Valines being reported as crosslinked by BS3?

[Vincent Groenewold]

Hi all,

I've got some nice results for the crosslinking of hemoglobin. However, some crosslinks with high confidence and good sequence coverage, are reported to be between 2 Valines. To my understanding, this is not possible when using BS3. Any thoughts on this?

Thanks, Vincent Groenewold

[Verena T.]

Hi,

I also get these results within hemoglobin. But it is always the first AA (V1), so I thought about the N-Terminus. In search definitions I always definde K and N-Terminus to be cross-linkable.

Which Valine you receive?

Best,
Verena

[Vincent Groenewold]

I can't actually believe I didn't think of the N-terminus. :) In my current data it seems like it's a hotspot for cross linking haemoglobin. VLSPADK is one with a very high confidence and occurrence rate. Is that the same you're getting? And it's cross linked to the same location as well, meaning it should be linked to the B chain of HBA. Funny enough with my crystal structure, those two are 35 Angstrom apart (I use 25 as a maximum), but I guess that could very well mean that in solution these parts were both 5 Angstrom closer to each other, which seems very reasonable to me.

Thanks! Vincent

 

[Bing]

Hi Vincent,

Do you get this result from pLink with the setting of cross-linker as BS3? I think it is strange, pLink should not show this kind of result. On the other hand, you can use pLabel, a powerful spectrum labeling tool to manually validate the identification of this spectrum. Maybe, there is another explanation of this spectrum.

Best
Bing 

[Vincent Groenewold]

Hi Bing,

Yes, when I've set it up using the BS3 setting I get them. But if it's the N-terminus, it makes perfect sense right? There's a free amine group there as well, so any amino-acid would be found then.

Thanks, Vincent

[Bing]

Hi Vincent,

Yes, It makes sense if they are N-terminus.

[Matthew Makowski]

Hi All,

Hopefully this isn't a silly question, but I've also been seeing similar results to this, in my case for cross-links with the second residue in my protein (proline) which, I guess, plink is treating as n-terminal. Just to be clear, does plink automatically consider n-terminal tryptic peptides (n-terminal to the protein) with cleaved methionine?

Thanks for your help,

Best,

Matthew