Hi all,

I've got some previous experience running Dionex U3000's as the front end to an LC-MS/MS proteomics workflow in a previous lab so I'm not entirely new to the field but I'm in need of some advice/reassurances.

I've started a new position and we're looking to use a U3000 system with an ICS-5000+ pump to fractionate native proteins to then run the fractions on ICP-MS and or LC-MS/MS proteomics approached to help us identify metalloproteins and the metals they bind.

I've identified the following things I could do with advice on -

1. Cleaning the system - as we'd be looking at metals we've bought in a system where the flow path is entirely PEEK and does not have any metal components coming into contact with the sample at. Before we try running samples on the system I want to thoroughly clean the system of any metal contamination, has anyone done this before? Have any advice to share?

2. We'll be doing a multi-tiered fractionation using the following hierarchy 'Size exclusion > SCX > SAX' does that sound reasonable? The idea is to be able to load a large amount of a whole cell proteome initially and then have it separated via multiple chemistries to give us optimal isolation separation/isolation.

3. I have in my possession an MFC-1 metal-free trap column (http://www.dionex.com/en-us/products/co ... 73287.html) that the guys at Thermo threw into the package. Does anyone know anything about these? What is the resin used? Do they work as advertised?

4. Hopefully a simple misunderstanding on my part for this one - I've been given an SCX guard column to be coupled with an SAX analytical. My limited knowledge on IC suggests this was a mistake by the vendors? I would have thought we'd have to have an SCX/SCX or an SAX/SAX guarad/analytical pair and that having an SAX/SCX combination would effectively make the guard column useless or am I missing something?

Any advice is really appreciated, hopefully the more I learn I can start helping others out on the forum.