Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as 15N. By utilising hydroponic media that contain 15N inorganic salts as the sole nitrogen source, near to 100% 15N-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled 14N- and 15N-hydroponically grown plants were fractionated by SDS–PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC–ESI–MS/MS). Proteins were identified and the spectra of 14N/15N peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the 14N and 15N peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct 14N and 15N peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2).