Seed Proteomics - Plant Proteomics: Methods and Protocols - page 356

Table 1
Sample pretreatment strategies for reduction of the seed storage proteins, and shortening
of the sample dynamic range

Method Ease of application Effi ciency Capacity Cost

Fractional solubility +++ + +++++ +

“Salting out” +++++ +++++ +++++ +

Lectin-chromatography ++ ++ ++ +++

Immunoremoval ++ ++ ++ +++++

Isoelectric precipitation + + +++ +++++

Random peptide-beads + +++ ++ +++++

Scale: Five is the easiest, has the greatest effi ciency, has the highest capacity, and is the most expensive

Fig. 4 Depletion of the supra-abundant seed storage proteins as a prelude to proteomic analyses. Lane A ,
fractional solubility of the maize endosperm prolamins. A1, total proteins; A2, dilute-buffer soluble proteins; A3,
extraction of the zein SSP with 35 % (v/v) ethanol. Lane B , “salting out” of soybean globulin SSP [ 3 ]. B1, total
proteins from mature seeds; B2, globulin-depleted proteins; B3, proteins precipitated by incubation with
10 mM CaCl 2 . Lane C , reduction in the beta-conglycinin SSP from a total protein fraction isolated from develop-
ing soybean seeds by immobilized-lectin chromatography [ 58 ]. Lane D , immunoremoval of the agglutinin
proteins from mature castor seed endosperm preparations. D1, total endosperm proteins; D2, ricin-depleted
endosperm proteins; D3, removed castor SSP [ 3 ]. The positions of size marker proteins are indicated to the left
and right of the Y -axes

However, if a “total protein” fraction has been initially isolated,
then fractional solubility of the globulins can be exploited by selec-
tive precipitation [ 56 , 57 ] (Fig. 4b ). If the interfering SSP are
N-glycosylated, it is possible to use immobilized-lectin affi nity
chromatography for their removal. This strategy was useful in