accompanying plant development, senescence, and response to biotic or abiotic stress. Among individual post-genomic methodological platforms, proteomics represents one of the most effective ones, giving access to cellular metabolism at the level of proteins. During the recent decades, multiple methodological advances were introduced in different branches of life science, although only some of them were established in seed proteomics so far. Therefore, here we discuss main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in this field of plant research, with a special emphasis on sample preparation, data acquisition, processing, and post-processing. Thereby, the overall goal of this review is to bring new methodologies emerging in different areas of proteomics research (clinical, food, ecological, microbial, and plant proteomics) to the broad society of seed biologists.

Protein glycation and drought response of pea (<em>Pisum sativum</em> L.) root nodule proteome: a proteomics approach

“…Although formation, metabolism and biological effects of AGEs are well-studied in animals and humans (Szoke and Gerich, 2005;Uribarri et al, 2005;Soboleva et al, 2017a, b), the role of the Maillard protein adducts in plants still remains mostly unknown, although intensively studied (Rabbani et al, 2020). Recently, the dynamics of protein-bound AGEs was characterized not only in leaves (Frolov et al, 2018), but also in seeds (Smolikova et al, 2020) and nodules of legumes (Matamoros et al, 2018). The role of protein glycation in drought-related metabolic shifts and nutritional properties of mature pea seeds (P. sativum) was addressed (Leonova et al, 2020).…”

Does Filter Aided Sample Preparation (FASP) Provide Sufficient Method Linearity for Quantitative Plant Shotgun Proteomics?

“…Thus the digestion protocol of the Mann group was transferred to the appropriate protein extraction method, which is currently considered as the most efficient one in terms of sample quality and protein identification rates. 2 The FASP approach proved to be an appropriate method for digestion of the pea seed proteins, i.e., it was ideally compatible with the phenol extraction method. Indeed, most of the total seed protein fraction (97.7%) was successfully digested and transferred through the cellulose membrane of the filter units (Figure 2A), whereas only 2.3% of the protein aliquot, subjected to proteolysis, remained on the membrane after 3x washing with 50 mmol/L aq.…”

“…1 During the last decade, gel-free LC-based shotgun proteomics became a gold standard of proteome analysis due to its higher throughput, superior proteome coverage, better analytical resolution and reproducibility. 2 However, because of the mechanistic limitations of electrospray ionization (ESI), shotgun proteomics is critically sensitive to detergents. 3 On the other hand, in contrast to in-gel proteolysis (which can be quantitatively accomplished in ammonium bicarbonate buffer), in-solution digestion for shotgun proteomics ultimately requires supplementation of detergents to ensure quantitative solubilization of protein isolates and their efficient proteolysis.…”

Plant Proteomic Research 3.0: Challenges and Perspectives

“…Four reviews deal with methodological aspects [ 3 , 4 ], mechanisms of ubiquitination [ 5 ], and the effect of and responses to nanoparticles treatments [ 6 ]. Smolikova et al [ 3 ] summarized the main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in plant research, such as sample preparation, data acquisition, processing, and post-processing.…”

“…Four reviews deal with methodological aspects [ 3 , 4 ], mechanisms of ubiquitination [ 5 ], and the effect of and responses to nanoparticles treatments [ 6 ]. Smolikova et al [ 3 ] summarized the main methodological approaches already employed in seed proteomics, as well as those still waiting for implementation in plant research, such as sample preparation, data acquisition, processing, and post-processing. Tartaglia et al [ 4 ] focused on several soil protein extraction protocols to highlight the methodological challenges for the application of proteomics to soil samples, which enhance the identification of proteins with low abundance or from non-dominant populations.…”