Proteomics - Chapter 3 Notes

Proteomics: Chapter 3 Notes

  • Proteomics: the proteome is the functional representation of the genome.

    • From the genome we know all genes present in an organism.

    • Central dogma recap: DNA -> RNA -> Protein.

    • The proteome identifies the proteins encoded by these genes.

    • Definition: Proteomics is the qualitative and quantitative comparison of proteomes under different conditions to unravel biological processes.

  • Learning objectives (quick reference):

    • Understand the principles of basic biochemical characterization techniques.

    • Be able to identify the specific biochemical technique appropriate for a given biochemical problem.

  • Topics covered (overview):

    • Protein purification

    • Protein sequencing

    • Immunology (detection of small amounts of proteins; in vivo imaging)

    • Artificial protein preparation (peptide synthesis)

    • 3-D structures of proteins

    • Mass spectrometry (MS) in proteomics

  • Why protein isolation/characterization matters:

    • Proteins perform key functions (catalysis, transcription, translation, signal transduction, etc.).

    • Physical identification and characterization help understand function, mechanism, and regulation.

  • Bioassay workflow (Stepwise):

    • Step 1: Isolate protein (preparatory steps).

    • Step 2: Assay to confirm the correct protein has been isolated or to understand protein function (analytical steps).

  • Differential Centrifugation

    • Separates cellular components and associated proteins based on the compartmental localization within cells.

    • Sequentially increases centrifugal force to partition organelles and protein complexes.

  • Protein isolation techniques (overview):

    • Salting out

    • Dialysis

    • Gel-filtration chromatography (size exclusion)

    • Ion-exchange chromatography (charge-based separation)

    • Affinity chromatography (specific binding to ligands or tags)

    • High-Performance Liquid Chromatography (HPLC)

    • Gel electrophoresis

    • Isoelectric focusing

    • (Core idea) Protein purification is based on physicochemical properties: solubility, size, charge, specific binding activity.

  • Salting out (principle and purpose)

    • Based on solubility and hydrophobicity changes with salt concentration.

    • Increasing salt disrupts water hydration shells; hydrophobic portions aggregate.

    • Proteins precipitate at different salt concentrations allowing fractionation.

    • Example: ammonium sulfate precipitation.

  • Dialysis

    • Uses a membrane with a molecular weight cut-off (MWCO).

    • Small molecules (salts, solutes) diffuse out; larger proteins stay inside.

    • Property exploited: size-based separation.

  • Chromatography: general concept

    • Separation based on differences in interaction with mobile vs. stationary phases.

    • Types of chromatography mentioned: Ion-exchange, Size-exclusion (gel-filtration), Affinity chromatography.

    • Liquid chromatography uses a liquid mobile phase and a solid stationary phase.

    • Common chromatographic columns: various chemistries enable different selectivities.

    • Retention time is a key readout in liquid chromatography.

  • Gel Filtration Chromatography (Size-based)

    • Principle: size exclusion; porous beads create aqueous internal spaces.

    • Large molecules cannot enter beads and elute earlier; small molecules penetrate beads and elute later.

    • Diagram concepts: flow through beads; separation by molecular size.

    • Useful for separating proteins from smaller molecules and for preliminary desalting/polishing.

  • Ion Exchange Chromatography (Charge-based)

    • Property: charge interaction with ion-exchange resins (exchanger).

    • Common stationary phases (examples):

    • Strong cation exchanger: Sulfopropyl (SP) with functional group -CH2CH2CH2SO3−

    • Weak cation exchanger: Carboxymethyl (CM) with -O-CH2COO−

    • Strong anion exchanger: Quaternary ammonium (Q) with -N+(CH3)3

    • Weak anion exchanger: Diethylaminoethyl (DEAE) / Diethylaminopropyl (ANX) with basic amine groups

    • Elution strategies:

    • Increase ionic strength to out-compete the analyte from the stationary phase.

    • Adjust pH to change the ionization state of the buffer, analyte, or stationary phase.

    • Note: Resin choice determines cationic vs. anionic separation and strength of binding.

