Raffi Hovasapian

Sequencing Larger Peptides & Proteins

Slide Duration:

Table of Contents

Section 1: Preliminaries on Aqueous Chemistry

Aqueous Solutions & Concentration

39m 57s

Intro

0:00

Aqueous Solutions and Concentration

0:46

Definition of Solution

1:28

Example: Sugar Dissolved in Water

2:19

Example: Salt Dissolved in Water

3:04

A Solute Does Not Have to Be a Solid

3:37

A Solvent Does Not Have to Be a Liquid

5:02

Covalent Compounds

6:55

Ionic Compounds

7:39

Example: Table Sugar

9:12

Example: MgCl₂

10:40

Expressing Concentration: Molarity

13:42

Example 1

14:47

Example 1: Question

14:50

Example 1: Solution

15:40

Another Way to Express Concentration

22:01

Example 2

24:00

Example 2: Question

24:01

Example 2: Solution

24:49

Some Other Ways of Expressing Concentration

27:52

Example 3

29:30

Example 3: Question

29:31

Example 3: Solution

31:02

Dilution & Osmotic Pressure

38m 53s

Intro

0:00

Dilution

0:45

Definition of Dilution

0:46

Example 1: Question

2:08

Example 1: Basic Dilution Equation

4:20

Example 1: Solution

5:31

Example 2: Alternative Approach

12:05

Osmotic Pressure

14:34

Colligative Properties

15:02

Recall: Covalent Compounds and Soluble Ionic Compounds

17:24

Properties of Pure Water

19:42

Addition of a Solute

21:56

Osmotic Pressure: Conceptual Example

24:00

Equation for Osmotic Pressure

29:30

Example of 'i'

31:38

Example 3

32:50

Biochemistry Sequencing Larger Peptides & Proteins

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Section 2: Amino Acids & Proteins: Primary Structure: Lecture 6 | 1:02:33 min

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	0 answers Post by Michael De La Rosa on November 20, 2021 Hello Prof. Hovasapian. Great Lecture. I am a little confused about what the actual wet-lab procedure for sequencing long chains of Amino Acids is. According to my understanding of your lecture the steps are as follows: 1. Break the Disulfide bonds 2. Fragement the long chain with one of the hydrolyzing reagents (enzyme or chemical) 2.1 Sequence the fragments with Edmon degradation 3. Fragment a copy of the long chain with a different hydrolyzing reagent 3.1 Sequence the fragments with Edmon degradation 4. Treat Fragments with FDNB 5. Combine information and deduce sequence of Amino Acid chain Now, what is confusing me, is step 4. If we treat the solution of the A/A fragments with FDNB, wouldn't that label all the N-terminal A/As of all the fragments? Towards the end of your lecture you walkthrough a practical application of this sequencing method, however, somehow you've managed to only label the first A/A of the chain with FDNB? This deviates from what I think you said was the order of sequencing, earlier in the lecture. Can you help me sort out the real-life wet lab procedure? Thank you.
	1 answer Last reply by: Professor Hovasapian Mon Jan 7, 2019 6:24 AM Post by Anthony Villarama on January 5, 2019 Professor Raffi, unfortunately, this lecture did not do a good justice. I was so confused.
	3 answers Last reply by: Professor Hovasapian Mon Jan 15, 2018 5:26 AM Post by Swati Sharma on December 21, 2017 Dear Dr Raffi Could you please clarify t me the difference between separation of the fragments versus sequencing the fragment. I know that we can determine the sequence of the fragments versus Edmans, but what is the difference between 1) Separation of the fragments 2)Hydrolysis of the fragments 3)From the sequence of the fragments. Is Hydrolysis same as separation? Please let me know I am lost on this Respectfully Swati
	2 answers Last reply by: Swati Sharma Thu Dec 21, 2017 8:43 PM Post by Swati Sharma on October 4, 2017 So Dr Rafi I am still very much confused between the edmans degradation nd the sangers reagents the 2 4 dinitro benzene. So it goes like the following :first we take the intact protein, we break the disulfide bonds via perfromic acid then we unfold the protein and arrange it in a linear fashion. You said we can also treat the intact protein with the sangers reagent to determine the n terminal of the amino acid and that would be our first amino acid. We use 6 molar of HCL to get the free amino acids so that they are countable. Now after that depending upon the different types of amino acids present we use enzymes to cleave them and we get the fragments with that first enzyme. Then again we take the whole peptide and cleave it with the second enzyme and get the second fragment. Now I am confused here, you said we now determine the sequence( What do you mean by the term sequence ??) Does it mean order ? so we determine the sequence of amino acids in the fragments via Edmans Degradation , but I thought that Edmans degradation was also used to identify the N terminus of the amino acid by using phenylisothiocynate , so edmans is used for identifying the n terminus and 1 floro 2 4 dinitrobenzene is also used for identifying the n terminus? So basically what is the difference between Edmans degradation and ! Floro 2 4 Dinitrobenzene followed by 6M HCL Could you please clarify I am really confused after listening the video two times and taking notes I don't understand the difference, besides that I understand everything. Respectfully, Swati Could you please explain more briefly what do you mean by That Edmans is used to determine the sequence of fragments why not Sangers?
	2 answers Last reply by: Billy Jabbar Thu Mar 6, 2014 1:25 AM Post by Billy Jabbar on March 5, 2014 @Marsha I would imagine the Sanger Reagent (which chops up the entire peptide to individual amino acids) is incredibly wasteful and requires more product than Edman's Reagent (which removes only the first amino acid leaving the rest of the peptide intact). Since both help determine the same thing (the first amino acid of the sequence), if I had to guess -- I think most biochemists would opt with Edman's Reagant. --- Dr. H I have two questions regarding the reagants you mentioned for removing disulfide bridges. My first question is in regards to Performic Acid (a strong oxidizing agent) -- wouldn't it also oxidize the sulfur on methionine amino acids? Secondly, for the second reagant mentioned (the thiol reducing agent), would basically any mercaptan reagant work? My instructor frequently uses 2-Mercaptoethanol. Thanks!
	1 answer Last reply by: Professor Hovasapian Tue Sep 24, 2013 1:28 AM Post by Ziheng Wang on September 22, 2013 Sir, why don't we just do the Edman degradation to find out first say twenty elements instead of using Sanger's reagent?
	1 answer Last reply by: Professor Hovasapian Sat May 25, 2013 12:32 AM Post by marsha prytz on May 24, 2013 I'm slightly confused, if we are using the E. degredation to sequence the protein large or small, wouldn't that give us the sequence of the amino acid chain? Why would we need to break them up into fragments also?

