What 2D peptide fractionation technique yields the highest number of IDs?

The field of proteomics has changed at a dazzling rate in a remarkably short period of time and it's an absolute challenge to keep up on instruments, software and methods. Separation science is also evolving and it's yet another factor to try to keep up on.

During my postdocs methods for in-solution isoelectric focusing of peptides were the cutting edge and that's what I used for all of my 2D-fractionations. 7-8 years later...not so much...I've seen one paper that used this separation methodology this year -- and I do watch out for it.

What does the current literature say about the best offline fractionation techniques for shotgun proteomics?  I know a lot of my friends are using high-pH reversed phase offline fractionation. But -- are they doing it for the same reason I was using IEF? Cause it's the cool thing right now?

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The first paper Scholar directed me to is this short review on the topic:

For studies where they have lots of sample, >10ug total peptides, these authors report dramatically higher peptide IDs (80% more peptides) when using offline high pH reversed phase fractionation rather than offline SCX. Interestingly, they mention that report that the desalting step post-SCX is a major point of sample loss, with up to 50% lost in their typical protocol.

However, they do find online SCX (MudPIT type methodologies) more sensitive when they are limited to less than 10ug of total peptides.

This review spends a lot of time stressing the importance of concatenation techniques. It's one shortcoming is that it doesn't give me much to work with in terms of technical details --resins and gradients and so forth.

However, it appears that all of these details can be found in this study:

The second paper Scholar directs me to is a phosphoproteomics one --

I know this group has been primarily using high pH revesed-phase (which they usefully abbreviate HpH, but I hadn't seen this technical note.

In terms of phosphopeptide identification, the work seems quite clear cut. Wow. Does the concatenation ever look like a pain in the...

Okay -- these studies have all compared HpH to SCX. What about isoelectric focusing (IEF)? Scholar?

First study that pops up doesn't have a very pro-IEF title...

I have to say, however, that the results aren't quite that drastic. HpH does outperform IEF in every way, but it isn't a night and day comparison. Interestingly the overlap isn't huge between the two techniques. They are sampling a pretty small percentage of the proteome (<3,000 proteins) so they're getting some interesting variations based on stochastic sampling of the whole.

This paper really goes beyond just a comparison of these two techniques. There is some really insightful charts showing peptide/protein distribution and relative protein coverage. Not that the other papers I link in this post aren't worth checking out -- but if you are just interested in peptide chemical properties -- this is a seriously interesting read.

Whoa...I've read too many papers for a Sunday morning. Time to get out and do something!

(No -- Ben -- do not investigate the impact all this concatenation has on LFQ....do that later...)