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Matt Padula
เข้าร่วมเมื่อ 24 ก.ค. 2016
The channel is dedicated to content delivery for the subject 91572: Proteomics, which is part of the Masters in Medical Biotechnology degree in the Faculty of Science, University of Technology Sydney.
Matt Padula, is an Associate Professor in the School of Life Sciences and Director of the Proteomics, Lipidomics and Metbolomics Core Facility, providing high level technical support and the instrumentation required for researchers to perform their research projects without having to spend long periods of time learning the complicated techniques and instruments needed to answer the scientific question they want answered. The Core is equipped with the knowledge, expertise and instrumentation to perform almost all proteomic, lipidomic and metabolomic experiments that could be of use to the faculty's researchers. In addition, Core staff have built relationships with other institutions and research groups to gain access to instrumentation and expertise not available within UTS.
Matt Padula, is an Associate Professor in the School of Life Sciences and Director of the Proteomics, Lipidomics and Metbolomics Core Facility, providing high level technical support and the instrumentation required for researchers to perform their research projects without having to spend long periods of time learning the complicated techniques and instruments needed to answer the scientific question they want answered. The Core is equipped with the knowledge, expertise and instrumentation to perform almost all proteomic, lipidomic and metabolomic experiments that could be of use to the faculty's researchers. In addition, Core staff have built relationships with other institutions and research groups to gain access to instrumentation and expertise not available within UTS.
วีดีโอ
Analytical Biochem Electrophoresis
มุมมอง 4223 ปีที่แล้ว
Everything about electrophoresis of biomolecules. Mainly focused on proteins, but DNA gets a good mention. Discusses agarose and polyacrylamide gels, separations by size (SDS-PAGE) and charge (isoelectric focusing), two dimensional separations, Native PAGE of protein complexes, gel staining and Western blotting.
Analytical Biochem MS Lecture Part 2
มุมมอง 1.1K3 ปีที่แล้ว
Part 2 of a series introducing students to how mass spectrometry works in the context of ionising biomolecules. This series contains freely available content from instrument manufacturers such as Thermo, Waters, Sciex, Agilent and we acknowledge their copyright.
Analytical Biochem MS lecture Part 1
มุมมอง 2.2K3 ปีที่แล้ว
Part 1 of a series introducing students to how mass spectrometry works in the context of ionising biomolecules. This series contains freely available content from instrument manufacturers such as Thermo, Waters, Sciex, Agilent and we acknowledge their copyright.
Analytical Biochem MS Lecture Part 3
มุมมอง 1.2K3 ปีที่แล้ว
Part 3 of a series introducing students to how mass spectrometry works in the context of ionising biomolecules. This series contains freely available content from instrument manufacturers such as Thermo, Waters, Sciex, Agilent and we acknowledge their copyright.
Mass spectrometry for proteomics - part one
มุมมอง 171K7 ปีที่แล้ว
Mass spectrometry for proteomics - part one
De novo peptide sequencing from mass spectrometry data
มุมมอง 36K8 ปีที่แล้ว
De novo peptide sequencing from mass spectrometry data
Mass spectrometry for proteomics - part 2
มุมมอง 91K8 ปีที่แล้ว
Mass spectrometry for proteomics - part 2
Searching databases for protein identification - part 1
มุมมอง 12K8 ปีที่แล้ว
Searching databases for protein identification - part 1
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
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.
Thanks ❤
I have a question about the difference in m/z values between peptides containing the C-12 or C-13 isotopes for isotopes - and this has driving me slightly insane for a bit because I encountered the same problem while reading a paper as well - at 5:08, when you go through the calculation of the m/z value for the peptide containing the C-13 isotope - why did you add the mass value of 1 Da corresponding to the one extra neutrino just once? Because this would mean that there is only one C-13 atom in the entire peptide, whereas, I thought all the carbon atoms in "isotopic peptide" would be the C-13 isotopes and therefore the mass of the "isotopic peptide" would be much larger due to the many extra neutrinos present there. Is my interpretation incorrect? When people refer to the peptide containing the C-13 isotope do they mean that there is only one C-13 carbon atom present in the peptide? Would be extremely grateful for a clarification, thanks!
In nature, C13 is ~1% of all of the carbon atoms available (that number isn't exact, but whatever). In a peptide, each carbon atom in the peptide has a 1% chance of being C13. A peptide has 10-1000's of carbon atoms depending on its length and the chance of one of them being C13 is 1% for each position, so it is highly likely that a single molecule of a peptide has at least one C13. Because different molecules of the same peptide can have a C13 at different locations in the molecule, those molecules will have the same m/z in a MS1 scan. It would be extremely rare that all of the carbons in a single molecule of a naturally occurring peptide would be C13. Then you need enough of these molecules of exactly the same thing to detect. So you would detect the molecules with no C13's first because they are the most abundant, then those with one C13, then those with 2 C13's etc as the abundance of those molecules of the same peptide sequence get less and less.