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Sequencing Larger Peptides & Proteins

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Intro

Sequencing Larger Peptides and Proteins

Example: Sequencing Larger Peptides and Proteins

Intro 0:00
Sequencing Larger Peptides and Proteins 0:28

Identifying the N-Terminal Amino Acids With the Reagent Fluorodinitrobenzene (FDNB)
Sequencing Longer Peptides & Proteins Overview
Breaking Peptide Bond: Proteases and Chemicals
Some Enzymes/Chemicals Used for Fragmentation: Trypsin
Some Enzymes/Chemicals Used for Fragmentation: Chymotrypsin
Some Enzymes/Chemicals Used for Fragmentation: Cyanogen Bromide
Some Enzymes/Chemicals Used for Fragmentation: Pepsin
Cleavage Location
Example: Chymotrypsin
Example: Pepsin
More on Sequencing Larger Peptides and Proteins
Breaking Disulfide Bonds: Performic Acid
Breaking Disulfide Bonds: Dithiothreitol Followed by Iodoacetate

Example: Sequencing Larger Peptides and Proteins 37:03

Part 1 - Breaking Disulfide Bonds, Hydrolysis and Separation
Part 2 - N-Terminal Identification
Part 3 - Sequencing Using Pepsin
Part 4 - Sequencing Using Cyanogen Bromide
Part 5 - Final Sequence

Biochemistry Online Course

Section 1: Preliminaries on Aqueous Chemistry
	Aqueous Solutions & Concentration	39:57
	Dilution & Osmotic Pressure	38:53
	More on Osmosis	29:01
	Acids & Bases	39:11
	Titrations and Buffers	41:33
	Example Problems with Acids, Bases & Buffers	44:19
	Hydrolysis & Condensation Reactions	18:45
Section 2: Amino Acids & Proteins: Primary Structure
	Amino Acids	38:19
	Amino Acids, Continued	27:14
	Acid/Base Behavior of Amino Acids	48:28
	Peptides & Proteins	45:18
	Amino Acid Sequencing of a Peptide Chain	42:47
	Sequencing Larger Peptides & Proteins	1:02:33
	Peptide Synthesis (Merrifield Process)	49:12
	More Examples with Amino Acids & Peptides	54:31
Section 3: Proteins: Secondary, Tertiary, and Quaternary Structure
	Alpha Helix & Beta Conformation	50:52
Section 4: Proteins: Function
	Protein Function I: Ligand Binding & Myoglobin	51:36
	Protein Function II: Hemoglobin	1:03:36
	Protein Function III: More on Hemoglobin	1:07:16
Section 5: Enzymes
	Enzymes I	41:38
	Enzymes II	44:02
	Enzymes III: Kinetics	56:40
	Enzymes IV: Lineweaver-Burk Plots	20:37
	Enzymes V: Enzyme Inhibition	51:37
	Enzymes VI: Regulatory Enzymes	51:23
	Enzymes VII: Km & Kcat	54:49
Section 6: Carbohydrates
	Monosaccharides	1:17:46
	Hexose Derivatives & Reducing Sugars	37:06
	Disaccharides	43:32
	Polysaccharides	39:25
	Polysaccharides, Part 2	44:15
	Glycoconjugates	44:23
	More Example Problems with Carbohydrates	40:22
Section 7: Lipids
	Fatty Acids & Triacylglycerols	54:55
	Membrane Lipids	38:51
	Membrane Lipids, Part 2	38:20
	The Biologically Active Lipids	48:36
Section 8: Energy & Biological Systems (Bioenergetics)
	Thermodynamics, Free Energy & Equilibrium	45:51
	More on Thermodynamics & Free Energy	37:06
	ATP & Other High-Energy Compounds	44:32
	Phosphoryl Group Transfers	30:08
	Oxidation-Reduction Reactions	49:46
	More On Oxidation-Reduction Reactions	56:34
	Example Problems For Bioenergetics	42:12
Section 9: Glycolysis and Gluconeogenesis
	Overview of Glycolysis I	43:32
	Glycolysis II	1:01:47
	Glycolysis III	59:17
	Glycolysis IV	39:47
	Gluconeogenesis I	41:34
	Gluconeogenesis II	34:18
	The Pentose Phosphate Pathway	42:52
Section 10: The Citric Acid Cycle (Krebs Cycle)
	Citric Acid Cycle I	36:10
	Citric Acid Cycle II	49:20
	Citric Acid Cycle III	44:11
Section 11: Catabolism of Fatty Acids
	Fatty Acid Catabolism I	48:11
	Fatty Acid Catabolism II	45:58
	Fatty Acid Catabolism III	33:18
Section 12: Catabolism of Amino Acids and the Urea Cycle
	Overview & The Aminotransferase Reaction	40:59
	Glutamine & Alanine: The Urea Cycle I	39:18
	Glutamine & Alanine: The Urea Cycle II	36:21
	Amino Acid Catabolism	47:58
Section 13: Oxidative Phosphorylation and ATP Synthesis
	Oxidative Phosphorylation I	41:11
	Oxidative Phosphorylation II	36:27

Transcription: Sequencing Larger Peptides & Proteins

Hello and welcome back to Educator.com, and welcome back to Biochemistry.0000

Today we're going to continue our discussion of sequencing a peptide or a protein, and we're going to talk about how to sequence larger proteins, and it's exactly what you might think it is.0004

