# Getting Ready for Large-Scale Proteomics in Crop Plants
## 1 Introduction  
Plants constitute the nutritional basis of virtually all life on Earth, and protein-rich foods from crop plants are essential for sustaining an increasing human population and counteracting climate change. While the genomes of crops are increasingly elucidated, little is known about their proteomes. The proteome is the entirety of all proteins expressed by a plant. It is these proteins that execute and control nearly every aspect of that plant's life. Proteomics is the large-scale study of proteomes and can examine the protein composition of a whole species such as tomato (_Solanum Lycpersicum_) or just a single organ such as its fruit. When compared to bacteria or animals, proteomic analyses of crop plants are often hindered by extreme concentrations of oxidative or proteolytic enzymes as well as often colorful secondary metabolites such as pigments, phenolic compounds, lipids, carbohydrates or terpenes. These are released from the plant material during protein extraction and often interfere with downstream processes such as protein solubilization, digestion into peptides or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) [1]. Complicating matters further, the generally low protein concentration in most plant tissues often necessitates the use of large quantities of starting material. As a result, crop proteome projects have, so far, demanded individually optimized protein extraction protocols to obtain samples of acceptable quality [1]. Many published protocols for plant proteomics include a protein precipitation step prior to proteolysis. Traditional methods such as TCA/acetone or phenol extraction efficiently remove a wide range of compounds soluble in the organic solvent [2; 3; 4]. However, precipitated proteins can be hard to re-solubilize, leading to the loss of valuable sample material. Inconsistent precipitation and re-solubilization from sample to sample also compromises quantitative precision and accuracy. The issue of protein re-solubilization has been largely solved by the introduction of methods that support the use and subsequent removal of strong detergents or chaotropes [5; 6]. Presumably because of the extreme molecular diversity of plant material and the many associated complications mentioned above, the plant proteomics community has relied on the use of two-dimensional gel electrophoresis (2-DE gel) in conjunction with mass spectrometry for protein identification and quantification for many years. This is in stark contrast to mainstream proteomics which rapidly replaced 2D gels by LC-MS/MS. The latter is by far superior to 2-DE gels in terms of proteome coverage, sensitivity, quantitative accuracy and the capacity to process and analyze large sets of samples [1; 7]. The dominance of gel-based methods in the plant field has recently started to vane as the adaptation and evaluation of methods originally developed for animal samples also showed promising results for plants. Today, it is possible to use strong detergents (sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC) or 3-((3-cholamidopropyl)dimethylammoniumio)-1-propanesulfonate (CHAPS)), chaotropes such as urea and thiourea or reducing agents such as dithiothreitol (DTT) for protein solubilization and denaturation from whole lysates. Combinations of these agents can further improve protein solubilization [8; 9; 10]. Another advantage is that one is no longer dependent on protein precipitation, which significantly reduces sample loss that can otherwise occur. SDS is perhaps the best and most widely used protein solubilization agent. However, it also inhibits protease activity, is incompatible with certain forms of liquid chromatography and suppresses peptide ionization during MS analysis [11; 12]. This necessitates the removal of SDS from a sample prior to protein digestion and LC-MS/MS analysis. Several methods have been developed over the past 10 years and are being increasingly adopted for plant samples.