Protein Separation & 2D-PAGE
From crude lysates to resolved proteomes: electrophoretic and chromatographic strategies for protein fractionation
9.1 Protein Extraction & Sample Preparation
Every proteomic experiment begins with the liberation of proteins from their biological context. Sample preparation is arguably the single most critical determinant of downstream analytical success: even the most sensitive mass spectrometer cannot compensate for a biased or degraded starting material. The goal is to solubilize the full complement of cellular proteins while preserving their integrity and, where relevant, their post-translational modifications.
Cell Lysis Strategies
Cell lysis can be achieved through mechanical, chemical, or enzymatic means. Mechanical methods include sonication, French press, bead beating, and freeze-thaw cycling. Chemical lysis relies on detergents and chaotropes to disrupt membranes and denature protein complexes. The choice of lysis buffer profoundly influences which proteins are recovered:
| Buffer System | Key Components | Best For | MS Compatible? |
|---|---|---|---|
| RIPA | NP-40, sodium deoxycholate, SDS (0.1%) | Whole-cell lysates, Western blot | Requires cleanup |
| Urea (8 M) | Urea, thiourea, CHAPS | 2D-PAGE, shotgun proteomics | Yes (after dilution) |
| SDS (4%) | SDS, DTT, Tris-HCl | FASP workflow, membrane proteins | Via FASP/SP3 |
| Triton X-100 | 1% Triton X-100, NaCl, EDTA | Soluble proteins, Co-IP | Yes |
| Native lysis | Digitonin or NP-40 (0.1-0.5%) | Protein complexes, enzyme assays | Yes |
Protease Inhibitors & Phosphatase Inhibitors
Once cells are lysed, endogenous proteases are released and can rapidly degrade the proteome. A cocktail of protease inhibitors must be added immediately. Common inhibitors include PMSF (serine proteases), leupeptin (serine/cysteine proteases), aprotinin (serine proteases), pepstatin A (aspartic proteases), and EDTA (metalloproteases). For phosphoproteomics, phosphatase inhibitors such as sodium orthovanadate (tyrosine phosphatases), sodium fluoride (serine/threonine phosphatases), and beta-glycerophosphate are essential. Commercial cocktails (e.g., Roche cOmplete, Thermo HALT) provide broad-spectrum protection.
Subcellular Fractionation
Fractionation reduces sample complexity and enriches for proteins localized to specific cellular compartments. Differential centrifugation separates organelles by sedimentation rate: nuclei pellet at 600-1,000 g, mitochondria at 10,000 g, microsomes at 100,000 g, and the cytosolic fraction remains in the final supernatant. Density gradient ultracentrifugation using sucrose or iodixanol (OptiPrep) provides finer resolution. Detergent-based sequential extraction (e.g., the ProteoExtract kit) uses buffers of increasing stringency to sequentially solubilize cytosolic, membrane-associated, nuclear, and cytoskeletal proteins.
9.2 SDS-PAGE: Principles & Practice
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is the workhorse of protein biochemistry. Developed by Ulrich Laemmli in 1970, this discontinuous buffer system separates denatured proteins strictly by molecular weight. SDS, an anionic detergent, binds to proteins at a roughly constant ratio of 1.4 g SDS per gram of protein, conferring a uniform negative charge density. The intrinsic charge of the protein is thereby overwhelmed, and electrophoretic mobility depends solely on the protein's hydrodynamic radius, which correlates with molecular mass.
The Discontinuous Buffer System
The Laemmli system uses a two-phase gel. The stacking gel (typically 4-5% acrylamide, pH 6.8) concentrates the sample into a thin band through an isotachophoresis effect: glycinate ions in the running buffer exist predominantly as zwitterions at pH 6.8 (trailing ions) while chloride ions from the Tris-HCl buffer act as leading ions. Proteins are sandwiched between these two ion boundaries and compressed. Upon entering the resolving gel (typically 8-15% acrylamide, pH 8.8), the glycinate becomes fully charged, the voltage gradient equalizes, and proteins separate by molecular sieving.
