I agree with Grzegorz

Sheps,

The ionisation process depends on the type of molecule, same the fragmentation. Thus, total ion current (TIC) will be different for different molecules.

" I want to have a better understanding of mass spectrometry in general " - Take a look for some good e-books on MS.

regards,

G

I agree with Grzegorz

Hello Sheps,

Mass Spectrometry is not inherently quantitative due to the different properties of our sample components. Therefore, the technique is qualitative unless you have calibration curves.

Considering you're going to work with proteomics, picture the different aminoacids! Some of them are more susceptible to enzymatic digestion and some are more easily ionized or detected in the equipment. Due to these aspects, in the absence of a previously made calibration curve (using a pure solution of your target), if you detect arginine and lysine (for instance) during your analysis, you can conclude that your sample is constituted by these two aminoacids (quality), but you cannot specify their amount/quantity in your analyte.

Best regards,

João Nina

Thanks! These responses are helpful!

Many hormones (in my case oxytocin) even under the same pH can be either oxidized or reduced (cysteine bridge open or closed). Thus, the same protein can have multiple peaks and even multiple ionization energies depending. Calibration will help but it should be the 'pure' protein in order to obtain a mass versus detector response curve.

Thanks Bruce Neagle

Thanks Markus Hartl

Dear Sheps,

You can safely state that mass spectrometry is quantitiative between the same peptide / phosphopeptide overall protein all treated under same conditions, for multiple experiments. Eg., time course, drug treated or not etc..

So, the quantitiative information you obtain is relative and not absolute. Peptides, depending on their recovery from digest and clean-up steps, will also have unique ionization and fragmentation patterns and behaviour. However, if you are comparing multiple samples and keep your conditions very controlled through-out, comparative analyses between the samples is possible. You have to keep all the experiments prepared in similar conditions with similar total protein contents, and prepared around the same time; proteins digested the same time, with the same batch of protease, LC-MS/MS run together, using the same analytical column etc. Finally, the final database searches can be performed on all, eg., using MaxQuant or Mascot.

Good luck,

Hediye.

Thanks Hediye Erdjument-Bromage !

Thanks Kou Hayakawa

In General, mass spectrometry is not inherently quantitative because ionisation efficiencies are variable. Further variables include adduct formation, ion suppression, in-source fragmentation, transmission losses in the mass analyser, post-ionisation fragmentation in the mass analyser (space charging, in drift-tube fragmentation), source fouling and detector degradation.

The only inherently quantitative mass spectrometry technique developed to date is Selected Ion Flow Tube Mass Spectrometry (SIFT-MS) where ionisation occurs at thermodynamic ground state wIth a Boltzmann distribution on ion energies. Ionization is governed solely by ion-molecule reaction kinetics and, as there is no additional energy input in the mass analyser, SIFT-MS is inherently quantitative.

Thanks Nathan Hawkins !

Since you are contemplating proteomics, you will probably be using electrospray ionization. In that case there is an additional reason that mass spectrometry is not inherently quantitative that you need to be aware of. In many cases the response is a non-linear function of the analyte concentration. Part of this relates to non-linearities that can sometimes occur in the ionization process. There is also an issue of detector saturation. Some types of mass spectrometer also suffer from non-linearities due to something called space charge effects.

For some applications these non-linearities are a minor issue. For others they are a major problem. The use of internal standards can often overcome these problems. However, internal standards are not always available or applicable, and even when they are they don't always work as well as hoped, though usually they do work well.

I would still argue the following regarding the project you are trying to undertake Sheps:

1.In case of quantitating the same phosphopeptide between samples (the same peptide with the same ionization property, elution time etc), as long as the total starting protein amount between samples is same, you can have a relative quantitation of this phospho-peptide between the samples. To achieve relative quantitation of peptides or proteins between similar samples or same sample undergoing different stimuli, is not difficult and it can be done or has been done many times with publications reflecting this kind of research in peer-reviewed journals.

2. It really depends on what kind of quantitiation you are talking about or you need to do; samples must be similar (eg., same proteome, same purification processes etc) and must have the same total protein concentration. These conditions are needed to be fulfilled for relatively quantitating levels of phosphorylation changes or levels of changes observed in a particular protein.

3. If you are after absolute quantitation of a phospho-peptide or a protein, it can be done by using synthetic and labeled modified / unmodified peptides using targetted mass spectrometric approach.

Good luck!

Hediye.

The better your own understanding of how MS actually works is (e.g. ESI in most applications), the better your own understanding will be in why it may not present a reliable method of quantitation. As stated earlier, ionization efficiencies may be variable with different samples (compounds), concentrations, instruments, instruments settings, methods, operators ...