Go Back   Science Forums Biology Forum Molecular Biology Forum Physics Chemistry Forum > Molecular Research Topics Forum > Proteomics Forum > Mass Spectrometry Forum
Register Search Today's Posts Mark Forums Read

Mass Spectrometry Forum Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


In solution

In solution - Mass Spectrometry Forum

In solution - Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


Reply
 
LinkBack Thread Tools Display Modes
  #1  
Old 03-17-2011, 10:04 AM
Pipette Filler
Points: 72, Level: 1 Points: 72, Level: 1 Points: 72, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2011
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default In solution



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.
Reply With Quote
  #2  
Old 03-17-2011, 05:40 PM
Pipette Filler
Points: 331, Level: 6 Points: 331, Level: 6 Points: 331, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2011
Location: Morgantown, WV
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: In solution

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
Reply With Quote
  #3  
Old 03-17-2011, 05:41 PM
Pipette Filler
Points: 331, Level: 6 Points: 331, Level: 6 Points: 331, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2011
Location: Morgantown, WV
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: In solution

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

Last edited by admin; 03-25-2011 at 06:39 AM. Reason: keyword spamming links removed
Reply With Quote
  #4  
Old 03-23-2011, 02:09 PM
Pipette Filler
Points: 72, Level: 1 Points: 72, Level: 1 Points: 72, Level: 1
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Mar 2011
Posts: 2
Thanks: 0
Thanked 0 Times in 0 Posts
Default Re: In solution

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
Reply With Quote
  #5  
Old 03-30-2011, 01:59 PM
Pipette Filler
Points: 331, Level: 6 Points: 331, Level: 6 Points: 331, Level: 6
Activity: 0% Activity: 0% Activity: 0%
 
Join Date: Feb 2011
Location: Morgantown, WV
Posts: 7
Thanks: 0
Thanked 1 Time in 1 Post
Default Re: In solution

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.
Reply With Quote
Reply

Tags
elution buffer , immunoprecipitation , mass spectrometry , sds , solution


Thread Tools
Display Modes

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off
Trackbacks are On
Pingbacks are On
Refbacks are On

Forum Jump

Similar Threads
Thread Thread Starter Forum Replies Last Post
question about making sodium ortho-vanadate stock solution Z.L. K Protocols and Methods Forum 4 10-20-2009 12:26 PM
Chemistry Acid-Base, Oxidation-Reduction Problems? Jellybean1 Chemistry Forum 0 10-25-2008 10:18 PM
Cylindrical wave, wave equation, and mistakes h_v_ansari@yahoo.com Physics Forum 2 10-26-2006 01:47 AM
probleme logique en equilibre acide base moruet Forum De Chimie 1 02-10-2006 09:11 PM
Sci.chem FAQ - Part 6 of 7 Bruce Hamilton Chemistry Forum 0 01-15-2004 09:12 AM


All times are GMT. The time now is 06:24 AM.


Powered by vBulletin® Version 3.8.4
Copyright ©2000 - 2014, Jelsoft Enterprises Ltd.
Copyright 2005 - 2012 Molecular Station | All Rights Reserved
Page generated in 0.15389 seconds with 16 queries