Does a peptide's intensity affect elution time?

[Gautam Saxena]

The main question is capture in the title: Does a peptide's intensity affect elution time?

The details (in case they are of interest) are as follows: Using only very intense peptides (~500 peptides spread across a ~2hr gradient on Thermo Fusion), there appears to be a clear alignment pattern that can be easily represented by a smoothing cubic spline, and almost all (eg 99.99%) of the peptides fall within a very narrow range (eg 20 seconds) of this smoothing cubic spline. HOWEVER, if lesser intense peptides are also used for this modeling exercise, then about 1% of the peptides are NOT anywhere close to within this 20 seconds error bar -- they can be as much as 1 to 3 minutes away. So I see two possibilities: one (the likely one) is that those peptides are simply outliers in my modelling algorithms. I consider this intuitively likely, since I can think of a number of reasons why my algorithm would incorrectly claim that these lower intense peptides are off by 1 to 3 minutes; BUT, the other possibility is that intensity affects elution (for perhaps 1% of the case) and that those peptides *truly* are 1 to 2 minutes misaligned (because of their intensity). 

So, is there are theoretical (or observational) reason to suggest that intensity may affect elution? 

I tried to come at the answer sideways by googling/skimming through papers -- mainly ones that discussed *prediction* of LC elution time -- and none suggested that intensity matter, though that is not a conclusive argument. (It could be that since no one knows the intensity of an analyte ahead of time, there's little point in attempting to consider it to predict elution LC time.)

[David Bouyssié]

Hi Gautam,

Here is a manuscript we published 2 years ago that should be useful for your research:

Ramus C, Hovasse A, Marcellin M, Hesse AM, Mouton-Barbosa E, Bouyssié D, Vaca  S, Carapito C, Chaoui K, Bruley C, Garin J, Cianférani S, Ferro M, Van Dorssaeler

A, Burlet-Schiltz O, Schaeffer C, Couté Y, Gonzalez de Peredo A. Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset. J Proteomics. 2016 Jan 30

The conclusions of this paper won't give an answer to your question but you could verify your hypotheses using the corresponding dataset:

http://proteomecentral.proteomexchange.org/cgi/GetDataset?ID=PXD001819

We indeed delivered here raw files corresponding to the analysis of a hybrid sample preparation:

* a background of Yeast lysate at fixed concentration

* the UPS1 standard (48 proteins) spiked at different concentrations

Thus you could check if UPS1 peptides at high and low concentrations have a different RT shift compared to the Yeast peptides.

In my experience, this is not what I observed when I was looking at RT shifts of UPS1 peptides but I may be wrong because I didn't make a thorough verification.

I hope this answer is useful.

Best,

David