SILAC = Stable Isotope Labeling by Amino Acids in Cell Culture

An in vivo metabolic labeling technique for quantitative proteomics.

Core principle:

• Uses stable isotopes (¹³C, ¹⁵N) — NOT radioactive
• Same chemical-physical properties as natural isotopes
• Isotopes incorporated into "heavy" amino acids
• Cells incorporate labeled amino acids during translation
• Label encoded directly into the proteome

Why Arginine and Lysine?

1. Essential/semi-essential: Cells must obtain them from media
2. Trypsin cleavage sites: Trypsin cleaves after K and R
3. Every tryptic peptide (except C-terminal) contains at least one K or R
4. Ensures all peptides are labeled

Also used: Leucine (present in ~70% of tryptic peptides)

1. Cell Cultures:
   • Two populations grown separately
   • One in "light" medium (normal amino acids)
   • One in "heavy" medium (¹³C/¹⁵N-labeled amino acids)
2. Protein Integration:
   • Cells incorporate amino acids during translation
   • Multiple cell divisions for complete labeling
3. Treatment:
   • Apply experimental condition (e.g., drug, stimulus)
4. Harvest & Mixing:
   • Samples mixed early (at cell level)
   • Minimizes experimental error
5. Lysis & Separation:
   • Cells lysed, proteins separated (SDS-PAGE or 2D-PAGE)
6. Digestion:
   • Trypsin digestion → peptides
7. MS Analysis:
   • Light and heavy peptides co-elute from LC
   • Two peak families in spectrum
   • Ratio of peak intensities = relative abundance

SILAC spectrum interpretation:

• Two families of peaks: "light" and "heavy"
• Heavy peaks shifted to the right (higher m/z)
• Peak intensity ratio = relative protein abundance

Calculation example:

• ¹³C₆-Lysine adds 6 Da mass difference
• With +2 charge state:
• m/z shift = Mass difference ÷ Charge
• m/z shift = 6 ÷ 2 = 3 m/z units

General formula:

Δm/z = ΔMass / z

Quantification:

• Compare peak heights or areas
• Heavy/Light ratio indicates fold change
• SILAC provides relative (not absolute) quantification

SILAC Limitations:

1. Requires living cells:
   • Cells must grow in culture
   • Must incorporate labeled amino acids
2. Time-consuming:
   • Multiple cell divisions needed for complete labeling
   • Typically 5-6 doublings
3. Limited multiplexing:
   • Maximum 2-3 samples (light, medium, heavy)
4. Arginine-to-Proline conversion:
   • Some cells convert Arg to Pro
   • Can cause labeling artifacts

Samples that CANNOT be used:

• Cell-free biological fluids:
  • Plasma/serum
  • Urine
  • Saliva
  • CSF
• Reason: No living cells to incorporate labels!

Samples that CAN be used:

• Cell lines
• Blood-derived leukocytes (if cultured)
• Biopsy-obtained cancer cells (if cultured)

SILAC Advantages:

1. Early mixing:
   • Samples mixed at cell level (earliest possible point)
   • Minimizes experimental error during sample preparation
2. Complete labeling:
   • Nearly 100% incorporation after sufficient doublings
3. No chemical modification:
   • Label is natural amino acid (just different isotope)
   • No affinity purification needed
4. High proteome coverage:
   • ~70% of peptides contain Leucine
   • All tryptic peptides contain K or R
5. Accurate quantification:
   • Light and heavy peptides co-elute
   • Analyzed simultaneously = same ionization conditions

ICAT = Isotope-Coded Affinity Tag

An in vitro chemical labeling technique targeting Cysteine residues.

Three functional components:

1. Reactive Group (Iodoacetamide):
   • Specifically binds to cysteine thiol groups (-SH)
   • Highly specific reaction
2. Isotope-Coded Linker (PEG):
   • Polyethylene glycol bridge
   • Light version: Normal hydrogen atoms
   • Heavy version: 8 hydrogens replaced with deuterium
   • Mass difference: 8 Da
3. Biotin Tag:
   • Affinity tag for purification
   • Strong binding to streptavidin/avidin
   • Enables selective isolation of labeled peptides

Structure: [Iodoacetamide]—[PEG linker]—[Biotin]

1. Denaturation & Reduction:
   • Unfold proteins
   • Break disulfide bonds to expose cysteines
2. Labeling:
   • Sample 1 → Light ICAT reagent
   • Sample 2 → Heavy ICAT reagent
   • Iodoacetamide reacts with Cys thiols
3. Mixing & Digestion:
   • Combine labeled samples
   • Trypsin digestion → peptides
4. Affinity Chromatography:
   • Add streptavidin-coated beads
   • Biotin-tagged peptides bind
   • Non-Cys peptides washed away
   • Reduces complexity!
5. Nano-HPLC & MS:
   • Separate and analyze peptides
   • Light/Heavy peaks separated by 8 Da
6. MS/MS:
   • Fragment for sequence identification
   • Database search (MASCOT)

Advantages:

• Reduced complexity: Only Cys-containing peptides selected → cleaner spectra
• Accuracy: ~10% accuracy in relative quantification
• Flexibility: Works on complex protein mixtures
• Clinical samples: Can use tissues, biopsies, fluids (unlike SILAC)

Disadvantages:

• Cysteine dependency:
  • Only ~25% of peptides contain Cys
  • Proteins without Cys cannot be identified!
• Accessibility issues:
  • Some Cys buried in protein structure
  • Cannot be labeled
• Limited multiplexing:
  • Only 2 samples (light vs heavy)
• Cost: Expensive reagents
• Yield concerns: Non-specific binding and incomplete labeling

Isobaric = Same total mass

All iTRAQ reagents have identical total mass (e.g., 145 Da for 4-plex).