Basically, you just take a larger protein, and you chop it up into fragments, and then you deal with those individual fragments, and then you just arrange the fragments.0015

Let's just go ahead and jump on in.0025

OK.0028

Before we actually get into sequencing the larger peptides and proteins, I want to talk about one other method that's used to identify the N-terminal amino acid because it is going to come up.0030

Another method for IDing the N-terminal amino acid, and again I'll just use AA for amino acid.0046

OK.0065

Frederick Sanger, who worked out the amino acid sequence of insulin, developed a reagent to identify the N-terminal amino acid.0068

OK.0103

It is called 1-flouro-2,4-dinitrobenzine.0104

I tend to separate the words; you'll see it as one word, dinitrobenzene, small b, but I tend to separate it.0115

It doesn't really matter.0121

We will just refer to it as FDNB- fluorodinitrobenzene.0123

OK.0129

Here is the procedure.0130

We will take our amino acid.0136

Let's just go ahead and go, let's see H₃N⁺ we have C, C, we have that, and over here, actually you know what, I'll put the O a little bit better, and over here I'll just write peptide, because again, we're just going to be concerned with the N-terminal amino acids, this thing right here; and I'll just make it generic so I'll put an R₁.0142

So, what we do is we react this with our FDNB, which I'll go ahead and draw the structure.0166

Let me just write FDNB, and the structure is this: 1-flouro or dinitrobenzene 1-flouro 2, 3, 4, 1, 2, 3, 4, 5, 6, numbered carbons.0172

We react it with FDNB, and what you do is you basically just create a derivative of this, so this is like a label, just like what we did before.0193

You are just labelling the amino acids so you can actually identify it.0200

What you end up with is the following.0203

Let's go N, C, C.0205

I'll write peptide, and we have a carbonyl here.0211

We have our R₁ group; we have our H, and we have this right here.0216

I'll go ahead and put the NO₂ over here.0229

I just flip this one around.0233

Basically, you are just replacing this fluorine with the N-terminal amino acid.0236

That is what you're doing; it just acts as a label.0240

That is it.0243

Once you go ahead and form this, once you've actually labelled this, so let me go ahead and put a little circle around our amino acid, so that's our N-terminal amino acid, and then what you do is you go ahead and react this with 6M HCl to hydrolyze, to completely break apart every single bond just to free-up every amino acid; and then when you do that, what you end up with is, of course, the labelled terminal amino acid.0244

You are going to end up with this; you're going to have the R₁.0279

You have H, and then, of course, you have your label, so NO₂, and I'll just put the NO₂ over here plus free amino acids.0283

That is it- free amino acids.0296

When you use strong acids like that 6M HCl, you’re just going to completely hydrolyze the protein into all of its free amino acids.0298

OK.0308

And, let me write here again, 6M HCl breaks all the peptide bonds, and, of course, this thing right here, this is what we're going to be using, we just want to identify the N-terminal side.0310

That is it.0330

That is all this Sanger reagent does is it identifies the N-terminal amino acid.0331

OK.0337

You are probably wondering to yourself, well, why would we even bother with this if we can just go ahead and use the procedure that we used before, the Edman degradation, and just identify all of them and sequence them that way.0338

You will see why it's actually important in a minute when we start sequencing larger proteins.0348

OK.0354

Now, let's go ahead and start talking about sequencing larger proteins and peptides.0355

That is fine; I’ll go ahead and keep it in black.0361

Well, it might be nice if I actually wrote down the words properly.0369

How's that?0373

Sequencing larger peptides and proteins.0380

OK.0391

As we said, in the intro, these have to be broken down into smaller fragments, and these fragments are sequenced just like before with the Edman degradation.0393

Let's say you have a protein or a peptide and you chop it up into 4 fragments.0427

Well, you sequence those 4, well, you'll see the procedure in just a minute, but essentially what you do then, you have to decide which fragment is in front of which fragment; and we have some techniques to deal with ordering those.0433

That is all that's going on.0446

You are doing the same thing, except you're doing it for more fragments.0448

OK.0453

Not only do the...because the protein is a peptide, you have to break it up into smaller fragments, but you remember these disulfide bonds, the disulfide bridges that exist between the cysteine residues, they are actually covalent.0454

We have to break those because they interfere with the sequence processing.0465

We also have to break the disulfide bonds in a protein, and that is usually the first thing you do, you break the disulfide bonds then you start chopping up the protein.0469

OK.0489

Now, let's talk about the chopping up the protein part, and we'll get back to the disulfide bonds in just a minute.0490

There are enzymes called proteases which catalyze the breakage of peptide bonds next to specific amino acids.0497

So, the fragmentation process or the actual fragmentation is predictable and reproducible.0542

This is important- predictable and reproducible.0552

We use enzymes called proteases to actually take that really, really long protein or peptide, and chop it up into specific places; and depending on what enzyme we use, it actually cuts the peptide chain next to specific amino acids, maybe 1 amino acid, maybe 2 amino acids, but it does not mess with all of the other bonds, not like the 6M HCl does.0560

So, these proteases are perfect, and because they cut the peptide chain next to specific amino acids, they're reproducible and they're predictable, so we know exactly where they're going to be cut, and that's what's really nice.0587

OK.0602

In addition to enzymes called proteases, we also have some chemical methods that do the same thing.0604

Chemical methods also exist.0612

Chemical methods also exist for breaking peptide bonds at specific points, for breaking peptide bonds next to specific amino acids.0618

OK.0643

Now, as a reminder of what I just said, note again that these enzymes, chemicals, they break single bonds to create short fragments.0644

