Chromatography Forum

Greetings!
Recently, I've been playing a bit with proteins analysis by LC-MS-MS (mostly for teaching, not for serious research). While Mw determination from charge series worked as charm, I have not managed to get any fragmentation at lower collision energies (< 50 V), and at higher (80 V and above) the molecules disintegrated to amino acids. Does anyone know why I was unable to obtain y- and b-ions?

I have used Agilent Technologies 6410A triple-quad with ESI. Samples (e.g. lysozyme) were injected via HPLC autosampler, directly connected to MS, using 0.1 % HCOOH/MeOH (1:1) as mobile phase, at flow of 0.5 mL/min.

ESI parameters were: nebulizer gas (N2) pressure 50 psi, drying gas (N2) temperature 350 °C and flow 10 L/min, capillary voltage 4000 V, fragmentor voltage 300 V (optimized for highest intensity of [M+H]+), positive ionization.

QqQ parameters were: Product Ion Scan mode, collision energies 10-140 V (in 20-30 V increments). Collision gas was N2.

Isn't it a case of too many degrees of freedom ? A very large molecule has a very large number of chemical bonds over which to disperse the energy.

I suspected as much. But, how to overcome that problem? CID *is* used for protein sequencing, so what can I change to force my proteins to fragment in controllable manner (i.e. via y and b ions)?

CID *is* used for protein sequencing

As you have discovered, this does NOT appear to be possible; CID is used for PEPTIDE sequencing. A 10-residue peptide (MW 1100) has relatively few degrees of freedom compared to a protein (MW 5000 and upwards).
I don't know enough about protein 3-D structure and chemistry to be able to suggest a solution. Your only variables appear to be collision energy, collision gas pressure and identity of collision gas. I have forgotten the details, but the energy imparted by collision is partly dependent on the MW of the gas.

If N2 & 80 eV gives AA, maybe He & 60 eV will give some sequence ions ????

Maybe a literature search and a study of the theory is in order. I have the feeling that this is of such fundamental interest, that it has been investigated more than once.

As a matter of interest, what is the highest MW peptide that has been (at least partially) sequenced by CID ?

The calculation is done with Peter Derricks Center of Mass CID equation which calculates available energy for uptake during singular collisions. It is indeed based on mass of the target gas, Parent MW and collision energy. We wrote some papers back in the early '80s looking at both high energy and low energy collision processes and parent MW dependencies; I believe we topped out, back then around 1500-1800amu, although its my impression the TOF-TOF folks are doing some higher MW, somewhat high energy collisions nowadays. The degrees of freedom argument was always complemented by 'preferred' route of energy pathway as well, that is some bonds are clearly more likely to fragment.

Thank you both for your replies. It appears that I have neglected the fact I've never saw fragmentation of large peptides in literature... I have never had to think about degrees of freedom before, since I only deal with small molecules (Mw < 700).