Please help me understand the LC/MS procedure in the lab

[Fig Neutron]

I need some help understanding the LC/MS process for proteomics.

I am writing a paper on using LC/MS to detect nut allergens in food. My problem is that I have never used LC or MS so I do not understand the process in the lab. The resources I have found are too complicated. I am a 3rd year college student.

I just need a basic description of the LC/MS steps to help me wrap my head around the process. I think that would help me better understand the details.

What happens first, second...?

I understand how LC (HPLC and UPLC) and MS (MS/MS) work and what they do. I do not understand when digestion of analyte is needed or when it happens. I do not understand the steps of the procedure. Can you make this more understandable?

I hope my request makes sense. Thanks much!

 

[DrJohn]

Liquid Chromatography is used to separate the components of a mixture. Separation depends on the column, its liquid phase, its carrier gas pressure, its temperature.
The components / output are fed into the mass spectrometry to analyse them, in the order that they come out of the end of the LC device.

In drug analysis, with a given LC column (it's actually a coil, usually in a heated container) at a given temperature and carrier gas pressure, a given drug will exit after a given time. (This will have been established before hand, by running just a pure sample of each drug you might want to analyse through the eLC column, to find out when they arrive at the detector.)

So you are looking at the sample after, say, six minutes and if there is any heroin in the original sample, it will come out at that time and have its MS recorded. But other things / impurities might come out at that time point, and so by looking at the MS you can see if it really is heroin, even if it was mixed with ten or twenty other things in the initial sample. You can detect things coming out the end of the LC in various ways, and record the MS of each one. So for total unknowns, you'd record the LC in the usual way, then now knowing the times to expect the components of sample, you record the MS of each one as it arrives. You might use two different columns to separate the components if you get a very complex MS that suggests you have a mixture coming through at some point in time. EG one column that binds polar compounds strongly, so they take ages to come through, and one that doesn't bind them strongly. Recording the MS of each component.

The sample might be prepared before the process is started so that a particular compound would react and decompose into known things, and detecting all the expected reaction products, each with the correct MS, confirms that a certain substance was present.

Which happens first, which second? The clue's in the name LC/MS. in that order!

 

[Fig Neutron]

@DrJohn Thank you very much for taking the time to break this process down for me. I really appreciate your help!