Biology Reference
In-Depth Information
Chapter 27
Back to Osborne. Sequential Protein Extraction
and LC-MS Analysis for the Characterization
of the Holm Oak Seed Proteome
M. Cristina Romero- RodrÃguez , Ana M. Maldonado-Alconada ,
Luis Valledor , and Jesus V. Jorrin-Novo
Abstract
It is impossible to capture in just one experiment all or most of the total set of protein species that constitute
the cell's proteome. Thus, according to our results, and even considering that they depend on the experi-
mental system carried out (plant, yeast, fungi, or bacteria), the best protein extraction protocol yielded less
than 20 % of the total amount of proteins, as determined by the Kjeldahl method. For this reason, protein
cataloguing and the whole proteome characterization require the use of fi rstly, fractionation techniques at
the cellular, subcellular, protein, or peptide level, and secondly, the use of complementary approaches.
Within our current research on Holm oak (
Quercus ilex
subsp.
ballota
), we aim to characterize its seed
proteome. For that we have optimized an experimental workfl ow in which the Osborne sequential protein
extraction (Osborne, Science 28:417-427, 1908) is combined with downstream electrophoretic protein
separation or shotgun MS analysis. In general, it can be used to study any plant seed, as well as to investi-
gate on seed maturation and germination, genotype characterization, allergens identifi cation, food trace-
ability, and substantial equivalence, among others.
Key words
Holm oak proteomics, Seed proteomics, Osborne
1
Introduction
Osborne (1924) classifi ed seed proteins into four categories according
to their solubility in various solutions: (1) albumins, soluble in
water, which primarily correspond to enzymes; (2) globulins,
insoluble in water but soluble in salt solutions (i.e., 1 M NaCl); (3)
prolamins, soluble in ethanol; and (4) glutelins, which can be
extracted with either very acidic or very basic solutions [
1
,
2
].
The major seed storage proteins are albumins, globulins, and
prolamins. All storage protein fractions are mixtures of compo-
nents that exhibit polymorphism both within single genotypes and
among genotypes of the same species according to species [
3
].
These protein classes are not present systematically in the seeds of all
