In solution

[amandine.viau]

Hi,

I need some help!
I want to identify the glycans that are bound to my protein of interest. For this, I need to have my protein as pure as possible without SDS nor salt because I want to digest with PNGase in solution. I work with mouse tissue and I immunoprecipitate my protein with protein G cross-linked to my antibody to avoid Ig in my sample. The problem is that I tested several elution buffer: SDS elution (with boiling), citrate elution pH3 (2 elutions of 10min at RT) and glycine elution pH2.5 (2 elutions of 10min at RT). The best is SDS with boiling.
Because SDS is not really compatible with mass spec how can I remove the SDS from my sample without loosing all my protein?
thanks for your help.

[proteaPam]

Hey amandine.viau,

I have had some experience with PNGaseF and glycans. I would run your protein of interest on an SDS-PAGE, perform in-gel Trypsin digestion and extraction. If you have enough sample, I would split your sample in half. a) One half I would clean up with C18 SpinTips for mass spec analysis. b) The other half of the sample I would incubate with PNGase F, clean up with C18 SpinTips for mass spec analysis.

If you don't have enough sample to split in half I would just do the latter recommendation "b" to identify your glycans.

I hope this helps and Good Luck!

Pamela Williams
Laboratory Technician at Protea Biosciences, Inc.
proteabio.com

[proteaPam]

Hey amandine.viau,

I have had some experience with PNGaseF and glycans. I would run your protein of interest on an SDS-PAGE, perform in-gel Trypsin digestion and extraction. If you have enough sample, I would split your sample in half. a) One half I would clean up with C18 Tips for mass spec analysis. b) The other half of the sample I would incubate with PNGase F, clean up with ZIC-HILIC tips for mass spec analysis.

If you don't have enough sample to split in half I would just do the latter recommendation "b" to identify your glycans.

I hope this helps and Good Luck!

Pamela Williams
Laboratory Technician at Protea Biosciences, Inc.
proteabio.com

[amandine.viau]

Hey Pamela,

Thanks for your quick reply. I'm sure your solution is the best one but the first problem is that I work on tissue. To see my protein in silver staining I need to immunoprecipitate 2mg of total protein extract. With 5mg, I don't even see my protein in coomasie staining.... I can overcome this problem by cutting an invisible band at the MW expected.
The other problem is that the guy whith whom I collaborate for mass spec wants to digest my sample with PNGase directly after immunoprecipitation and then analyzed only the glycans. The problem with that is that to elute my protein I need SDS and to analyse by mass spec I need to avoid SDS. The other problem will be that I will probably eluate also all the proteins that interact with my protein of interest.....
Thanks for your help.

Amandine

[proteaPam]

Hey Amandine,

I recommend trying Protea’s anionic acid labile surfactant (AALS surfactant cat#AALS-100) as an alternative to SDS for your IP elution buffer (if your elution buffer can have a pH of 6-11). AALS is a degradable surfactant that is easily degraded after IP by changing the pH to 2-3 using TFA or formic acid. The surfactant breakdown products can be easily removing by reversed phase SPE or ultrafiltration devices, after which the proteins can be digested with trypsin and PNGase prior to LC-MS.
Alternatively, you can try acetone precipitation of the IP Eluate to pellet–out the proteins. This is effective in removing the bulk of the SDS for successful digestion with trypsin and PGNase, and also allowing MS analysis.

I hope this helps and Good Luck!

Pamela Williams
Laboratory Technician at Protea Biosciences, Inc.