Relative Mobility (Rf)
where $d_{\text{protein}}$ is the distance migrated by the protein band and $d_{\text{front}}$ is the distance migrated by the tracking dye (bromophenol blue). For a given gel percentage, there is a linear relationship between $\log(M_r)$ and $R_f$ over a defined molecular weight range.
The Ferguson Plot
The Ferguson plot provides a rigorous method for determining whether differences in electrophoretic mobility are due to size, charge, or both. By running the same protein on gels of varying acrylamide concentration (%T), one obtains a linear relationship between the logarithm of mobility and gel concentration:
Ferguson Equation
where $\mu$ is electrophoretic mobility at gel concentration $T$ (total acrylamide %),$\mu_0$ is the free mobility (y-intercept, reflecting charge), and $K_R$ is the retardation coefficient (slope, reflecting size). Proteins with the same $K_R$ have the same size; those with the same $\mu_0$ have the same charge-to-mass ratio.
Native PAGE vs. SDS-PAGE
In native (non-denaturing) PAGE, proteins migrate according to their intrinsic charge, size, and shape. No SDS or reducing agents are used, so proteins retain their native conformation and subunit interactions. This is valuable for studying protein complexes, enzyme activity (zymography), and charge variants. Blue native PAGE (BN-PAGE) uses Coomassie G-250 as a mild charge-shift molecule to drive migration of membrane protein complexes while preserving their quaternary structure. Clear native PAGE (CN-PAGE) omits any charge-shift agent entirely.
Polyacrylamide Gel Composition
The gel matrix is formed by co-polymerization of acrylamide monomers and the cross-linker N,N'-methylenebisacrylamide (bis), catalyzed by ammonium persulfate (APS) and TEMED. The pore size is controlled by two parameters:
Gel Composition Parameters
where $a$ is the mass of acrylamide (g), $b$ is the mass of bisacrylamide (g), and $V$ is the total volume (mL). Typical resolving gels use %C = 2.6-3.3%. Higher %T produces smaller pores and better resolution of low-molecular-weight proteins.
9.3 Two-Dimensional Gel Electrophoresis (2D-PAGE)
Two-dimensional polyacrylamide gel electrophoresis, introduced by O'Farrell and Klose in 1975, remains one of the most powerful techniques for resolving complex protein mixtures. By combining two orthogonal separation dimensions — isoelectric focusing (IEF) in the first dimension and SDS-PAGE in the second — 2D-PAGE can resolve thousands of protein species on a single gel. Each spot ideally represents a single proteoform, defined by its unique combination of isoelectric point (pI) and molecular weight.
First Dimension: Isoelectric Focusing (IEF)
In IEF, proteins migrate through a pH gradient under an applied electric field until they reach the position where the ambient pH equals their isoelectric point (pI) — the pH at which the net charge is zero. At this point, electrophoretic mobility ceases and the protein is "focused" into a sharp band. The pI of a protein is governed by its ionizable amino acid side chains, the alpha-amino and alpha-carboxyl groups, and any post-translational modifications that introduce charged groups (e.g., phosphorylation, sialylation).
Henderson-Hasselbalch Equation & pI Estimation
The isoelectric point is estimated as the average of the two pKa values flanking the zwitterionic species:
For multi-ionizable proteins, the pI is computed numerically by finding the pH at which the sum of all partial charges equals zero: $\sum_i q_i(\text{pH}) = 0$.
Modern IEF uses immobilized pH gradient (IPG) strips rather than carrier ampholyte gels. IPG strips contain acrylamido buffers (Immobilines) co-polymerized into the gel matrix, creating a stable, reproducible pH gradient. Available gradients span broad ranges (pH 3-10, 3-11 NL) or narrow ranges (e.g., pH 4-7) for higher resolution. The sample is loaded by in-gel rehydration or cup loading, and focusing is performed on a dedicated IEF unit (e.g., IPGphor) at high voltages (up to 10,000 V) for a total of 50,000-100,000 Vh.
Second Dimension: SDS-PAGE
After IEF, the IPG strip is equilibrated in SDS buffer containing DTT (to reduce disulfide bonds) and then iodoacetamide (to alkylate free thiols and prevent re-oxidation). The strip is placed atop a standard SDS-PAGE slab gel and sealed with agarose. Electrophoresis in the second dimension separates proteins by molecular weight, orthogonal to the pI-based separation.
