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2-D, LC/MSMS Nano-SCX/RPC LC-MSMS proteomics fractionation

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Use of pH and salt gradients for 2-D, Nano-SCX/RPC-LC/MSMS fractionation of tryptic digests.

Data courtesy of Witold Winnik, US EPA
(This work does not necessarily reflect EPA policy, and it is not an endorsement of any commercial products.)

ABRF 2005 poster P-115: Analysis of Protein Digests by Nano-SCX/RP/MSMS with pH/salt Gradient SCX Elution.

The above pdf copy of Winnik's poster may require these additional comments regarding his methodology and how it might be customized for different MUDPIT or 2-D LC/MSMS applications (look for the full article in Analytical Chemistry).


Protein samples can be solubilized in 6-8M urea. Urea should be purified. I recrystalize urea in my lab by dissolving it in a small amount of warm methanol first, and by pouring it into boiling heptane. Precipitated urea is collected by filtration. Commercial urea is contaminated with isocyanate and will produce protein and peptide carbamylation. Tris buffer should be used if one uses urea to help scavenge traces of isocyanate. In addition, I remove DTT, iodoacetamide, tris, urea, etc by acidifying the sample at the end of the reduction-alkylation procedure, trapping alkylated proteins on your macro protein trap and eluting them with water / acetonitrile / acetic acid / TFA

After this solid - phase extraction step, the eluted protein sample is evaporated to almost dryness using a Speedvac evaporator, is reconstituted in 50 mM ammonium bicarbonate and digested with mass spectrometry-grade trypsin. The sample is evaporated using the Speedvac evaporator again to almost dryness to remove most of the volatile ammonium bicarbonate buffer. Then the sample is acidified with 2M acetic acid to the starting SCX pH conditions (3.7 in my case). A pH lower than 3.7 would also be acceptable. It is important to remove most of ionic material from the sample or else some peptides might not be retained on the SCX column.

I injected about 20 µg of the peptide mixture on the SCX column. Overloading the SCX column will result in competitive displacement of acidic peptides by basic peptides.

Factory pre-cut 20 µM ID PeekSil material was used for transfer lines for microfluidics.

Two Michrom peptide micro traps were used in the ten-port switch valve to trap SCX - eluted peptides.

Acetonitrile (2%) was used as a SCX solvent additive. You might try dropping it to 1% or lower if need to retain very hydrophilic peptides on the micro trap.

After each 5-min SCX gradient segment, while waiting for the end of the 120 min RP elution, the SCX Surveyor LC pump flow rate was lowered to almost zero (peak parking) or else a "no-flow" error message will be generated by the software if one attempts to stop the flow completely. Surveyor autosampler injector loop needs to be immediately switched back to LOAD position in all 2-D runs except for the first one, in order to divert the "dummy 10 µL loop injection" that is necessary to initiate each LC/MSMS run.

My automated pH-SCX-gradient 2-D Nano-LC/MSMS approach is quite flexible: for instance, you can use ammonium formate buffer for "A" should lower - pH elution starting conditions be necessary to capture very acidic peptides, or if more SCX gradient steps are necessary in the low pH region to fine-fractionate acidic peptides (such as phosphorylated peptides, for example). The 200Å, PolySULFOETHYL A material is recommended for separation of phosphopeptides. But if you use 3µm PolySULFOETHYL A material instead of the 5µM, much higher back pressure will require a significant split line adjustment.

The SCX peptide elution pattern is consistent with the number and position of basic vs. acidic residues in a peptide sequence. My new SCX - elution vs pH peptide score will be described at ASMS 2005 and in the full journal article.

As far as the protein Macrotrap, this was a Michrom Bioresources' Macro Protein Trap, polymeric, C4-like material, syringe-mounted trap in a holder. This trap was used for the solid-phase extraction sample prep step.

The two micro peptide cap-traps (the smallest size available from Michrom Bioresources, polymeric) (Ed.Note: Although a PLRP-S-100 or 300, in any of the 300nL to 5µL Piccolo™ guard/trap column formats could be substituted here) were positioned in the 10 - port valve for SCX fraction collection and desalting by RPC before the nano-LC/MSMS analysis.

Also tested were the MiniSpin peptide and protein spin columns. They work just fine for sample purification and could have been used for this project. The main reason why I ended up using the Hamilton syringe - operated polymeric trap for protein separation in this particular project is its low dead volume (the trap material is compressed like in a HPLC column), and also I wanted to observe the wash solution eluting off the protein trap column to see when it stops "foaming", because I needed to completely remove an "APTA" quaternary amine CYS - labeling reagent before feeding the purified protein onto the 2-D LC SCX column (to prevent competitive displacement of cations).

Last Updated: Friday, 02-Dec-2016 13:05:08 EST
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