Off-line fractionation using Strong Cation eXchange (SCX).

Thusfar, best results in fractionation of complex peptide mixtures  are obtained with off-line 2D methods using SCX as the first dimension.  This is ascribed to o.a the use of acetonitrile in the salt buffer (35% v/v) a condition which is not compatible in a on-line set-up with reversed phase in the second dimension. In addition, off-line fractionations employ a gradient elution whereas on-line fractionation are accomplished by means of step elutions.

In our set-up, the sample is injected on a hypercarb trap column and subsequently transferred to the SCX column by a plug injection (1 min = 2 uL) of 50% ACN+0.1 M acetic acid. Next, the peptides are eluted from the SCX column (200umID x 10 cmL; flowrate 2 uL/min) using a gradient from 0-100% C (C =500 mM KCL + 0.1M acetic acid in 35% ACN). Fractions are collected every 2 min (4 uL), evaporated to dryness and reconstituted in water/dimethylsulphoxide/acetic acid (95/5/0.1, v/v/v) for analysis.
KRQHPGKR 5.1+ @ pH=3
RVYIHPF 2.1+ @ pH=3
CYIQNCPLG  0.1+ @ pH=3
MHC class II elution sample (yellow); fractionated in 10 unique fractions