Reductive dimethylation labelingq

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edelman
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Reductive dimethylation labelingq

Postby edelman » Sat Feb 02, 2013 2:05 pm

Hey all,

I am experiencing a loss of half of my unique peptide identifications after I chemically label with reductive dimethylation protocol. My question is what side reactions/ other mods should I search for to find the missing peptides? Any advice would be greatly appreciated.!

Thanks!

Bill

I typically use t 5mg of peptides for each label in separate in-solution reactions:

Light Reaction

+ 200 ul 4% d0-formaldehyde
+ 200 ul 600 mM NaCNBH3
Heavy Reaction

+ 200 ul 4% d2-formaldehyde
+ 200ul 600 mM NaCNBD3
Add formaldehyde, mix well by flicking the tube and incubate for 2 mins. Then add NaCNBH3/D3, mix well and incubate for 10 mins.
Repeat the previous step but incubate for 1 hr.
Quench with 10% TFA/FA to pH of 3-4 (ca. 5 ml of 10% TFA/FA)
Incubate at low pH for 1 hr (bubbles may occur). Large volumes should be quenced in the hood due to cyanide gas formation

Desalt on appropriate SEP-PAK for the total peptide amount (here it is 10 mg so use at least a 200 mg resin).

hodi
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Postby hodi » Tue Feb 05, 2013 2:43 pm

It depends on your experimental workflow and LC-MS analysis, but couldn't it be just because of the doubling of complexity and thus undersampling? A typical problem of non-isobaric labeling techniques such as dimethyl, SILAC and ICPL.
Assuming you have two "real" and complex samples which are typically >90% equal, after pooling you will have each peptide in two forms which will be selected for MS/MS and then the number of unique peptide IDs will decrease.

edelman
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Postby edelman » Tue Feb 05, 2013 9:50 pm

That is true, however, I did intend to restrict my question to one label. So, in this example I am not mixing. I'm only speaking of labeling one sample as light or heavy and analyzing it.

odedk
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Postby odedk » Wed Feb 06, 2013 4:41 am

Hi,
I am not sure what volumes you are using hence what are the final concentrations of the formaldehyde and NaCNBH3.
Some issues might be related to the digest itself was generated (i.e buffer, pH, leftovers of DTT, etc): we have seen odd results few times when the dimethylation was done after directly after Cys alkylation which seemed to be related to the presence of IAA and since than we quench the IAA with extra DTT prior to the labeling with FA and that seems to solve this issue.
I never quenched the labeling with TFA/FA alone but we always need to first quench with large excess of primary amines (i.e glycine or just Ammonium bicarbonate) and then acidify for desalting.
[not directly related to dimethylation itself we also had some issues with C18 Seppak that led to sample losses (quite severe losses every now and then) so if possible we look for alternatives (Stage tips etc)]
Somehow related to the point raised before - do you actually observed less spectra or it is more of a decrease in the number of identified peptides? if so it might be related to your search parameters (i.e fixed vs variable diMet etc).

edelman
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Postby edelman » Wed Feb 06, 2013 9:28 pm

Odedk, thanks for your reply. In the worst case the volume was 5mL with the above volumes. Those peptides that I did find were almost 100% labeled, so the efficiency was great. These peptides were reduced alkylated digested and desalted on C18 columns, then split into non-labele and labeled samples. So until labeling they were treated equally. So I think the DTT and IAA are the least concern. I do think you are onto something, with the ammonium, I should try this. Also, I do retain the same amount of spectra from run to run. I think will try this and post.

Infinity
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Postby Infinity » Thu Feb 07, 2013 3:47 pm

I was using very nice protocol from this paper: http://www.ncbi.nlm.nih.gov/pubmed/19300442
In fact it is quite convenient to perform labeling while your peptides are retained on C18 column, - you will not have to perform desalting after and it works really good. Just follow their protocol.


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