# Getting Ready for Large-Scale Proteomics in Crop Plants
## 1 Introduction
These include filter-aided sample preparation (FASP) [9; 13; 14; 15] introduced in 2009, protein suspension trapping (S-Trap) reported in 2014 [16] and single-pot-solid-phase-enhanced sample preparation (SP3) published in 2019 [17; 18]. FASP makes use of ultrafiltration columns that contain membranes with a 30 kDa molecular weight cut-off. When fully denatured, even small proteins have hydrodynamic volumes large enough to be retained while small organic molecules such as SDS are washed out during centrifugation. The proteins can then be directly digested on the membrane and peptides can be easily recovered for LC-MS/MS analysis [13]. That said, the FASP workflow can be rather time-consuming, and filter membranes have been known to develop blockages inexplicably, which poses a risk of complete sample loss. Full removal of SDS from FASP filters may also require an additional extraction step using ethyl acetate, thus increasing time demands and potential for sample losses [19]. A variation of the FASP idea is the protein suspension trapping (S-Trap) method. Here, proteins are solubilized with SDS and then acidified, leading to a suspension of protein precipitate in an aqueous buffer containing traces of SDS. This suspension is transferred into commercial S-Trap tips containing a filter to retain proteins and remove small molecules via centrifugation. Following protein digestion on the filter, peptides are retained by the hydrophobic component in the S-Traps, allowing direct peptide clean-up after digestion. As S-Traps are available in 96-well format and are compatible with different extraction buffers, they have become popular in mainstream proteomics [16; 20; 21]. In the recently introduced SP3 method, proteins are precipitated onto (paramagnetic) beads by the addition of high percentages of organic solvents, notably acetonitrile. SDS and other small molecules remain in the organic phase and are easily removed by washing beads with organic solvent. Digestion of proteins is performed directly on the beads, and prior work on single _Drosophila_ embryos showed that SP3 can handle even minute amountsof protein [17; 22]. The latter should facilitate the analysis of scarce plant materials such as pollen [23]. SP3 can also be automated in 96-well format on robotic platforms to increase throughput and reproducibility (termed AutoSP3) [24]. The method has been successfully applied to the analysis of _Arabidopsis_ leaves and has the potential to become a standard approach for crop plants [18]. Results from two studies using leaf material from barley and _Arabidopsis_ suggested that FASP is superior to the classic in-solution digest (ISD), but the study did not include the S-Trap or SP3 methods for comparison [9; 10]. A recent paper comparing FASP, S-Trap and two different SP3 protocols using _Arabidopsis_ leaves reported that S-Trap was inferior to FASP and SP3 with regard to the number of identified peptides and protein groups. In the same study, SP3 outperformed FASP for low sample input and gave comparable results for large sample amounts. Because SP3 is less time-consuming, cheaper and more easily automated, the authors projected that SP3 may become the preferred method for plant proteomics in the future [18]. Inevitably, the identification of peptides and proteins is performed by LC-MS/MS. For many years, the prevailing approach used nano-flow liquid chromatography coupled to electrospray ionization (ESI) tandem mass spectrometry. This is because of the superb sensitivity offered by this combination. However, the often extremely high concentrations of certain proteins in plant tissue, such as Rubisco in green tissues, degrades chromatographic performance and, in turn, quantitative precision. More recently, we have shown that chromatography operating at 50 \(\mu\)L/min (micro-flow LC-MS/MS) can alleviate this issue at a moderate cost of sensitivity [25; 26]. We further showed that nearly 9000 proteins from Arabidopsis could be identified by such a system in a single 3-hour LC-MS/MS experiment [25; 26].