Spot Detection & Analysis
After staining, gel images are digitized and analyzed with software such as PDQuest, Melanie, or Progenesis SameSpots. The workflow includes spot detection, background subtraction, normalization, spot matching across gels, and statistical analysis of spot volumes. Differential expression is typically assessed using fold-change thresholds combined with ANOVA or Student's t-test, corrected for multiple testing (e.g., Benjamini-Hochberg FDR).
Key Advantages & Limitations of 2D-PAGE
Advantages
- Resolves proteoforms (isoforms, PTMs)
- Visual representation of the proteome
- Intact protein mass preserved
- Quantitative with DIGE (2D-DIGE)
- Compatible with spot excision & MS identification
Limitations
- Poor representation of membrane proteins
- Limited dynamic range (~104)
- Excludes very acidic/basic proteins
- Low throughput, labor-intensive
- Difficult to automate fully
9.4 Western Blotting & Immunodetection
Western blotting (immunoblotting), developed by Towbin et al. (1979), combines the resolving power of SDS-PAGE with the specificity of antibody-antigen recognition. Proteins separated by electrophoresis are transferred (blotted) onto a solid membrane — typically nitrocellulose (0.2 or 0.45 $\mu$m pore) or polyvinylidene difluoride (PVDF) — where they become immobilized and accessible to antibody probing.
Transfer Methods
| Method | Mechanism | Time | Efficiency |
|---|---|---|---|
| Wet (tank) transfer | Submerged in buffer, constant current | 1-16 hours | Highest (especially for large proteins) |
| Semi-dry | Buffer-soaked filter papers, plate electrodes | 30-60 min | Good for <150 kDa |
| Turbo (rapid dry) | Pre-assembled stacks, high current | 3-10 min | Good for most targets |
Detection & Quantification
The membrane is blocked (BSA or non-fat milk) to prevent non-specific antibody binding, then probed with a primary antibody targeting the protein of interest, followed by a secondary antibody conjugated to a reporter. Detection methods include chemiluminescence (HRP + ECL substrate), fluorescence (IR dye-conjugated secondaries for multiplexing on Odyssey systems), and colorimetric (AP + BCIP/NBT). Quantitative Western blotting requires validation that signals are within the linear dynamic range of the detection system, normalization to a total protein stain (e.g., Ponceau S, Stain-Free) rather than a single housekeeping gene, and replicate analysis.
9.5 Chromatographic Protein Separation
Liquid chromatography exploits differences in physicochemical properties to fractionate proteins in solution. Unlike gel electrophoresis, chromatography can be performed under native or denaturing conditions, is readily scalable from analytical to preparative volumes, and is directly interfaceable with mass spectrometry. Four principal modes are employed in proteomics.
Size Exclusion Chromatography (SEC)
Also called gel filtration. Proteins are separated by hydrodynamic radius as they pass through a porous bead matrix. Large proteins elute first (they are excluded from pores), while smaller proteins are retarded. SEC is non-adsorptive and preserves native interactions, making it ideal for complex characterization and buffer exchange. The partition coefficient $K_{av}$ is related to size:$K_{av} = (V_e - V_0)/(V_t - V_0)$, where $V_e$ is elution volume, $V_0$ is void volume, and $V_t$ is total column volume.
Ion Exchange Chromatography (IEX)
Separates proteins based on net surface charge. Anion exchangers (e.g., Q Sepharose, DEAE) bind negatively charged proteins; cation exchangers (e.g., SP Sepharose, CM) bind positively charged ones. Bound proteins are eluted with a salt gradient (increasing ionic strength) or pH step. Strong exchangers maintain constant charge across pH; weak exchangers change ionization state. IEX has high capacity and resolution and is commonly used as the first chromatographic step in purification.
Affinity Chromatography
Exploits specific biological interactions: antibody-antigen, enzyme-substrate, receptor-ligand, or engineered tags (His-tag/Ni-NTA, GST/glutathione, Strep-tag/Strep-Tactin, FLAG/anti-FLAG). Immobilized metal affinity chromatography (IMAC) using Ni2+, Co2+, or Fe3+ is also essential for phosphopeptide enrichment. Affinity chromatography provides the highest selectivity of any chromatographic mode but requires specific knowledge of the target.
