Phosphopeptides can be separated according to their phosphorylation level by sequential elution with an increasing concentration of potassium hydrogen phosphate in the elution buffer (Mamone et al. 2010).
The lipid was eluted from the column by sequential elution with 100 ml each of 1, 1, 1-trichloroethane, acetone, and methanol and the fractions collected.
Proteins were eluted from the matrix by sequential elution with the peptides GKKRARAADLE ('RARA'), GKKRVRWADLE ('RVRW'), and finally, 3M sodium thiocyanate.
The extract was then adsorbed onto a 10-ml Bond Elut SI solid-phase (SP) fractionation cartridge (Agilent Technologies, Sweden), followed by sequential elution with cyclohexane, EtOAc, and methanol (MeOH).
We also fed GO into the column pre-equilibrated by Cu as sequential elution experiments and found that the later introduced GO can complex the pre-adsorbed Cu from the sand surface because GO has a higher adsorption affinity for Cu.
The column was preconditioned by sequential elution with 200mL of methanol and 200mL of buffered methanol.
Many proteins were found both in ATP- and NaCl-eluted fractions, some of them possibly due to the sequential elution first by ATP and second by NaCl.
Sequential elution of peptides was accomplished by using a flow rate of 250 nl/min and a linear gradient from Solution A (2% acetonitrile; 0.1% formic acid) to 50% of Solution B (98% acetonitrile; 0.1% formic acid) in 40 minutes over the pre-column in-line with a homemade 15 cm resolving column (75 μm I.D.; 375 μm O.D.; Zorbax 300-SB C18, Agilent Technologies).
Soluble extracts were prepared from whole wild type C57Bl/6 mouse brain and purified on a diethylaminoethyl (DEAE Fast Floww column (GE Healthcare Life Sciences, Pittsburgh, PA) with sequential elution with increasing concentrations of NaCl as described [ 24, 25].
The sequential elution is another advantage, since having a greater amount of less complex phosphopeptide fractions heightened the probability of their ionization and identification by MS. Taken together, the protocols combining more methods and re-enriching the flow-through can be considered promising.
