Understanding the global and dynamic nature of plant developmental processes requires not only the study of the transcriptome, but also of the proteome, including its largely uncharacterized peptidome fraction. Recent advances in proteomics and high-throughput analyses of translating RNAs (ribosome profiling) have begun to address this issue, evidencing the existence of novel, uncharacterized, and possibly functional peptides. To validate the accumulation in tissues of sORF-encoded polypeptides (SEPs), the basic setup of proteomic analyses (i.e., LC-MS/MS) can be followed. However, the detection of peptides that are small (up to ~100 aa, 6-7 kDa) and novel (i.e., not annotated in reference databases) presents specific challenges that need to be addressed both experimentally and with computational biology resources. Several methods have been developed in recent years to isolate and identify peptides from plant tissues. In this chapter, we outline two different peptide extraction protocols and the subsequent peptide identification by mass spectrometry using the database search or the de novo identification methods.
Keywords: Ammonium sulphate; Arabidopsis; C-18; Database; Mass spectrometry; Peptidome; Reverse-phase chromatography; Ultrafiltration.
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