Chromatography Forum

Hi, I have been searching for a long time to get a basic protocol for protein identification on LC-MS/MS.
I know how to prep gel pieces from 2D gel electrophoresis. But im looking for several data on LC-MS/MS.

-Recommended column and column size
-recommended buffers
-recommended ul/min
-recommended factors on MS/MS
gas flow, temp, etc etc etc...

There are so many variables and i just do not want to go in blind.
Info: We have done 2D gels with silver staining on human plasma samples pH 4-7 used the classic shevschenko method.

I a beginner on both LC and MS/MS so tips are always welcome!!

We have a TSQ Quantum Access Max from thermo. I know its not the ideal for protein identification but i still want to try.

tnx/kind regards a master student i need...

Hi Mad4Sci,

Let me start by highlighting that I am by no means an expert in this myself. That being said, can I confirm whether you want a general method for identifying any proteins that you put through your LC-MS system, or whether you were looking for a basic protocol to allow you to identify a single (or a select few) proteins?

Also, would you be able to confirm the type of LC system you currently have, as well as the pumps on the system? This information will be useful to anyone offering a method, as certain columns generate pressures that are incompatible with certain LC systems.

A further point of interest is the sample cleanup approach you will use. Different ways exist by which to extract your protein (immunoaffinity chromatography, magnetically tagged immunoglobulins etc), or to clean up the solution (protein depletion) before introducing it to the LC system. Considering this will be important in deciding the column length (and need for a guard column) for your analysis.

All the best.

Yay someone answered =)

Ill answer the questions i can right now and will get back with more info/details about the machine later on.

Well my first goal is just to identify proteins in a certain spot on a 2-D gel. I have a protocol on how to digest them and get the proteins out of the gel piece dry them and then freeze in -20 C.

So first step would be to reconstitute them with something. 0,1% TFA in water is one i have seen.

But in the future i want to learn how to take a protein band from a 1-D gel and separate proteins with the help of LC (2DLC?) and then identify with MS/MS.

So basic protocol to identify a single (or a select few) unknown! proteins for now =)

I have been told that our system is not suited for the thing we are after but that it is possible.

We have and Accela 1250 pump with a TSQ quantum access max (triple quadropole).

I will try to find out more information on the LC-system.

kind regards AC

I'm not an expert either but I can give you few generic remarks:
1. LC-MS of intact protein would give you only m/z which can be used to calculate MW by deconvolution but is not specific enough for identification.
2. LC-MS/MS of intact protein would give you some details on structure and sequence. I think this kind of information can be used for database search.
3. Digestion with specific agent (chemical or enzyme) followed by LC-MS or LC-MS/MS may give you a lot of information on primary structure of your protein. For determining primary structure of new protein it may be required to digest with at least two different agents to get two series of sequences which can be arranged in proper order.

-Recommended column and column size

C18 (there's a lot of them) or C12 (eg. Jupiter Proteo from Phenomenex). Narrow bore would increase ESI response.

-recommended buffers

A: <0.1% formic acid or TFA in water
B: <0.1% formic acid or TFA in acetonitrile or methanol
TFA - less tailing, more supression of ionisation
FA - other way around

-recommended ul/min

As low as possible to increase ESI response.

Info: We have done 2D gels with silver staining

Not recommended, unless you have used some very special MS-compatible silver stain. (In this case I want manufacturer name and catalogue number or full protocol. ) If not - use colloidal coomassie instead eg. "blue silver" or similar.

To add to the above post:

If you wish to analyse intact proteins, or peptides that are derived from your proteins (proteotypic peptides), you will generally be looking to purchase a column with a large pore size (> 150 Angstrom; I use 300 Angstrom).

If you wish to eventually move to 2D-LC, then you will need to be very careful in your selection of columns. I imagine you would combine a C18 or C12 (as suggested) with some form of cation/anion exchange. You might do well to look in to HILIC columns to achieve your goal - these are very interesting column types for biomolecules.

If you intend to screen for multiple proteins, I guess we can assume that you'd want to run a gradient LC method (in reversed phase mode). Make sure to start at a low, but not 0%, organic content. Perhaps start around 97% A and 3% B (A and B being either of the options suggested in the above post).

I'm unsure of the availability of MS/MS libraries for small & large molecules. The particle designs of different instruments mean that the relative intensity of ions often shifts, as may the fragments generated (traps versus triple quadrupoles). Tryptic digest libraries are probably a safe starting point.

All the best.