Right, that makes a lot of sense, thanks for the context!
nice to learn about these "wet" lab things as well, thanks! wondering what are the downstream impact on LC MS MS IDs? Is there an review article on what kinds and amounts of proteins one gets with different reagents and workflows in general?
The short answer is that anything in your sample that is NOT a peptide is going to affect your ID numbers and quantitative accuracy. Lipids often ionise better than peptides. I am not aware of a comprehensive review article about all of this. There is a good one about detergents (that I can't find right now). The other excellent resource is this: github.com/jessegmeyerlab/proteomics-tutorial. However, it does contain some omissions (like sulfonate based stage tips).
@@MatthewPadula thanks again for sharing which looks like a wonderful resource! i will surely take a look, meanwhile could these "impurities" be the reason that we see not-so-great correlation between DIA and DDA ID and Quant values?
@@AnimeshSharma1977 No. That's a different problem based in the way each of those methods acquire the data. The sample being analysed is almost always prepared in the same way.
Great video🫢
This is SUCH. A. GOOD. VIDEO!
The lecture was incredibly informative and clear.
Physical Biochem exam today, you are a saviour dude :D
HOW DID YOU IDENTIFIED THESE ARE ISOTOPES OF CARBON
Super great! Thank you very much. It is very helpful to me, a beginner of MS user
like and subscribed 👍
Thank you so much! I needed this for my uni course.
Great video, really nice and understandable introduction.
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?
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?
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+.
Bloody legend mate
This guy is extremely good, super kind.. it is such a huge sacrifice to be able to dispense such finer details to the seekers of this knowledge. I am so grateful to you, Sir. I wish you good health and long life and a happy family and a tranquil working atmosphere wherever you are; you are a great treasure to me and many more. You are the best I have ever encountered here.
Thanks for the kind words. I'll use them on the next promotion application. :)
You're a legend! This is currently helping me with my mass spec lab in my biochemistry degree 💪🏽📊
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.
Hi, thanks for the great lecture series. Could you share the link to the interactive learning module for ETD at 27:20?
It's a flash animation. If you send me an email, I'll send you the file.
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!
link.springer.com/article/10.1007/s13361-016-1341-0 - see scheme 6
@@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
@@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.
Thank you so much, I definitely owe you one 👏🙌
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
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.
@@MatthewPadula Hi, Please i urgently need help, where do i find your email?
Its fine found it, I am going to send you an email right now thanks a lot!!!
@@FunTime-hd4zz searching my name and "UTS" in Google is a good start.
Really appreciate it, I have sent the email! 🙂
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.
Excellent video, thanks very much!
brilliant lecture! thank you so much!
Can you please name that 2010 paper?
Can you be more specific?
@@MatthewPadula I wanted to read the paper at 13:20 but I could not find it on the internet.
@@leoalmeida2583 Nesvizhskii, A. I. (2010). "A survey of computational methods and error rate estimation procedures for peptide and protein identification in shotgun proteomics." J Proteomics 73(11): 2092-2123.
@@MatthewPadula thank you very much. I started my journey into proteomics and your channel has been very useful.
@@leoalmeida2583 Excellent. Don't be afraid to ask more questions. Either here or via email.
The Thermo video (th-cam.com/video/KjUQYuy3msa/w-d-xo.html) is no longer available.
Remind me where this is referred to in the video.
@@MatthewPadula 20:17
@@SethCrosby Thanks. I will fix it for the next version.
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.
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.
Best explanation I saw. Congrats!
Which software you have used for matching the fragment ion of peptide?
They are mainly manually annotated. Otherwise, I have used Peaks Studio since v4.5 (~10+ years).
Thanku
pls share the Karen Jhon Shaw link
Helping a ton for my proteomics course final! Thanks!!
This was very helpful! Thank you!
Could you pls sometime, if possible, run a sample and optimize the Mass Spectrometry conditions. Basically develop a method. That would be the best.
Yes. I am working on this. It is a huge subject and I need to decide what is important. And it needs to be focused differently for peptides compared to proteins compared to metabolites compared to lipids.
Is there a resource preferably videos for beginners to learn exhaustively.
Other then the rest of the videos here? It depends what you want to know specifically.
Thanks Matthew for this very nice and comprehensive lecture series. I learned a lot. It would be great if you could paste the links to other sources that you mentioned in your videos down in the description. This makes it easier to access the other videos.
I'll try and get it done once marking is finished. Keep reminding me.
Really enjoying your videos! Thank you for uploading these; such high quality lectures and slides.
This is a really good lecture. Thank you very much!
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.
Great...
Do you do shotgun proteomics? If so can I ask you a few questions about the instruments for this purpose?
Yes. You can email me if you like. Search 'UTS Matt Padula' and you will find my staff profile and email address.
@@MatthewPadula Thank you very much. I sent you the email with the questions.
From the two instruments, you showed at the beginning (Q Exactive Plus and Synapt XS), which one is better for shotgun proteomics? (The type of proteomics when you want to identify as many proteins as possible from a sample...)
A comprehensive overview of Mass spectrometry. Good job Matthew.
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!
Specta is plural. One is a mass spectrum.
thanks so much
thanks for sharing bro