They do not hydrolyze the entire peptide.0682

They do not hydrolyze -that's a Y -the entire protein.0690

OK.0696

We are creating fragments, short peptides.0697

We are not just breaking it up into free amino acids like HCl.0699

OK.0704

Let's go ahead and list some of these.0705

Let me go ahead and do this one in red.0706

Some of the enzymes and/or chemicals - I'm going to do mostly enzymes, only 1 chemical - used for fragmentation, some enzymes and chemicals used for the fragmentation process.0711

OK.0735

We will go ahead and list the name, and we'll go ahead and list the cleavage point, in other words, where it actually breaks the peptide.0736

OK.0752

There is an enzyme called trypsin, and it breaks lysine and arginine C.0753

This enzyme trypsin, when we use that to break up a protein or a very large peptide, it will actually cut the peptide chain next to a lysine, next to an arginine.0763

I will explain what the C means in just a minute.0773

I just want to go through the list, and then I'll tell you where this tells us...well, I'll tell you in just a second.0776

Chymotrypsin, OK, I'll put enzyme so you know this is also an enzyme.0785

It breaks it up at phenylalanine, tryptophan and tyrosine next to the aromatic amino acids and C.0795

OK.0809

Cyanogen bromide, this is a chemical procedure, and it breaks it up next to a methionine, and last, pepsin.0812

It is an enzyme and it's a phenyl tryptophan tyrosine with an N.0831

Now, I'll tell you what the C and the N mean.0840

I will tell you what they are, then I'll go ahead and write them down.0843

Trypsin, when it cuts a peptide chain, it cuts it either next to a lysine or an arginine, but what it does is it cuts the peptide bond on the carbon side, the carboxyl carbon, in other words, to the right of this amino acid.0845

OK.0861

Now, notice this one right here, pepsin.0862

Pepsin phenyl tryptophan tyrosine N - this means that it actually cuts the peptide next to a phenylalanine, a tryptophan or a tyrosine, but it cuts the peptide bond to the left of these amino acids.0865

In other words, it breaks the N-terminal, the N side, the nitrogen side, right?0881

So, what you have is something like this.0888

What you have is some amino acid residue that looks like that.0889

This is going to go on on a peptide that way; this is going to go on on a peptide that way.0895

Let's say what we are dealing with is a phenylalanine.0901

Well, if I use trypsin, oh I'm sorry, chymotrypsin, and this is phenylalanine, it is going to break it up on the C side, so it is going to end up breaking that bond; and then we will have this fragment on the left and we will have this fragment, but if we use pepsin and it's phenylalanine, it is going to break that bond.0904

It is still next to a phenylalanine, but it is going to be phenylalanine the N side of phenylalanine.0926

That is all that means.0932

These Ns and Cs, it tells you which peptide bond that is actually breaking because you have 2 of them for each amino acid, the one on the right, the one on the left, the carboxyl, and the amino bond.0933

OK.0945

Let me just go ahead and write that down formally, go back to blue.0948

C, it means it cleaves the peptide bond at the C end of the amino acid, and N means it cleaves the peptide bond at the nitrogen end of the amino acid.0957

That is all that means, so either to the right, to the C end, or to the left of that particular amino acid.0997

OK.1004

Let's just do a couple of examples really quickly just to make sure we absolutely get this.1006

Let's say we have this following H₃N⁺.1016

Let's see, we have Ala, we have Gly, we have Phe, and Met, and Leu, and Leu, and Tyr, and Ala, and of course we have that.1023

Let's say we have this little peptide right here.1045

Well, if we use chymotrypsin - I'll write it down below - if I'll use chymotrypsin, well, I notice that the chymotrypsin is the one that cleaves next to the aromatics, the phenylalanine, the tyrosine and the tryptophan, so I look here and I notice that I have a phenylalanine and I have a tyrosine and it clips it on the carbon side of the peptide bond.1048

So, chymotrypsin is going to break the bond here; and it is going to break the bond there to the right- that one and that one.1080

It is going to clip it that way.1088

So, we are going to create 3 fragments: this fragment, the middle fragment, and this fragment- if we use chymotrypsin.1091

OK.1098

Now, let me go ahead and rewrite this.1099

This time let me write it in blue again.1102

H₃N⁺, we have Ala, we have Gly, we have Phe, we have Met, Leu, Leu, Tyr, Ala, and we have our final carboxyl group over there.1105

Now, if I use pepsin, let me go to red, well, pepsin does the same thing.1126

It still breaks it up next to the phenyl alanine and the tyrosine.1131

There is no tryptophan in here, but it breaks it up on the nitrogen side, on the left, the amino peptide bond.1136

So, we are going to break that bond, and we are going to break tyrosine to the left of that bond.1144

I have this fragment, and I have this fragment, and I have this fragment.1151

Again, 3 fragments, but I've snipped it in a different place.1154

That is all that's going on here.1159

OK.1161

Now, let's see.1164

OK, let's continue on.1166

OK.1171

Once we have a set of fragments whose sequence we have deduced by using the Edman degradation, so I break up my protein, I have my separate fragments, and then I go ahead and I sequence those fragments, once I know that, we use another hydrolyzing reagent, another enzyme, we use a different hydrolyzing reagent, in other words, an enzyme on that list or some chemical on that list, to create a different set of fragments with sequences we deduced.1175

Just like we did for the example a second ago, we used the chymotrypsin and then we used pepsin, we broke it up into separate fragments.1264

We do the first one, we break up those fragments, we sequence those 3 fragments, and then we use another enzyme or a chemical to get a different set of fragments, we sequence those, and we compare the two, and that is how we set up the order, and that's all we are doing.1276

Let me see whose sequences we deduced.1291

OK.1295

We then compare the 2 sets of fragments because now, we have to arrange the fragments in a given order to find overlap, and when we have overlap, thereby establishing the overall sequence, and I'll draw this out in just a minute.1299

This is actually really, really amazing that we can do this.1334

Let's say for example - let me do this in red - so, we have this whole peptide chain, so I’ll just write whole; let's say we use one of those chemicals on that list to hydrolyze this protein at specific points, to break it up.1338

