Chromatography Forum

Hi there,
Sorry for asking this very basic question. I want to determine the molecular weight of a peptide (20 AAs), using Agilent LC/MS. Can I use SIM mode to determine the exact MW of my peptide. Sometimes, I am not able to get exact mass using scan mode, but with SIM mode, it mostly gives the correct mass. Is SIM mode acceptable for publication.
Thank you all for your response.

what instrument are you using? "Exact mass" usually implies a ToF or similar, but SIM/scan usually implies a quadrupole...

Hi, I am using Agilent 6125 LC/MSD Single Quadrupole, having both Scan and SIM mode options. I prefer to use SIM mode. Not sure if I can rely only on SIM mode. Thanks

SIM is good for quantification. For mass identification you should scan the spectra.

this is a test reply before I type yet another huge reply that gets deleted

you can also reply to my email: sokarvi@kfshrc.edu.sa

I just wanted to clarify a few things:

Your instrument is a quadrupole, so its mass accuracy is to about m/z 0.1. You may find that the software will allow you to specify masses to a much higher precision, but a quadrupole filters masses with a width of about m/z 0.7, depending on how it's tuned, so a SIM centred on 1004.1234 will give the same chromatogram as a SIM centred on 1004.1000 (and frankly it won't make much difference if you enter 1004 or 1004.4). Nevertheless, the software might report the mass its found as 1004.1234 because that's what you asked it to look for! It has not actually measured the mass, it's just set a (wide) filter to permit ions through, if they have a mass approximately around what you asked for!

In Agilent-world, the window is usually from about 0.3Da below to about 0.7Da above, because Agilent know that people often enter an integer, and most small organic molecules are a bit heavier than an integer.

Worse, when you collect a SIM ion, you don't actually know whether it's the parent ion, an isotope peak of a parent that's lighter, a fragment from a parent that's heavier, an isotope peak of a fragment, or a non-covalent cluster ion of analyte plus something else. So yes, you do need scans.

The awkward thing is that quadrupoles are very biased towards low mass. Even when the manufacturer claims they go up to high mass, they'll be low efficiency up there, so a 20 amino acid peptide is likely to be low sensitivity, particularly if you're looking for the singly charged ion.

Exact mass generally means mass measured to better than 5ppm, or approximately m/z 0.001. This is good enough to distinguish many near-isobaric ions (situations where two ions add up to the same integer, but differ int he decimal places; sulphate and phosphate are an example). Unfortunately, if you want to confirm the exact mass of your peptide, you may need to find someone with a Q-ToF or Orbitrap. Good luck!

hi try to acquire an MS spectrum in profile and then deconvolute

Hi Antonio,

Can you please explain how to do this? Thank you

You're using an Agilent single quad. This explains how to do it:

https://www.agilent.com/cs/library/tech ... x4dyh7KAxU