Why this matters:

• Identical peptides from different samples appear as ONE peak in MS1
• Keeps spectrum simple and clean
• No peak splitting like in SILAC

How it works:

• Different isotope distribution within the reagent
• Reporter group + Balance group = constant mass
• When reporter is heavier → balancer is lighter

Example (4-plex):

Reagent	Reporter	Balance	Total
1	114 Da	31 Da	145 Da
2	115 Da	30 Da	145 Da
3	116 Da	29 Da	145 Da
4	117 Da	28 Da	145 Da

iTRAQ reagent has three parts:

1. Reporter Group:
   • Unique "ID" for each sample
   • 4-plex: 114, 115, 116, 117 Da
   • 8-plex: 113-121 Da
   • Released during MS/MS fragmentation
   • Used for quantification!
2. Balance Group:
   • Compensates for reporter mass
   • Ensures total mass is constant
   • Lost during fragmentation
3. Reactive Group (NHS ester):
   • Binds to N-terminus and Lysine side chains
   • Labels all peptides (not just Cys like ICAT)

Structure: [Reporter]—[Balance]—[NHS-ester]

iTRAQ Workflow:

1. Extraction & Preparation: Purify, denature, reduce proteins
2. Digestion: Trypsin → peptides BEFORE labeling
3. Labeling: Each sample labeled with specific iTRAQ reagent
4. Pooling: Combine all labeled samples into one
5. HPLC Separation: Treat as single sample
6. MS1: Single peak per peptide (isobaric!)
7. MS/MS (CID): Fragmentation breaks Reporter-Balance bond
8. Reporter ions released: 114-117 region shows intensities

Quantification occurs at MS/MS (MS2) level!

Method	Quantification Stage
SILAC	MS1 (peak ratios)
ICAT	MS1 (peak ratios)
iTRAQ	MS2 (reporter ions)

Ratio Compression Effect: Measured differences in protein abundance appear smaller than actual biological values, compressing observed ratios toward 1:1.

Cause: Co-Isolation Challenge

• During MS2, mass spectrometer isolates precursor ion for fragmentation
• Peptides with similar m/z that co-elute are co-isolated
• These "contaminating" peptides also fragment
• Their reporter ions merge with target signal
• Background peptides at different concentrations → dilute the true signal
• Result: Systematic underestimation of fold-change

Mitigation strategies:

1. Better chromatography: Reduce co-elution
2. MS3 analysis: Additional fragmentation stage (gold standard)
3. Narrower isolation windows: Reduce co-isolated species

Advantages:

• High multiplexing: Up to 8 samples (4-plex or 8-plex)
• Statistical power: More samples = better p-values, less noise
• Clean MS1 spectra: Isobaric tags → single peaks
• High coverage: Labels N-terminus + Lys (most peptides)
• Relative & absolute: Can include standards

Limitations:

• Ratio compression: Background interference underestimates differences
• Expensive reagents: High cost compared to label-free
• High sample concentration needed:
• Complex preparation: Risk of sample loss, incomplete labeling
• Sophisticated software needed: ProQuant, etc.

Label-Free Quantification: Quantitative proteomics without isotope labels or chemical tags.

Key characteristics:

• Direct comparison of individual LC-MS/MS runs
• No expensive reagents needed
• Samples never mixed — analyzed separately
• Requires strict experimental standardization

Comparison to label-based:

Feature	Label-Based	Label-Free
Sample mixing	Combined before MS	Analyzed separately
Cost	Higher (reagents)	Lower
Multiplexing	Limited by reagents	Unlimited samples
Variability	Lower (same run)	Higher (run-to-run)
Complexity	Sample prep	Data analysis

1. Spectral Counting:

• Principle: More protein → more peptides → more MS/MS spectra
• Data level: MS2
• Measures: Number of spectra, unique peptides, sequence coverage
• Advantages: Easy to implement, no special algorithms
• Best for: High-abundance proteins

2. Precursor Signal Intensity (AUC):

• Principle: Measure Area Under the Curve of chromatographic peaks
• Data level: MS1
• Measures: Peak intensity/height as peptides elute
• Advantages: More accurate for subtle changes
• Best for: Low-abundance proteins

Feature	Spectral Counting	AUC
Data Level	MS2	MS1
Complexity	Low	High (needs alignment)
Sensitivity	Better for abundant	Better for low-abundance

Technical challenges:

1. Experimental Drift:
   • Fluctuations in retention time (RT) between runs
   • m/z drift over time
   • Hard to align same peptide across samples
   • Solution: Alignment algorithms that "stretch/shrink" chromatograms
2. Run-to-Run Variability:
   • Even identical samples show intensity differences
   • ESI efficiency fluctuations
   • Column performance variation
   • Solution: Internal standards, global normalization
3. Data Complexity:
   • Massive data volume from separate runs
   • Requires sophisticated bioinformatics pipelines
   • Automated alignment, normalization, statistics
4. No internal standard:
   • Unlike labeled methods, no built-in reference

Label-Free Advantages:

• Cost-effective: No expensive reagents
• Simple sample prep: No labeling steps
• Unlimited multiplexing: Compare any number of samples
• Works with any sample: Tissues, fluids, cells
• Lower sample amount: No sample loss during labeling
• Dynamic range: Can detect wider range of changes
• No ratio compression: Unlike iTRAQ

Best applications:

• Large-scale studies (many samples)
• Clinical cohorts
• When sample is limited
• Initial screening studies