Let's say we break it up into that fragment and then that fragment, and then maybe that fragment, and let's say that fragment.1351

OK.1362

Here we have 4 fragments, something like that, used in one of the enzymes or one of the chemicals on that list to break it up.1364

Now, we take this whole peptide protein; we do it again, but this time with a different hydrolyzing reagent, so maybe we end up with this.1376

We end up with that fragment, that fragment, and let's say maybe that fragment.1384

Now, we have 3 fragments.1393

Well, now, the thing is, I have a bunch of fragments, and I know the sequences of each of these fragments.1397

I know those sequences, but I don't know what order they go in.1403

I have to arrange the order.1406

What I do is I compare this with this, and I set up overlaps.1409

That's it.1416

Notice here, I have some overlap.1418

There is overlap there; I have here.1425

There is a bunch of overlap here and here.1427

OK.1430

Of course, here there is overlap between those two, this one and this one.1431

This overlaps these two; this one overlaps this, this, and this.1436

This one overlaps this and this.1440

By comparing and seeing what the overlap is because I know the sequences, I know where every amino acid is, I can establish which fragment goes where.1442

Does this one go first?1450

Does this one go first?1452

Now, that way I've drawn it, I've cut it in certain places; but once I’ve cut the fragments, they are just randomly ordered, the fragments are.1453

I have to see which fragment goes first, which fragment goes second.1462

That is all we are doing.1465

OK.1467

Now, using Sanger’s FTNB, like we talked about at the beginning of this lesson, we know the N-terminal amino acid.1469

That information, that info gives us our first fragment, gives us our first fragment.1499

In other words, let say I come up with these four fragments here, when I break it up with my first cleavage enzyme and let say I run a Sanger procedure on these fragments and I end up discovering that the N-terminal happens to be some amino acids, well, let’s say it ends up over here, well, that automatically tells me that this fragment is my first fragment.1511

That is the one that goes on the far left, so that's why this is important.1536

I hope that makes sense.1542

And again, if it doesn't, it's ok.1544

We are actually going to be going through a process, going through in detail what it is that we're actually doing here for a specific example.1546

OK.1555

Now, let's go back and talk about breaking the disulfide bonds, how we do that chemically, and then we'll go ahead and run through an example of a protein sequencing bifragmentation.1557

All right.1568

Breaking disulfide bonds.1571

OK.1581

I'm going to list 2 procedures for breaking disulfide bonds.1583

OK, and recall what a disulfide bond is.1599

It is a covalent bond between these sulfurs on cysteine residues along a protein chain.1606

They don't need to be next to each other.1633

They can be anywhere, but if there is a cysteine and a cysteine, they'll tend to come together, which helps in the folding process but actually a covalent bond.1634

OK.1645

The first procedure is using a chemical called performic acid, and the chemical structure of performic acid is OOHH.1646

It is a peroxy acid using performic acid.1665

OK.1667

Let's go ahead and do this.1668

Let me draw out…let me do this in blue.1670

I've got N, C, C, and peptide goes this way, peptide goes this way, and, of course, this is CH₂, this is S, and this is S.1677

Let me make this S a little bit clearer.1692

So, we have this disulfide bond between two cysteine residues.1695

This is C, this is C, this is O, and this peptide bond goes on, this is N, this peptide bond goes on.1702

This is the bond that we want to break right here.1709

Ok.1713

This is the disulfide bond; that's the one that we want to break.1714

OK.1716

We do that with performic acid, so let me go ahead and draw that.1717

Let me go ahead do H, C, O, O, O, H- this is oxidation that's taking place here.1724

I'm oxidizing this, OK, in order to break it; and when I do break that bond, this bond right here, here is what I end up with.1734

I end up with N, C, C, this is O^- - oops, that's not O^-, this is - I haven’t broken the peptide bond here.1741

This goes on to a peptide; this goes on to a peptide.1755

I've got CH₂, and then I have S, double bond O, double bond O, O^-, that is one of them, and then I have O^-.1758

I hope I have enough room here S, double bond O, double bond O, CH₂, C, C, O, that goes on that way, and this goes on this way.1772

What I've done, I have broken this bond, and I’ve created, I’ve basically added a bunch of oxygens to the sulfur.1784

I have just broken the bond in order to free things up, so that things don't stay attached.1792

Again, because there is this covalent attachment between 2 sulfurs on the cysteine residues, I have to break that; I have to free it up, so that the chain is completely free to be sequenced like a string.1798

I can’t have it folded.1809

I can’t have 2 disulfurs attached to each other.1811

That won't give me anything.1815

This is one way to break a disulfide bond.1817

This particular process is oxidation.1819

What I end up producing here is 2 cysteic acid.1823

This dysfunctional group here, this is a cysteic acid.1831

OK.1836

I guess I can write that: 2 cysteic acid residues.1839

That is it.1850

I have broken it, and now, because they are like this, the bonds are not going to reform.1851

These are fully oxidized.1855

They are not going to actually go back the other way at this point.1858

This is one method using performic acid to break the disulfide bond.1860

OK.1864

Now, the second procedure for breaking the disulfide bond is using a reagent called dithiothreitol followed by iodoacetate.1865

Dithiothreitol looks like this: C, CH...actually you know what, that's fine, I'll go ahead and do CH₂, I'll go C, I'll put my H there, I'll put my H there, and I'll go CH₂.1887

This is OH; this is OH.1910

This is SH, and this is SH.1912

So, this is the dithiothreitol, and my iodoacetate.1916

Well, acetate looks like that.1920

I'll go ahead and write the acid, iodoacetic acid.1924

It has...I'll put an H there; I'll put an H there.1929

One of the Hs is actually just replaced by an iodine.1935

That is all it is.1938

This is just acetic acid and you have an iodine.1939

OK.1942

Here, chemically it is going to look like this.1943

Let's go ahead and do this one in blue again.1946

We have got N, C, C.1950

We have CH₂, and we have S, and we have S.1956

We have CH₂, we have C, we have N, we have C.1964

Peptide bond goes that way; peptide bond goes that way, peptide bond, peptide bond.1972

