Is there a way to determine the start/stop LC time when "real" peptides are eluting?

[Gautam Saxena]

My understanding is that there is a time at the beginning and the end of the LC elution in which the peptides from a sample of interest are *not* eluting, and it's some "overhead" time (approx ~10 minutes on a 2 hour gradient?) for the LC to do some "prep" activity. Is this correct? If so, is there a way to determine what those times are by inspecting the MS file, in particular a Thermo .raw file from a Fusion instrument (possibly by using the new Thermo API that gives us access to all the metadata within a raw file)? Or, if that information is not explicitly captured as metadata in the raw files (from Thermo or Sciex, for example), is it possible to accurately infer that information by inspecting the MS1/MS2 chromatograms?

[David Bouyssié]

Hi Gautam,

It totally depends on the gradient method, but usually indeed peptides do not elute at the very beginning.

The easiest way to determine approximately the peptide elution range would be to take the time of the first and last MS/MS events.

This should work at least for DDA acquisitions if MS/MS scans are triggered using the "peptide detection" option (existing in many acquisition methods).

The MS1 TIC may also be a good indicator but you will have to define the boundaries yourself, either by using a fixed intensity threshold or by trying to perform a more fined-grained data analysis of the shape of the TIC.

Unfortunately, I do not know Thermo meta-data that could provide this kind of information.

Best,

David

[Vladimir Gorshkov]

Adding to what David wrote. I can definetly say that there is no obvious signal in Thermo RAW file (including metadata) for peptide elution start and peptide elution end. The primary reason is that this will depend a lot on the gradient, LC setup (dead-volume of the system, method, etc), and sample itself (for example, peptide hydrophobicity range, if one think of regular reverse phase LC). It is common to assume that most of peptides elute under 50% ACN (i.e. everything eluting from the column after that is likely non-peptidic). Of course, you need to know the gradient delay, i.e. the time that is necessary for the solution to reach the electrospray from the pump, to calculate the retention time border when you have all peptides eluted with 50% ACN arriving in the MS. This time can be determined experimentaly, by switching the solvent composition in steps and monitoring the pressure in the system (I would recommend using rather big steps of 20 - 50% so the change in the pressure is easy to see).

Having only a single raw file one can detect the elution window by either monitoring TIC and/or MS2 scan density. Typically, in the beginning of the run there is there is a constant TIC - the background level (the exact value is instrument dependent) and MS2 scans are rather sparse (by experience I can say that some MS2 scans will happen even before the peptide elution window). In the beginning of peptide elution window the TIC should become well above the background and the MS2 scans should be much more frequent (asuming enough material was injected and some kind of DDA method with TopN or TopSpeed is used). The detection of peptide elution end time is more challanging, since after the peptide elution the TIC and MS2 frequency still remain above the background level, though usually lower, that in the middle of elution.