Plant proteomics: Methods and protocols. Class III peroxidases

Abstract

In this fi rst, introductory chapter, it is intended to summarize from a methodological point of view the state of the art in plant proteomics, focusing on mass spectrometry-based strategies. Thus, this chapter is mainly directed at beginners or at those trying to get into the fi eld, rather than at those with real experience or a long trajectory in plant proteomics research. The different alternative workfl ows, methods, techniques, and protocols from the experimental design to the data analysis will be briefl y commented, with cross references to previous monographs and reviews, as well as to the rest of the book chapters. The diffi culty of working with proteins, together with the power, limitations, and challenges of the approach will also be briefl y discussed. Proteins, as molecular entities, and the cell proteome, as a whole, are much more complex than what we thought in the past and can be studied in a single experiment. Because of that, fractionation and complementary strategies are required for its study. The MS analysis of complex samples may result in up to 100,000-peptide spectra that cannot be easily analyzed with standard procedures. Therefore, proteomics, more than other -omics, needs a dry lab, time, and an effort in data mining. As main conclusion, it can be stated that proteomics is in its beginnings. It is starting to make important contributions to a proper gene annotation, identifi cation, and characterization of gene products or protein species and to the knowledge of living organisms, having also an enormous application potential to translational research. However, and despite its great potential, and as in any other experimental approach, it is far from being a Pandora’s Box. In the case of plant research, the full potential of proteomics is quite far from being totally exploited, and second-, third-, and fourth-generation proteomics techniques are still of very limited use. Most of the plant proteomics papers so far published belong to the descriptive, subcellular, and comparative proteomics subgroup, mainly using a few experimental model systems—those whose genome has been sequenced—and being from a biological point of view quite descriptive and speculative. From now on we should put more emphasis on the study of posttranslational proteomics and interactomics, and move to targeted, hypothesis-driven approaches. Furthermore, and even more important, we should move to data validation through other -omics or classical biochemical strategies, in an attempt to get a deeper, real, and more accurate view and understanding of cell biology. In the modern Systems Biology concept, proteomics must be considered as a part of a global, multidisciplinary approach. Making biological sense of a proteomics experiment requires a proper experimental design, data validation, interpretation, and publication policy