That is the protein chain.1976

This is a carbonyl here.1979

This bond, again, is the...oops...do this in red.1982

Again, the disulfide bond that we are trying to break is that bond.1987

OK.1991

The first thing that we do is we react it with the dithiothreitol, and this process is actually a reduction.1992

OK.2009

When you reduce this, it is going to end up looking like this.2010

I wonder if I have any room here.2015

Yes, that's fine; I should be able to do this.2018

So, we have got N, C, C.2022

It is going to be that way, CH₂, SH.2026

What you do is you break it up into cysteine residues again.2031

OK.2037

That is the first part, CH₂, C, C.2039

That goes that way, and, so, the peptide, peptide.2044

So, you have broken this bond.2048

That bond is now broken.2050

And now, what you have to do, is you actually have to react this with the iodoacetate, and the reason you do that is because if I did not do that then these 2 will just react again and form the disulfide bond again.2054

So, once I've actually reduced it to the thiol, the 2 individual cystein residues that are not covalently attached, I have to do something to these, so they don't go backwards; because this reaction actually wants to be over here.2076

It would prefer to be this way, not this way.2088

So, I have to do something to it.2091

When I do that, I end up with the following.2093

I'm going to do one of them over here, and I'm going to do the other over here, see what they look like.2095

You are going to end up with N, C, C; and you are going to end up with C, H, S, CH₂, COO^-.2101

Basically, I have just added an acetic acid.2120

I have attached an acetic acid.2123

I have acetylated it.2125

I have attached an acetic acid to this, so that this S and this S can't react anymore.2127

That is all that I've done.2131

I have created basically 2 molecules of that.2133

The peptide goes on this way; the peptide goes on that way, and, of course, this one over here, you’ve got N, C, C.2135

The peptide goes on this way; the peptide goes on this way.2146

You have CH, you have an S.2150

You have CH₂, and you have COO^-.2154

That is it.2158

You have taken your protein that is stuck together with a disulfide bond.2159

You have broken that disulfide bond.2164

Now, the protein can unfold, and now, you have a nice straight chain that you can go ahead and sequence.2166

That is what we have done.2172

So, 2 ways of breaking a disulfide bond, you have the performic acid, or you have the dithiothreitol; and these are the reactions that take place.2174

And again, you probably don't have to worry too much about reproducing these.2184

You just have to actually know what happens is that this disulfide bond breaks, you end up forming the thiols; and then here, you go ahead and you acetylate them.2189

You add these groups to it.2200

It is OK if you can't reproduce these, but I did want you to see it.2202

It is very, very important that you actually see these chemical structures over and over and over again.2206

That is how we develop the familiarity with these things because again, there is a lot going on here.2213

OK.2218

Let's see what we can do.2220

Now, let's go ahead and actually follow a process of taking a protein, breaking the disulfide bond, hydrolyzing it, splitting it up into fragments, sequencing those fragments, and seeing if we can arrange it in order.2222

We are going to run through this process, so let's just jump on in.2239

Let's see; let me go to...OK.2245

Here we have this random protein, and I'm going to go ahead and draw a couple of disulfide bonds in here.2249

I don't know if you can actually see, or I wonder if we can actually do it on here.2256

No, that's OK; it doesn't really matter.2266

Let me go ahead and this one in blue.2269

We have this random protein that we start with.2272

The first thing that we are going to do is we are going to break the disulfide bond, and we are going to hydrolyze this protein completely, so we can count the amino acids.2274

So, this is the process we are going to use.2282

We want to sequence this protein.2285

That is what we are doing, trying to find the sequence of this really long protein.2287

Alright.2290

Here is what we are going to do.2292

We are going to...oops...should I do it on this page?2295

Yes, that's fine.2302

The first thing we want to do is we want to break any disulfide bonds.2303

We want to free-up the chains, so it is just one long straight chain.2309

Break any disulfide bonds- that is the first step.2313

OK.2316

The next thing we are going to do is we are going to hydrolyze it.2318

We are going to hydrolyze it completely.2322

OK.2324

We are going to use probably some really, really heavy acid like 6 mono-hydrochloric acid.2325

We want to completely break up this thing, so hydrolyze.2329

We want to separate all the amino acids, and we want to be able to count them.2338

And, we can actually do this with other techniques.2346

We don't need to necessarily go through an Edman degradation for that.2347

We have other techniques for separating individual amino acids, proteins.2350

This is just one thing that we can do, one step that we can do.2356

We are going to count which amino acids are there.2359

This is an extra process that we can run.2361

When we go ahead and do this, break the disulfide bonds, we hydrolyze the protein, in other words, break it up into completely free amino acids, separate the amino acids using whatever technique that we have at our disposal, and then, count to see what amino acids are there, this is what we end up with.2367

Again, this is just an example, protein; and this is the data we end up with.2384

We end up with 5 As; we end up with 2 Hs, 1 R.2390

We have 2 Cs, we have 3 Is; these are amino acids, the A, H, R, C, I, right?2399

The arginine, cysteine, isoleucine, things like that.2405

I'm just using these single-letter designations instead of the 3-letter designations.2409

S, we have 2 of them, D, we have 4 of them.2415

Yes.2423

K, we have 2 of them, L, we have 2, M, we have 2, P, we have 3, R, we have 1.2427

Wait, A, H, R, C, I, S, yes, there we go.2441

And then we have S, we have 2 of them, T, let me do it over here.2446

T, we have 1 of them, V, we have of them, and Y, we have 2 of them.2456

We have taken this protein; we've broken the disulfide bonds.2465

We have completely hydrolyzed it, broken every single peptide bond.2468

We have separated the free amino acids, and we have counted them, and these are the counts we get.2471

So, these are the amino acids, their single-letter designations, and this is how many we have.2475

