Two peaks from pure protein

[davidfan05]

I am now working on Transferrin, a glycosylation protein using Aglilent 6550 iFunnel Q-TOF LC/MS. It seems that I got two peaks for even pure transferrin. These two peaks can not be separated well. The following are related settings:

Injection Volume 500 ng
Flow Rate 0.4 mL/min
Solvent A 0.1% formic acid in LC/MS H2O
Solvent B 0.1% formic acid in acetonitrile

Wash & Purge Solution 50% Methanol
Seal Wash 10% isopropanol
Column Acquity UPLC BEH C4 1.7 µm, 2.1 x 100 mm column
Column Temperature 80˚C
Running time 10 min

Time (min) Flow Rate (mL/min) % Mobile Phase A % Mobile Phase B
Initial 0.4 97 3
5 0.4 10 90
6 0.4 10 90
6.1 0.4 97 3
7 0.4 97 3

Drying Gas Temp (˚C) 270
Drying Gas Flow (L/min) 14
Nebulizer (psi) 20
Sheath Gas Temp (˚C) 400
Sheath Gas Flow (L/min) 12
Capillary (V) 5000
Nozzle Voltage (V) 2000
iFunnel parameters H pressure RF: 200
L pressure RF: 100
MS TOF Fragmentor 365 v
Oct1 F Vpp 750 v

It may be the formation of salt and solvent clusters with my protein. How can I minimize or remove this effect? I tried using 0.1% formic acid (mobile A) to load samples instead of water. It helped, and the first peak become higher, but there is still the 2nd peak.

I am quite new for MS, and I have a lot to learn. This is the first big problem I encounter for me and my team. I hope that I could get helpful suggestions and input from experts here. Thanks in advance!

David

[tom jupille]

By "two peaks" do you mean peaks at different chromatographic retention times or one retention time with two major m/z on the MS ?

[HPLC chemist]

This is not uncommon! In the case of proteins/enzymes/hormones that have a cysteine bridge (in my case Oxytocin), you can have the bridge open ready for the next reactant or closed (reduced).

[davidfan05]

By "two peaks" do you mean peaks at different chromatographic retention times or one retention time with two major m/z on the MS ?

I meant peaks at two different, but close, retention times.

[tom jupille]

That means that you have a chromatography issue; it has nothing to do with the MS part if the system.

As HPLC chemist pointed out, it is quite possible for the same protein to be present in multiple forms. In addition to the disulfide bridging mentioned, you can also have multiple conformational states (tertiary structure) for the same protein. The chromatography system can only interact with the outside of the molecule, so the conformers can have different retention times.

For assay purposes, you can minimize the problem by fully denaturing the protein before injection, and by running under denaturing conditions; the idea is the "fully denatured" is the most stable conformational state.

[davidfan05]

Thank you for your suggestion. How can I denature the protein for MS assay? Do you have related protocol?

That means that you have a chromatography issue; it has nothing to do with the MS part if the system.

As HPLC chemist pointed out, it is quite possible for the same protein to be present in multiple forms. In addition to the disulfide bridging mentioned, you can also have multiple conformational states (tertiary structure) for the same protein. The chromatography system can only interact with the outside of the molecule, so the conformers can have different retention times.

For assay purposes, you can minimize the problem by fully denaturing the protein before injection, and by running under denaturing conditions; the idea is the "fully denatured" is the most stable conformational state.