Very easy to follow along and clear overall material presented in the video. Thank you very much, it is a pleasure to learn with you explaining these novel to me concepts.
You are such a life-saver! I am doing the final thesis for my master course and I am having the worst tutor/mentor ever. It is too late and too risky for me to change the mentor now. Your videos really help me a lot. Hope that in a year, I can have everything sorted out.
You should have a long discussion with your tutor about what you need from a mentor because they need to learn as well. However, you can more than one mentor. I have had several throughout my career as our situations have changed and have had different mentors for different aspects of my career.
your explanatory videos are ultra helpful! I am a masters student in biochemistry lab and these now form the basis of my understanding of MS. I have a question however: Why do we subtract another 18 amu (a H2O molecule) from the intact peptide in addition to the largest b ion in order to determine the first (and why only the first?) aa from the carboxyl-terminal (lys/arg), as in: intact - b3 - 18 = lys/arg ? instead of: intact + 18 = b3 + lys/arg => intact + 18 - b3 = lys/arg ? Aren't we referring to the water molecule that breaks the peptide bond between b3 and lys/arg or am I missing something ?
The water is lost from what becomes the y-ion (whichever y-ion) during fragmentation and rearrangement in the gas phase. Because there are thousands of peptide molecules, a collection become y1, another collection become y2, another collection become y3. But when we are determining sequence, each of those collections has lost water so the mass difference between y2 and y3 is the amino acid mass.
@@mattpadula837 thank you for the quick and enlightening response! You really help me a lot with your top quality videos. Congratulations for your research output too!
I thought I mentioned a cool guide/pdf by Karen Jonscher, which has a "cheat sheet" in the back of the de novo sequencing rules. If you can't find it by Googling, send me an email and I will send it to you.
at 7:00, you say that the difference between 1337.5 and 1276.5 is lesser than 57, hence its not a y ion and therefore a b ion. But, the difference between them is actually 61. Hence the reason why you took it as a b ion is because there is no amino acid with a weight of 61 right?
61 is not the mass of any amino acid so it is not part of the series that was trying to be deciphered. It may be part of the other series, sequencing the peptide from the other termini, but this would have to be checked. In this example, there are no modifications or water losses or ammonia losses from fragmentation, but they could be confounding in a more complicated example.
Leucine and Isoleucine are isobaric and cannot be differentiated using MS. When de novo sequencing a peptide one merely has to assume it could be either.
Hi Matthew, If the spectra is from a doubly charged mass precursor, do we look for the masses below the precursor mass or true mass of the precursor? If the precursor was 650 (doubly charged) - should i look for masses below 650 Da or nominal mass of 1300 Da?
Do determine the sequence of the amino acids, you start from the true mass of the precursor. For the large b or y ions, look for peaks that are 57 - 186Da below the precursor's singly charged mass.
Thanks for that. Just to clarify, so in the video where you use the spectra from 1449.38 precursor, you said it was doubly charged. Does this mean the actual ion fragmented was 724.69 (+2), am I correct?
A poor pause in my speaking. A quadrupole TOF instrument has two quadrupoles in series (actually more, but lets not get confusing). Quadrupoles are excellent at selecting all of the molecules of a single m/z (ion) and transmitting them to another part of the instrument, in this case the next quadrupole. All others ions exit the quad out the sides and are sucked out by the vacuum system. The selected ions of the same m/z then enter the second quadrupole which is the collision cell, where the ions are accelerated and collide with gas molecules (typically nitrogen) that has been pumped into the collision cell. The ions fragment and the fragments transmitted to the TOF analyser to measure their mass. Answer the question?
I haven't gone looking for courses online, focusing mainly on the online teaching for our coursework students. I can recommend Sciex University for mass spectrometry based information. Also, Ben Orsburn's blog is an awesome place to keep up to date (proteomicsnews.blogspot.com/). I am slowly updating the videos here for next semesters teaching, so keep an eye out. Also, if there are specific questions you need answering, just contact me.
I use Peaks Studio from Bioinformatics Solutions to get initial sequences from the MS2 scans. It is very good, but has problems when there are too many modifications. There is no substitute for a print out and a calculator.
Hello, thank you for the video, I learned a lot. but in th-cam.com/video/L6MHSb7I820/w-d-xo.html , the reason that the second peak is not for an amino acid is that its difference from the first peak is 61 and it is between the mass of Gly (57) and Ala (71), however, you said that its difference is less than 57.
I'll have to fix that when I get around to updating this. There is a newer version in the Analytical Biochem playlist that hopefully doesn't have the same mistake.
Helping a ton for my proteomics course final! Thanks!!
This is SUCH. A. GOOD. VIDEO!
It's too wonderful and helpful. It's the time when I need. Thank you so much.
Very easy to follow along and clear overall material presented in the video. Thank you very much, it is a pleasure to learn with you explaining these novel to me concepts.
You are such a life-saver! I am doing the final thesis for my master course and I am having the worst tutor/mentor ever. It is too late and too risky for me to change the mentor now. Your videos really help me a lot.
Hope that in a year, I can have everything sorted out.
You should have a long discussion with your tutor about what you need from a mentor because they need to learn as well. However, you can more than one mentor. I have had several throughout my career as our situations have changed and have had different mentors for different aspects of my career.