There is some information that we can extract from this.2481

Let's see what sort of information we can extract from this.2484

OK.2486

Now, this gives us a total of 38 amino acids in our protein.2489

This protein that we now know contains 38 amino acids.2499

We basically take a look at this and see what it is that we have, because now, what we want to do the next step, we want to break this up.2505

We want to take this protein, and we want to chop it up.2516

Well, notice I've got 2 lysines.2517

I have 1 arginine, 2 methionines.2526

OK.2531

If we use trypsin...let me do this in red.2533

Using the enzyme trypsin, trypsin breaks peptides next to lysine and arginine.2540

So, using trypsin we have 2 lysines and 1 arginine, that means we're going to make 1, 2, 3 cuts, right?2553

1, 2, 3 cuts, it’s going to give us 4 fragments.2564

So, using trypsin, we can recover 4 fragments.2567

That is pretty good.2575

OK.2578

And now, if we use cyanogen bromide, cyanogen bromide is that chemical way of clipping a peptide chain next to a methionine residue.2579

So, if I use cyanogen bromide, well, I have 2 methionines, so I have 2 points where I'm going to clip this protein; if I clip it in two places, I'm going to end up with a first, a middle and a third.2592

This one will give us 3 fragments.2609

This should be pretty good.2613

I'm going to go ahead and do a trypsin cleavage, and then I'm going to do a cyanogen bromide cleavage, and then I'm going to compare those fragments, and see if there is an overlap or see what kind of information they give me, and see if I can arrange the final sequence, what is the sequence of all these free amino acids.2614

And again, I got that just by sort of taking a look at this list.2633

There are other proteins I could have used and other reagents, but I notice the arginine, I notice the lysine, I notice the methionine, so trypsin and cyanogen bromide, they just happen to be the ones that I have picked in this particular case.2637

OK.2649

Now, let's go ahead and run through the fragmentation process.2651

All right.2656

Now, the first thing I'm going to do, however, is I want to find out what the N-terminal amino acid of this protein is.2658

So, this protein ends somewhere.2665

There is an amino side, and there is a carboxyl side.2667

I want to know what the N-terminal is.2670

All right.2673

I take my intact protein, and I react it with the Sanger reagent, the FDNB- fluorodinitrobenzene.2674

OK.2690

Hydrolysis separation and then, of course, the N-terminal has been labelled with the FDNB, so N-terminal identification.2696

This is the identi…oops let me write this out…identification.2713

My results are, when I do everything, dinitrophenol glutamate is detected.2724

When I run this process, I label it with FDNB; I hydrolyze it.2742

I separate it, and now, I Identify the N-terminal.2747

It happens to be, the molecule that I detect is 2,4-dinitrophenol glutamate, so glutamate is my N-terminal amino acid.2750

So, E, which is glutamate, is the N-terminus.2760

Now, next step of the process that I take when I actually do my fragmentation, one of my fragments on the left is going to have an E; I know that is my first fragment automatically.2775

I don’t have to worry about that; no matter whether it's short or long, I’ve identified the left hand side of the protein to which nothing else is attached.2785

That is why this process is good.2794

OK.2795

Identified glutamate as the N-terminus, now, I can go ahead and start with the breaking up process.2797

OK.2803

I take my intact protein again.2804

First thing I do is, of course, let me see; let me write it over here, yes, so my first step, I’m going to break the disulfide bonds- that's the first step.2808

OK, the second step.2831

Here's what I'm going to do, now, I'm going to run my fragmentation process.2833

I'm going to use trypsin first.2837

Trypsin cleaves the protein on the carbon side, the carboxyl side of lysine and arginine, OK, an R.2840

I separate the fragments; I sequence each individual fragment, sequence the 4 fragments, and my results are as follows.2857

OK.2876

My T1 fragment - T1 for trypsin - my T1 fragment consists of the following sequence: D, C, V, S, H, D.2877

My second fragment is as follows: L, Y, I, A, C, G, P, M, T, K.2892

I'm doing now the results of this process, added the trypsin; I've cut I up.2906

I've separated the fragments; I've sequined the 4 fragments, the Edman, and these are the results: T3, I have E, A, G, G, H, Y, F, E, D, P, I, D, P, R, that's my third fragment.2912

And, of course, I have a fourth fragment, right?2940

Four fragments because we cut it in 3 places.2944

I have got G, A, A, M, I, L, A, K.2948

Notice, there is my K, that's my lysine.2957

There is my R, that's my arginine; and there is my K, that's my other lysine.2960

So, these 3 fragments are where I clipped, right, because it’s going to clip it next to a lysine or an arginine- the K or an R.2968

These are going to show up at one end of the fragments.2976

Notice, this particular fragment doesn't have a K or an R.2980

Chances are, this is our final fragment, the one at the end.2983

OK.2988

Now, let's sort of take a look and see if we can extract some information from what it is that we've done here and we can; let’s see.2989

Here's our E, remember we said that E glutamate we know that that's our N-terminal amino acid on the protein.3002

So, T3 is our first fragment.3011

Let me write that over here.3014

T3 this is the far left fragment.3016

I know that the T3 is our first fragment because the N-terminal is E glutamate.3025

T1, I can also make a comment about T1.3043

T1 is the far right fragment.3048

It is our last fragment in the order.3051

I've got 4 fragments.3053

I need to place them in some sort of an order.3054

I know that T3 is the first; I know that T1 is the last.3057

I know that T1 is the last fragment because it does not end with a lysine or an arginine.3059

In other words, when we cut this, we're cutting it to the…we have our lysine or our arginine residue, we're cutting it to the right of it, right, because we're cutting, we're snipping the carboxyl side, we're breaking this bond and this bond.3082

That means anything over here is not going to have anything attached to it.3102

There is going to be no lysine and no arginine anywhere because this one doesn’t have a lysine or an arginine that tells me that it's the final fragment.3106

