LC–MS separation of peptidesContinuing Part II of the Q&A featured at the end of the webinar titled, Preparation and High Resolution Separation of Peptides Using Two-dimensional LC–MS. The webinar is still available as an on-demand webcast and requires a quick registration. For Part I of the Q&A, check out this blog post: LC-MS: Two-Dimensional Analysis of Peptides Q&A (Part I).

The Q&A was moderated by Alasdair Matheson, Editor, LCGC Europe and Tony Edge, Technical Manager, Chromatography Consumables, Thermo Fisher Scientific provided the answers.

You talked about chaotropic agents (i.e., guanadine vs. urea) could help peptide signal in MS. Is that always true? I am thinking could that also suppress some peptide ion formation in MS?

Yes, that is exactly right. When we are looking to optimize the sample preparation, it is specific to a particular peptide, and there are tens of thousands to hundreds of thousands of potential peptides that could be analyzed and, as a result, each of those can react slightly differently. Each of these individual sample preparation techniques does need to be looked at in isolation for the peptides that the scientist is actually looking at.

It seems like the higher the temperature, the better on the chromatograms. What is the ceiling for high temperature before denaturation?

In terms of denaturing, clearly as we go to higher temperatures there is a greater propensity for the protein to denature and that can cause catastrophic effects in terms of the columns performance. The compound starts to denature in the column and cause blockage etc. So in terms of how long or what temperature that it goes at depends on two key components. It depends on the proteins that you are analyzing and the length of time the protein spends in the column. If you can elute that particular protein quickly then it can be beneficial. I would suggest in the case that temperatures, above 90 – 100 Celsius, you will have some significant issues in terms of the sample stability. When you are looking at the protein analysis, if the analysis isn’t quick and, because of the complexity of the samples, you will need to give a longer time for the analysis to occur.

What arrangements must we undertake to avoid the contamination of nano-ESI capillaries and needle?

If you are looking at contamination of needles, it really does depend on the type of components you are looking at.

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What format of columns do you see as being predominant in this field?

I believe reverse phase columns will be predominant; in particular the C18 type stationary phases. Looking at the 2-dimensional work, strong cation exchange really has developed a place, and size exclusion to a certain extent. The data that the guys from the Pattinson Institute obtained demonstrates quite admirably that there is actually a place for graphite carbons among the mix. So I think going forward, I can see development for the C18 material will have substantial effects in particular particle size. But, in terms of other columns I am really quite hopeful that we will see resurgence in a column that was invented, designed, and developed by John Knox.

Is this approach suitable for nano-flow systems?

The sample preparation is applicable and in terms of the chromatography, again the approach is very applicable, so yes is the short answer.

What columns and mobile phases were used for the separation of peptides?

I think it is best to answer this individually but in terms of some of the peptides that we separated, most C18 columns can get good separation of peptides and, if you talk to your manufacturer they should be able to give you advice on what can give you the best separation. There are a whole variety of tests that manufacturers are performing on their columns to characterize column performance. I think once this information is available to users, it will make column choice a lot easier to do and it will also dispel some of the myths that happen with a lot of the traditional C18 type columns as well. But in this particular case, these are our HLPC GOLD columns available in 1.9 µm , 3 µm, and 5µm formats. For an overview plus technical details, check out this technical guide on GOLD columns (downloadable PDF).

For more information, check out the Protein Separation, Identification and Characterization. Any Methodology, Any Scale page on our website.

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