Reversed-Phase HPLC (RP-HPLC)
The backbone of LC-MS/MS proteomics. Stationary phase: C18 (octadecyl) or C4 (butyl) silica. Mobile phase: water/acetonitrile with 0.1% formic acid. Peptides bind via hydrophobic interactions and elute with increasing organic solvent. Sub-2-$\mu$m particles and ultra-high-pressure (UHPLC) systems achieve extraordinary peak capacities (>500 in 2-hour gradients). Nano-flow RP-LC (200-500 nL/min) is standard for high-sensitivity shotgun proteomics.
9.6 Protein Staining & Quantification Assays
Visualization and accurate quantification of proteins are essential at multiple stages of the proteomic workflow. In-gel staining reveals separated protein bands or spots, while colorimetric and fluorometric assays measure total protein concentration in solution.
In-Gel Staining Methods
| Stain | Sensitivity | Dynamic Range | MS Compatible? | Notes |
|---|---|---|---|---|
| Coomassie R-250 | ~100 ng | ~10-fold | Yes | Simple, inexpensive, fixation required |
| Colloidal Coomassie G-250 | ~10 ng | ~30-fold | Yes | Lower background, no destaining needed |
| Silver stain | ~1 ng | ~10-fold | Modified protocols | Glutaraldehyde-free for MS compatibility |
| SYPRO Ruby | ~1-2 ng | ~1000-fold | Yes | Fluorescent, UV transillumination |
| CyDye (DIGE) | ~0.5 ng | >10,000-fold | Yes | Multiplexed, pre-labeling, best quantification |
Solution-Phase Protein Assays
All spectrophotometric protein assays ultimately rely on the Beer-Lambert law to relate absorbance to concentration:
Beer-Lambert Law
where $A$ is absorbance (dimensionless), $\varepsilon$ is the molar extinction coefficient (M$^{-1}$ cm$^{-1}$), $l$ is the path length (cm), and $c$ is the molar concentration. Direct UV absorbance at 280 nm exploits the aromatic side chains of tryptophan ($\varepsilon_{280} \approx 5,500$ M$^{-1}$ cm$^{-1}$) and tyrosine ($\varepsilon_{280} \approx 1,490$ M$^{-1}$ cm$^{-1}$).
Bradford Assay
Based on the binding of Coomassie Brilliant Blue G-250 to proteins, which shifts the dye's absorption maximum from 465 nm (red/brown) to 595 nm (blue). The assay is fast (5 minutes), requires only 1-20$\mu$g protein, and is relatively tolerant of many buffer components. However, it shows protein-to-protein variability (basic and aromatic residues bind preferentially) and is incompatible with detergents above 0.1% SDS. A standard curve with BSA is required.
BCA (Bicinchoninic Acid) Assay
A two-step reaction: (1) Cu2+ is reduced to Cu+ by peptide bonds in alkaline conditions (biuret reaction); (2) two molecules of BCA chelate each Cu+ ion, forming a purple complex absorbing at 562 nm. The BCA assay has a broader linear range (20-2,000 $\mu$g/mL), is more tolerant of detergents than Bradford, and shows less protein-to-protein variation. However, it is incompatible with reducing agents (DTT, beta-mercaptoethanol) and chelating agents (EDTA).
Chapter Summary: Key Concepts
- ◈Sample preparation — including lysis, protease inhibition, and fractionation — dictates the quality and coverage of all downstream proteomic analyses.
- ◈SDS-PAGE separates proteins by molecular weight via uniform charge coating by SDS; the Ferguson plot enables size/charge deconvolution.
- ◈2D-PAGE resolves proteoforms by combining IEF (charge/pI) and SDS-PAGE (mass), offering unparalleled resolution for intact protein analysis.
- ◈Western blotting provides antibody-based confirmation of specific proteins from electrophoretically separated mixtures.
- ◈Chromatographic methods (SEC, IEX, affinity, RP-HPLC) offer complementary separation mechanisms and are essential for both purification and LC-MS/MS workflows.