These cannot be the final fragments.3113

I hope that makes sense.3116

If not, just stop and think about what it is that I've just said.3117

OK.3121

Now, we have our first fragmentation procedure.3122

Now, I'm going to redo this procedure, this whole thing, except instead of trypsin, I’m going to use cyanogen bromide to create a new set of fragments that I can compare to the first.3124

OK.3135

Let's go again.3136

Let's do this in blue.3141

Again, our first step is to break the disulfide bonds, and, of course, our second step, let's go ahead and use cyanogen bromide.3142

Now, once we go ahead and we clip it with cyanogen bromide, we're going to need to separate the fragments by whatever separation procedure we happen to have at our disposal in that particular laboratory, separate the fragments, and then we're going to sequence the fragments using our Edman degradation, or again, whatever other procedure you happen to have in your laboratory.3161

Maybe you have some mass spectrometry machines, those work also, sequence the fragments, but in this case, let's just say using Edman, and now, these are the results of this particular analysis.3190

Our C1 fragment consists of the following E, A, G, G, H, Y, F, E, D, P, I, D, P, R, G, A, A, M- that's our first fragment.3203

OK.3226

Notice, here's our E.3227

We notice that immediately, so we can almost automatically say this is our first fragment, C1, C2. 53:58 And remember, cyanogen bromide, it breaks things; it clips right next to a methionine, it clips the carboxyl end of the methionine.3230

We had 2 methionines, so in our long peptide, this protein, when I stretch it out after I’ve broken the disulfide bonds, there are going to be 2 methionines.3250

I know that already from the initial amino acid count, because there are 2 in there, I'm going to clip it in two places; I’m going to break it into 1, 2, 3 fragments, so I normally get 3 fragments.3261

This one is T, K, D, C, V, S, H, D, and our C3 fragment is going to be I, L, A, K, L, Y, I, A, C, G, P, M.3271

These are the results of my analysis.3290

Now, let's see what we've got: something to the right of C1, something to the right of C3.3293

Well, I have my methionine here and here.3307

Well, because I'm cutting it to the right of methionine, right, that's what cyanogen bromide does, I know that there is going to be something to the right of C1.3313

Well, because of these, I also know there is going to be something to the right of C3.3321

So, my results: something to the right of both C1 and C3, but I notice that C1 has the E, which is the N-terminus, therefore, I know that C1 is the far left fragment.3327

I know that.3373

OK.3374

We're almost there.3375

Now, in this case, I notice that I've got 3 fragments here.3377

Actually, what's happened is, in this case, I don’t necessarily need to compare overlaps, but I’m going to go ahead and compare the overlaps just so you see it.3384

Let's go ahead and do that; and then we’ll discuss how it is that this cyanogen bromide procedure actually go ahead and gives me my final answer.3391

I didn't actually need to go through the trypsin procedure, but again, live and learn.3399

You don't really know this in the lab because you are just given a protein and they say sequence it.3402

OK.3408

Now, let's go ahead and write out this sequence, and hopefully I can do it in one line here.3409

So, lets' go E, A, G, G, H, Y, F, E, D, P, I, D, P, R, G, A, A, M, I, L, A, K, L, Y, I, A, C, G, P, M, T, K, D, C, V, S, H, N, D.3414

Yes, so what we have is the following from E all the way to the first R.3455

Let me do this one in black.3460

From E to R, this was our T3 fragment.3464

From here to M, that was our C1 fragment.3472

From G to K, this was our T4 fragment.3477

And from I to G, P, M, T, this here to here, this was our C3 fragment.3484

T, K, from here to here, this was our T2 fragment, and then, of course, our T1 fragment, and then from here to here is, of course, our C2 fragment; and notice, we have a C, and a C, so this is our disulfide bond.3499

Disulfide bond is there between those 2 Cs, and notice the overlap between the T3, T4 and the C1, the overlap between the T4, T2, the C3, and the overlap between the T2, T1 and the C2.3527

Here are our points of overlap.3544

Now, in this particular case, I don’t actually need to compare these fragments, the ones from trypsin to the ones that I got from the cyanogen bromide, and the reason I don't need to do that, the cyanogen bromide gave me 3 fragments.3546

Well, those 3 fragments told me that what's going to be on the right one, what's going to be on the right of the other, and the fact that I knew that glutamate was my N-terminal amino acid, I knew that that was going to be my far left fragment, so that automatically put me, I was already able to arrange this overall sequence based on just the cyanogen bromide.3561

OK.3585

Let me go ahead and write this out, and we will go ahead and close out this lesson.3586

In this particular case, since we already knew from the first hydrolysis, remember when we did our initial count, we knew from the first hydrolysis that we had 2 methionines, and also knew that glutamate was the N-terminus, was the N-terminal amino acid.3591

OK.3657

Since we already knew from first hydrolysis that we had 2 methionine, and also knew that glutamate was the N-terminal amino acid, we knew we would have 2 cleavages, which implies 3 fragments.3658

Well, would you have 3 fragments, 3 fragments are easy to order.3687

If you know the far left one, you know what the other 2 are because you know something has to be to the right of the methionine, and something has to be to the right of the other methionine.3690

Well, that's it.3699

We can actually use the cyanogen bromide just to go ahead come up with the final sequence.3701

However, we're not always that lucky where we have 3 fragments.3707

If we don’t, 4 fragments, 5 fragments, or if something else is getting in the way, then we have to use the other information from the other fragmentation procedure to actually set up an overlap.3712

So, what we do is we take these fragments and we see where overlaps occur.3722

When we find those overlaps, we just rearrange those fragments; it’s just like putting a puzzle together and we come up with our final sequence.3726

So that's it.3735

I know that this appears a little bit complicated.3737

Hopefully, you'll see a couple of these problems in your book and you'll run through them.3739

It does take a while to go through, but it is worthwhile to go through it.3744

I promise you.3748

Thank you again for joining us here at Educator.com.3749

We'll see you next time, bye-bye.3752

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