The Q&A presented in this blog post was featured at the end of a webinar we presented on the leap day this year: Feb 29.I thought to feature the Q&A in this blog post because of very interesting questions about the advances in peptide identification and the type of columns being predominant in this area. The webinar titled, Preparation and High Resolution Separation of Peptides Using Two-dimensional LC–MS is still available as an on-demand webcast and requires a quick registration.
The Q&A was moderated by Alasdair Matheson, Editor, LCGC Europe and Tony Edge, Technical Manager, Chromatography Consumables, Thermo Fisher Scientific provided the answers.
Here is Part I of the Q&A session!
Are there any other orthoganol chromatographic techniques that could be considered?
Yes, techniques such as restricted access hydrophobic interaction chromatography (downloadable PDF) and size exclusion chromatography have been applied to the analysis of peptides and proteins in a two-dimensional sense.
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Where do you see significant advances for peptide identification in the near future?
Clearly Mass Spectrometry (MS) has had a massive influence in this area. Before the advent of MS, the whole area of proteomics was really struggling substantially. But, what we have demonstrated in this method is that we can actually start using the chromatography to get better selectivity and I think that the last plot that I demonstrated this, where clustering with the ion exchange material but by using a different column technology with different selectivity we got a more continuous spread of compounds. That to me suggests that we can look at the selectivity option of the column chemistry in order to optimize things. I think there have been great advantages in terms of the production of smaller and smaller particles within the chromatographic manufacturing industry, and I think that means a massive improvement in terms of efficiency. The introduction of UHPLC sub 2 micron particles really has led the way there. These high efficiency columns have led to situations where we can now get hundred of thousands of plates for our separations, so capacity factors in the thousands which really do aid the scientists to identify a wider range of individual peptides etc. I think that is very beneficial.
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.
Next blog post will cover the remaining questions from this Q&A!
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