De novo peptide sequencing from mass spectrometry data

[@TJ-do4wt]

You're a legend! This is currently helping me with my mass spec lab in my biochemistry degree 💪🏽📊

[@jingjingxu8845]

Super great! Thank you very much. It is very helpful to me, a beginner of MS user

[@mamarain8225]

This saved my skin, thank you so much! Succinct and easy to understand.

[@niloofarfam4294]

This video was really a life-saver. Thank you!!!

[@xuejie5507]

This Video is extremely helpful, thank you so so much!

[@둥글-s2y]

Thank you for your kind explanation :) This is very helpful

[@acell1618]

Thanks a lot for these videos. However, a doubt remains. The peptic fragments are just being blasted with inert gas to break them down into b and y ions. Where from are the extra 1 or rather 2 H+ at the N terminal of 'y-ions' coming from, giving them overall +1 charge?

[@MatthewPadula]

The peptide ions are accelerated into the collision cell and collide with the inert gas molecules, which causes the fragmentation. There are no extra H+. The peptide starts out 2H+ and the fragments are 1H+.

[@isabelacopetti404]

This was very helpful! Thank you!

[@TheMistakentruth]

Just the information i needed! Thank you sir :)

[@doudna2020]

when digest with trypsin, it cuts at the c terminal side of Arg and Lys, so the Arg and Lys should be at the terminus of b ion, isn't it. I don't get how is it at the y ion? Could you please explain

[@MatthewPadula]

This isn't to do with trypsin. If the detected fragment is from the C-terminal end of the peptide, it is a y-ion. If the detected fragment is from the N-terminal end, it is a b-ion. This applies to ALL peptides, no matter if generated from an enzyme cutting a protein or if the peptide is endogenous.

[@arjentenhave8961]

Excellent!

[@axalnas15]

this is really informative video, thank you so much, can we have the ppt version i want to study it as well

[@lucab012]

This was very helpful but I still have one question. I don't get why the relative intensities of complementary b and y fragments are different, like b2 and y10 in your example. I mean, when the parent ion fragments on a specific bond you get the same amount of the two complementary fragments, so I really don't get this. could you help me please?

[@ucrjc]

Why and how does the quadrupole select only C-12 isotope?

[@MatthewPadula]

The quadrupole isolation width determines this and the user sets it. Most instruments for DDA are set to 1.4 m/z, which is 0.7 m/z either side of the C-12 isotope. This lest the C-13 ion through to increase the number of fragments and thus MSMS fragment intensity. In DIA, the isolation width is typically 25 m/z but can be variable.

[@ucrjc]

@@MatthewPadula Thank you and a follow-up question is that I noticed other than the y and b ions you had Z, X, and A what do those represent?

[@MatthewPadula]

@@ucrjc They are fragments produced through breaking the peptide through different bond positions. See here: www.ionsource.com/tutorial/DeNovo/full_anno.htm

[@ucrjc]

@@MatthewPadula OH! Right! Thank you so much!

[@AnniePrettyFace]

Hey sir! Thanks for the help- question I had is how would you draw a simple mechanism for the y and b fragmentation? How do both the nitrogen np oxygen become positively charged? Thanks for the vid!

[@MatthewPadula]

link.springer.com/article/10.1007/s13361-016-1341-0 - see scheme 6

[@erikhenze2339]

@@MatthewPadula How does that scheme account for the first b ion? according to that you would never get a N-terminal single amino acid, would always have at least two side-chains incorporated

[@MatthewPadula]

@@erikhenze2339 b1 is the singly charged parent ion mass minus the largest y-ion. With CID you probably will see b1 but not a lot more of the b-series. This depends on the amount of peptide. I don't understand about the side-chains comment.

[@FunTime-hd4zz]

I have a questions I did all this, b ions I got sequence of RYAAYKYA and for y ions I have the sequence of IKAYKYAA I am not sure what's my final sequence going to be

[@MatthewPadula]

You did all of what? Is it a tryptic peptide? If so, it should end in K or R. You should probably email me with more detailed info.

[@FunTime-hd4zz]

@@MatthewPadula Hi, Please i urgently need help, where do i find your email?

[@FunTime-hd4zz]

Its fine found it, I am going to send you an email right now thanks a lot!!!

[@MatthewPadula]

@@FunTime-hd4zz searching my name and "UTS" in Google is a good start.

[@FunTime-hd4zz]

Really appreciate it, I have sent the email! 🙂