Evaluation of Optimized Tube-Gel Methods of Sample Preparation for Large-Scale Plant Proteomics

Abstract

The so-called tube-gel method is a sample preparation protocol allowing for management of SDS for protein solubilization through in-gel protein trapping. Because of its simplicity, we assumed that once miniaturized, this method could become a standard for large scale experiments. We evaluated the performances of two variants of the miniaturized version of the tube-gel method based on different solubilization buffers (Tris-SDS or urea-SDS). To this end, we compared them to two other digestion methods: (i) liquid digestion after protein solubilization in the absence of SDS (liquid method) and (ii) filter-aided sample preparation (FASP). As large-scale experiments may require long term gel storage, we also examined to which extent gel aging affected the results of the proteomics analysis. We showed that both tube-gel and FASP methods extracted membrane proteins better than the liquid method, while the latter allowed the identification and quantification of a greater number of proteins. All methods were equivalent regarding quantitative stability. However, important differences were observed regarding post-translational modifications. In particular, methionine oxidation was higher with the tube-gel method than with the other methods. Based on these results, and considering time, simplicity, and cost aspects, we conclude that the miniaturized tube-gel method is suitable for sample preparation in the context of large-scale experiments.

Keywords: FASP; plant proteomics; protein digestion; protein extraction; shotgun proteomics; tube-gel.

Figure 1

Schema of the experimental design. A single protein pellet was divided into 16 aliquots (A). Proteins were solubilized by using four different methods with four replicates per method. Two independent digestions (D) were performed per protein sample. For the two methods based on tube-gel digestion, two additional digestions were performed on 1 month old gels (dark tube gel symbols).

Figure 2

Protein yields obtained with the four tested methods of sample preparation.

Figure 3

Numbers of MS² spectra (A) and of assigned spectra (B) obtained for FASP, liquid digestion and the variants of the tube-gel methods.

Figure 4

Protein clustering according to spectral counts. Data were scaled: 0 corresponds to the general mean of each protein in all samples.

Figure 5

Principal component analysis performed on XIC data. F: Fasp; L: Liquid; Tto: tube-gel-Told; Ttn: tube-gel-Tnew; Tuo: tube-gel-Uold; Tun: tube-gel-Unew.

Figure 6

Effect of the methods on PTM frequencies. Only major effects are shown. All graphs are available in Figure S3. Ratio: modified peptides/(modified + unmodified peptides). Modified peptides per injection: mean number of modified peptides per injection.