Hello Matthew,
amazing video!!! Thank you for sharing
love u mathew i wish u keep posting the stuff
Thank you, i understand very very well! You save me before my exam
your explanatory videos are ultra helpful! I am a masters student in biochemistry lab and these now form the basis of my understanding of MS. I have a question however:
Why do we subtract another 18 amu (a H2O molecule) from the intact peptide in addition to the largest b ion in order to determine the first (and why only the first?) aa from the carboxyl-terminal (lys/arg), as in:
intact - b3 - 18 = lys/arg ?
instead of: intact + 18 = b3 + lys/arg => intact + 18 - b3 = lys/arg ?
Aren't we referring to the water molecule that breaks the peptide bond between b3 and lys/arg or am I missing something ?
The water is lost from what becomes the y-ion (whichever y-ion) during fragmentation and rearrangement in the gas phase. Because there are thousands of peptide molecules, a collection become y1, another collection become y2, another collection become y3. But when we are determining sequence, each of those collections has lost water so the mass difference between y2 and y3 is the amino acid mass.
Watch the de novo sequencing video for a better explanation.
@@mattpadula837 thank you for the quick and enlightening response! You really help me a lot with your top quality videos. Congratulations for your research output too!
Very helpful ! Thank you so much
Thanks ❤
At 3:42, you said “ in electrospray , protein an d peptide having multiple (what?) can be protonated,” , thanx
multiplied amines?
multiple amine groups where hydrogen ions can be attached to the molecule.
Great video🫢
I wonder how you know if you need to subtract a water molecule or not from the fragment.
I thought I mentioned a cool guide/pdf by Karen Jonscher, which has a "cheat sheet" in the back of the de novo sequencing rules. If you can't find it by Googling, send me an email and I will send it to you.
Correction: Phenylalanine is 147. The largest mass amino acid is Tryptophan with the mass of 186.
is this for a correction at 6:17? I was wondering where he got the AMU lesser 1440
at 7:00, you say that the difference between 1337.5 and 1276.5 is lesser than 57, hence its not a y ion and therefore a b ion. But, the difference between them is actually 61. Hence the reason why you took it as a b ion is because there is no amino acid with a weight of 61 right?
61 is not the mass of any amino acid so it is not part of the series that was trying to be deciphered. It may be part of the other series, sequencing the peptide from the other termini, but this would have to be checked. In this example, there are no modifications or water losses or ammonia losses from fragmentation, but they could be confounding in a more complicated example.
Hi, where can I find the original figure you use at 3:02?
I think its from one of Luis' presentations. I'll have to hunt for it.
brilliant lecture! thank you so much!
I have a question. How can you differentiate leucine from isoleucine using this mass spectrum?
I don't think you can. I think that's one of the limits of MS.
Leucine and Isoleucine are isobaric and cannot be differentiated using MS. When de novo sequencing a peptide one merely has to assume it could be either.
Specta is plural. One is a mass spectrum.
Hi Matthew, If the spectra is from a doubly charged mass precursor, do we look for the masses below the precursor mass or true mass of the precursor? If the precursor was 650 (doubly charged) - should i look for masses below 650 Da or nominal mass of 1300 Da?
Do determine the sequence of the amino acids, you start from the true mass of the precursor. For the large b or y ions, look for peaks that are 57 - 186Da below the precursor's singly charged mass.
Thanks for that. Just to clarify, so in the video where you use the spectra from 1449.38 precursor, you said it was doubly charged. Does this mean the actual ion fragmented was 724.69 (+2), am I correct?
What do you mean by Quadrupole one in the isolation step in 2:37?
Oh it is clear you explained it in the first part thank you these are very helpful videos
A poor pause in my speaking. A quadrupole TOF instrument has two quadrupoles in series (actually more, but lets not get confusing). Quadrupoles are excellent at selecting all of the molecules of a single m/z (ion) and transmitting them to another part of the instrument, in this case the next quadrupole. All others ions exit the quad out the sides and are sucked out by the vacuum system. The selected ions of the same m/z then enter the second quadrupole which is the collision cell, where the ions are accelerated and collide with gas molecules (typically nitrogen) that has been pumped into the collision cell. The ions fragment and the fragments transmitted to the TOF analyser to measure their mass.
Answer the question?
Anyone notice how part 2 came out a year before part 1
Thank you for the explanation Matthew.
Could someone recommend a good online course for proteomics?
Thank you :)
I haven't gone looking for courses online, focusing mainly on the online teaching for our coursework students. I can recommend Sciex University for mass spectrometry based information. Also, Ben Orsburn's blog is an awesome place to keep up to date (proteomicsnews.blogspot.com/). I am slowly updating the videos here for next semesters teaching, so keep an eye out. Also, if there are specific questions you need answering, just contact me.
hey if you still need help let me know i am also student but i have great lectures may could be useful for you too
Thank you for this amazing tutorial. Do you know what software I can use to determine the sequence of MS2 as explained around 7:00?
I use Peaks Studio from Bioinformatics Solutions to get initial sequences from the MS2 scans. It is very good, but has problems when there are too many modifications. There is no substitute for a print out and a calculator.
Hello, thank you for the video, I learned a lot.
but in th-cam.com/video/L6MHSb7I820/w-d-xo.html , the reason that the second peak is not for an amino acid is that its difference from the first peak is 61 and it is between the mass of Gly (57) and Ala (71), however, you said that its difference is less than 57.
I'll have to fix that when I get around to updating this. There is a newer version in the Analytical Biochem playlist that hopefully doesn't have the same mistake.