🧬 1. How Do We Determine Protein Primary Structure?

First choice: DNA databases

Today, we usually determine protein sequence from:

1. Genome / cDNA databases
2. Translate DNA → protein sequence

But this does not reveal:

• Post-translational modifications (PTMs)
• Processing events (cleavages, signal peptides removed)

When DNA info is missing or incomplete → we must determine sequence experimentally.

✂️ 2. Protein Digestion: Why Do We Cut Proteins?

Large proteins are too big to sequence directly. So we:

1. Digest into smaller peptides
2. Separate peptides (HPLC)
3. Sequence peptides (Edman or MS)
4. Reconstruct full sequence

🔪 Enzymatic Cleavage (Very Important)

Each protease cuts at specific residues:

Also chemical cleavage:

• CNBr → after Met
• Mild acid → Asp–Pro
• Hydroxylamine → Asn–Gly

These different cleavage patterns are crucial for determining sequence order.

🧪 3. Edman Degradation (Stepwise N-terminal sequencing)

How it works

1. Label N-terminal amino acid with phenyl isothiocyanate (PITC)
2. Cleave off labeled amino acid
3. Identify it by reverse-phase HPLC
4. Repeat cycle

❗ Why can we only sequence ~10 amino acids?

You asked:

Edman only sequences few AA because labeling is not 100% degraded?

Yes — correct, and here is the full explanation:

Each cycle is ~99% efficient (example).

Cycle 1:

• 99% correct removal
• 1% remains uncleaved

Cycle 2:

• Now mixture contains:
  • Mostly second AA
  • 1% leftover from first cycle

Cycle 3:

• Even more mixture

After many rounds:

• Signal becomes too noisy
• Peaks overlap
• Sequence becomes ambiguous

So we can only reliably sequence ~10 residues.

This is cumulative inefficiency.

🔁 4. Why Use Trypsin AND Chymotrypsin?

You asked:

To know the order, do we use trypsin & chymotrypsin?

Yes — this is essential logic.

If we digest only with trypsin:

Example fragments:

A - B - C - K D - E - F - R G - H - I - K

We don’t know their order in full protein.

Now digest same protein with chymotrypsin:

Different fragments:

C - K - D F - R - G I - K - J

Now we find overlaps:

• Fragment 1 overlaps with fragment 2
• Like solving a puzzle

This is called overlapping peptide mapping.

Without two different digestions → cannot reconstruct full order.

This applies to BOTH:

• Edman sequencing
• Mass spectrometry

⚡ 5. Tandem Mass Spectrometry (MS/MS)

Modern method of choice.

Step 1 – Ionization

Two soft ionization methods:

🟢 MALDI-TOF

Matrix Assisted Laser Desorption Ionization Usually produces:

• Mostly singly charged ions (+1)
• Sometimes +2

🔵 ESI (Electrospray Ionization)

Sprays protein into charged droplets.

Produces:

• Multiple charge states (+5, +10, +20…)
• One molecule can carry many charges

❓ Why does ESI produce more charges than MALDI?

Because:

• MALDI → desorption event usually creates single protonation
• ESI → droplet evaporation leaves many protons on protein surface
• Large proteins expose many basic residues
• More sites → more protonation

So ESI gives a charge distribution spectrum.

🧩 6. Tandem MS: How Sequence Is Determined

MS1:

Measure mass of intact peptide.

Collision Cell:

Peptide fragmented.

MS2:

Measure masses of fragments.

💥 Peptide Fragmentation

Fragmentation happens at peptide backbone.

Main ions:

Most common in CID:

• b-ions
• y-ions

How sequence is read

If peptide is:

A–B–C–D–E

You get:

• b1 (A)
• b2 (A–B)
• b3 (A–B–C)
• etc.

Differences between peaks correspond to one amino acid mass.

So sequence is deduced by: Mass difference between consecutive fragments.

❗ 7. Can MS Distinguish Lysine and Isoleucine?

You asked:

MS can't determine lysine and isoleucine because masses are same?

Correction:

It is Leucine (L) and Isoleucine (I) that have identical mass.

Both:

• Same elemental composition
• Same molecular weight
• Structural isomers

Mass spectrometry cannot distinguish them by mass alone.

However:

• HPLC separation can sometimes distinguish due to structural differences.
• Advanced MS fragmentation methods sometimes help.

But classical MS → cannot distinguish L and I.

📏 8. Protein Size Determination Methods

1️⃣ SDS-PAGE

• SDS denatures proteins
• Coats protein with negative charge
• ~1 SDS per 2 amino acids
• Mobility depends mainly on size

Smaller proteins → move faster.

Used for 1–200 kDa.

2️⃣ Mass Spectrometry

Most accurate molecular weight method.

Can detect:

• Monomers
• Dimers
• Multiple charge states

3️⃣ Size Exclusion Chromatography (SEC)

You asked:

Smaller molecules have longer travel time? Why?

Yes — and this is correct.

Column contains porous beads.

Large proteins:

• Cannot enter pores
• Travel straight through
• Elute earlier

Small proteins:

• Enter pores
• Take longer path
• Elute later

So:

Separation based on hydrodynamic radius.

⚖️ 9. Comparison of Methods

🔬 10. Post-Translational Modifications (PTMs)

Mass spectrometry can detect:

• Phosphorylation
• Glycosylation
• Other modifications

Because: Fragment masses shift according to modification.

DNA sequencing cannot detect PTMs.

🧠 Final Big Picture

To determine protein sequence experimentally:

1. Digest protein
2. Separate peptides (HPLC)
3. Ionize (ESI or MALDI)
4. MS1 → select peptide
5. Fragment in collision cell
6. MS2 → read b/y ion ladder
7. Repeat with different protease
8. Align overlapping fragments

🔎 Summary of Your Specific Questions

✔ Edman limited due to cumulative inefficiency ✔ Two proteases needed for overlap mapping ✔ Tandem MS determines sequence via fragment mass differences ✔ MS cannot distinguish Leu/Ile by mass ✔ b, y, c, z+1 ions result from backbone cleavage ✔ ESI produces multiple charge states due to droplet protonation ✔ SEC: small molecules take longer because they enter pores