Glass peptide adsorption

Although the separation of amino acids and small peptides by reversed-phase HPLC is becoming an accepted procedure, the application of this technique to the purification of proteins still requires careful evaluation. The biological activities of many proteins are sensitive to denaturation by extremes in pH, by contact with organic solvents or high salt concentrations, by adsorption into glass or hydrophobic moieties, or at an air-water interface (2/). 

Because of the favorable sorptive properties of the reversed-phase supports, batch adsorption and desorption can be a very effective way to desalt a chromatographed sample or to partially fractionate a peptide mixture during a purification procedure. For example, 1-2 gm of an oc-tadecyl silica packed into a silanized glass or plastic pipette can be used for the batch fractionation of small amounts of a crude peptide extract from tissues, such as the pancreas or pituitary, or from a synthetic experiment. A number of commercial products, such as the Waters Sep-Pak, have found use in this manner 10) as a purification or sample preparation aid. Protocols for batch extraction procedures on alkyl silicas have been discussed 17a,b) and applied to neuropeptides 10, 158, 166) and other hormonal peptides 88, 162, 167, 168). With these methods recoveries of peptides present in a tissue extract are generally higher than those found with classical fractionation techniques due in part to the fact that proteolytic degradation is minimized. 

The main goal of the sample preparation protocol is to purify and enrich the analyte molecules and to dehver them in a format that best fits the ionization process. In addition, it should be simple, reproducible, and suitable for automation. For peptides, the best technique to purify them and recover them afterward in a small voliune is RP chromatography. If not coupled with an LC system, small coliunns integrated at the outlet of a glass capillary or pipette tip have proven efficient for this task [65-68]. In this way, analyte loss due to surface adsorption is minimized and sample recovery is possible with only one microHter, which is sufficient for nano-ESI MS (see below) and more than needed for MALDl MS. The latter would benefit from even smaller volumes, the handling of which, however, can be difficult. 

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