🧬 MS for Structural Proteomics – Lecture 2 (Full Summary)

📄 Page 1 – Title

• Introduction to MS-based structural proteomics
• Focus: extracting structural and interaction information from proteins using MS

🎯 Page 2 – Learning Objectives

You should be able to:

• Apply MS to protein structure analysis
• Understand HDX, XL-MS, LiP-MS
• Evaluate advantages + limitations
• Interpret real research data
• Solve method-based problems

🧭 Page 3 – Lecture Outline

• Lecture 1: Basics (BUP, TDP, PTMs, disulfides)
• Lecture 2: → HDX (Hydrogen-Deuterium Exchange) → XL-MS (Cross-Linking MS) → LiP-MS (Limited Proteolysis MS)

🧠 Page 4 – Big Picture (IMAGE)

The diagram shows:

• A central protein system
• Multiple MS techniques branching out:
  • HDX → dynamics
  • XL-MS → distances/interactions
  • LiP-MS → conformational changes
  • Others → PTMs, topology

💡 Key idea: No single method gives full structure → combine methods for systems-level structural proteomics

🔬 PART 1: SURFACE PROBING

🧪 Page 5 – Surface Labeling

• Intact MS = gives mass only, not structure
• Solution: chemical labeling of exposed residues

Types:

• Amine-specific (Lys)
• Carboxyl-specific
• DEPC labeling
  • Targets: His, Lys, Tyr, Ser, Thr, Cys
  • Adds +72 Da

💡 Interpretation:

• Only accessible residues react → gives surface exposure info

🌊 PART 2: HDX-MS (Hydrogen-Deuterium Exchange)

💧 Page 6 – HDX Basics

• Measures exchange: N–H → N–D (backbone amides)

💡 Why backbone?

• Present in all residues → gives global structure info

⚙️ Page 7 – HDX Workflow (IMAGE)

Step-by-step workflow:

1. Protein in H₂O
2. Transfer to D₂O buffer
3. Exchange occurs (H → D)
4. Quench (low pH + cold)
5. Digest → peptides
6. Analyze by MS

Important buffers:

• D₂O buffer → labeling
• Quench buffer (pH 2.3, cold) → stops exchange

💡 Key concept:

• You “freeze” structural information at specific times

⏱ Page 8 – HDX Kinetics

Two regimes:

EX1:

• Fast labeling after unfolding
• All-or-none behavior

EX2:

• Slow exchange
• Depends on probability of opening

💡 Interpretation:

• EX1 → cooperative unfolding
• EX2 → local fluctuations

🔍 Page 9 – Structural Interpretation (IMAGE)

• Heatmaps + spectra show exchange rates

💡 Key rule:

• Fast exchange = exposed / flexible
• Slow exchange = buried / stable

→ You map protein dynamics + solvent accessibility

🌡 Page 10 – Conditions Matter (IMAGE)

Exchange depends on:

• Temperature ↑ → faster exchange
• pH (especially basic conditions)
• Time

💡 Important:

• You must carefully control experimental conditions

⚡ Page 11 – Fragmentation Effects

• ECD (Electron Capture Dissociation) → low scrambling (good)
• CID (Collision-Induced Dissociation) → high scrambling (bad)

💡 Why?

• Scrambling = losing spatial info

Also:

• Back-exchange occurs → reduces signal

🔄 Page 12 – Comparing Proteoforms (IMAGE)

Example: phosphorylation at D55

• Different HDX patterns → structural differences

💡 Use case:

• Detect conformational changes caused by:
  • PTMs
  • mutations
  • ligand binding

🔁 Page 13 – HDX Summary Workflow (IMAGE)

Reinforces:

• D₂O labeling
• Quenching
• Digestion
• MS analysis

🔗 Page 14 – Studying Interactions (IMAGE)

HDX can detect:

• Protein–protein binding
• Ligand binding
• Environmental effects

💡 Interpretation:

• Binding → reduced exchange at interface

☕ Page 15 – Break

(no content)

🔗 PART 3: XL-MS (Cross-Linking MS)

🔗 Page 16 – XL-MS Basics

• Uses bi-functional cross-linkers
• Links two residues

Provides:

• Distance constraints
• Interaction info

💡 Important:

• XL-MS ≠ standalone → used for modeling + network analysis

⚙️ Page 17–18 – XL-MS Workflow (IMAGES)

Workflow:

1. Add cross-linker
2. Cross-link forms (intra/inter protein)
3. Digest into peptides
4. MS analysis
5. Identify cross-linked peptides

💡 Outcome:

• Distance constraints (~10–30 Å)

⚠️ Page 19 – Complexity

• Many linkers exist
• Trade-off:
  • More flexibility → more complexity

🧩 Page 20 – Data Interpretation

Two strategies:

1. Simple linker:

• Mixed fragment spectra → complex analysis

2. Isotope-labeled linker:

• Light + heavy peaks
• Easier to identify cross-links

💡 Trick:

• Mass shift = identify linker-containing fragments

🔀 Page 21 – Inter vs Intra (IMAGE)

• Intra-link → within same protein
• Inter-link → between proteins

💡 Why important?

• Defines interaction networks

✂️ Page 22 – Cleavable Linkers

Example: DSBU

• Breaks during MS
• Reveals individual peptide masses

💡 Advantage:

• Simplifies identification

🧬 Page 23 – MS-Cleavable Linkers

Two modes:

• MS2 identification
• MS3 sequencing

Also:

• Many linkers target Lys (NHS esters)

🔗 Page 24 – Multi-site Crosslinking

• New linkers connect >2 sites

💡 Trade-off:

• More info
• Much harder analysis

🌐 Page 25–26 – XL-MS Applications (IMAGE)

Used for:

• Protein complexes
• Interaction networks
• Structural modeling

🔪 PART 4: LiP-MS (Limited Proteolysis MS)

✂️ Page 27 – LiP-MS Basics

• Use limited proteolysis before digestion

Enzymes:

• Proteinase K (broad specificity)

Key idea:

• Structure determines protease accessibility

💡 Output:

• Structural differences in native state

⚙️ Page 28 – Workflow

1. Native protein
2. Limited proteolysis
3. Full digestion (trypsin)
4. MS analysis

💡 Requires:

• Semi-tryptic analysis

🔢 Page 29 – Multiplexing

• Compare multiple conditions in parallel

🧠 FINAL INTEGRATION

🧬 Page 30 – Putting It All Together

Core MS structural methods:

• HDX-MS → dynamics + accessibility
• XL-MS → distances + interactions
• LiP-MS → conformational changes

Complementary methods:

• AP / co-IP → interactions
• TPP → stability
• FPOP → oxidation footprinting
• CPP → surface exposure

💡 Key takeaway: 👉 Structural proteomics = multi-method approach

🎯 Page 31 – Learning Objectives (Revisited)

Confirms:

• You now understand:
  • Methods
  • Workflows
  • Interpretation

📝 Page 32 – Assignment

• Apply concepts

🧩 BIG PICTURE SUMMARY

🧪 What each method tells you:

🧠 Key Concepts to Remember

• Faster HDX → more exposed region
• Cross-links → spatial constraints
• LiP cleavage → flexible regions
• No single method is enough → integration is key