  • Affinity Chromatography

    • Principle: isolate proteins based on specific interactions with a ligand attached to column beads.

    • If a protein binds a ligand X, you can attach X to the column and then elute by changing conditions or using a competitor.

    • High affinity, non-covalent interactions ensure specificity.

    • Common strategies:

    • Tags: His, GST, FLAG, biotin (for affinity tags)

    • Immuno-affinity (antibody-based capture)

    • Practical idea: very high purity can sometimes be achieved in a single step when a strong specific interaction is available.

  • High-Performance Liquid Chromatography (HPLC)

    • Finer column material requires higher pressures for separation; yields better resolution with smaller sample requirements.

    • Adsorption chromatography variants: normal phase, reverse phase, hydrophobic interactions, HILIC.

    • Typically used for analytical separations; preparative scale separations may require scale-up.

  • Reverse-Phase HPLC (RP-HPLC)

    • Column phase is hydrophobic; nonpolar residues interact with the stationary phase.

    • Large polypeptides often do not partition deeply; they adsorb to the hydrophobic surface and remain there until organic solvent concentration increases enough to desorb them.

    • Organic solvent example: CH3CN (acetonitrile).

    • Mechanistic summary:

    • Polypeptide enters column in mobile phase

    • It adsorbs to RP surface

    • It desorbs when the organic solvent concentration reaches a critical level

    • Visualization idea: peptides sit on the stationary phase with hydrophobic interactions driving retention.

  • Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

    • Used to assess protein purity and approximate size.

    • Key variables: v (velocity of migration), E (electric field strength), z (net charge), F (frictional coefficient), η (viscosity).

    • Property: primarily size-based separation under denaturing conditions (SDS-coated proteins behave as spheres with uniform charge-to-mass ratio).

    • Mobility concept: electrophoretic mobility is inversely related to protein size.

    • Detection options:

    • Coomassie Blue staining (sensitive to ~0.1 μg or ~2 pmol)

    • Silver staining (more sensitive, ~0.02 μg)

    • Key relation to remember: electrophoretic mobility μ ∝ 1/log(Mw) for proteins in SDS-PAGE context.

  • Isoelectric Focusing (IEF)

    • Concept: separates proteins by isoelectric point (pI, where net charge is zero).

    • In a pH gradient, proteins focus at pH where their net charge is zero.

    • Diagram idea: polyampholytes create a pH gradient; proteins migrate to their pI and stop.

    • Important for resolving proteins with similar sizes but different charges.

  • Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE)

    • Combines IEF (first dimension) with SDS-PAGE (second dimension).

    • Provides high-resolution separation of complex mixtures by separating first by pI and then by size.

    • Conceptual visualization: a map of proteins separated across two orthogonal axes.

  • Quantification of a purification protocol (Table 3.1, summary interpretation)

    • Purpose: illustrate how total protein, total activity, specific activity, yield, and purification level change across purification steps.

    • Example steps (from table):

    • Homogenization: total protein ~15,000 mg; total activity ~150,000 units; specific activity ~10 units/mg; yield 100%; purification level 1.

    • Salt fractionation: total protein ~4,600 mg; total activity ~138,000 units; specific activity ~30 units/mg; yield ~92%; purification level 3.

    • Ion-exchange chromatography: total protein ~1,278 mg; total activity ~115,500 units; specific activity ~90 units/mg; yield ~77%.

    • Gel-filtration chromatography: total protein ~68.8 mg; total activity ~75,000 units; specific activity ~1,100 units/mg; yield ~52%.

    • Affinity chromatography: total protein ~1.75 mg; total activity ~52,500 units; specific activity ~30,000 units/mg; yield ~110%; purification level ~35 (illustrative).

    • Overall takeaway: successive steps increase specific activity and purification while reducing total protein; yields typically decline with each step.

  • Protein purification and sequencing topics recap (Page 21 & 32)

    • Reiterates six key topics:

    • Protein purification

    • Protein sequencing

    • Immunology (detection of small amounts; in vivo imaging)

    • Artificial protein prep (peptide synthesis)

    • Mass spectrometry

    • 3-D structures

  • Amino acid sequencing: initial approach (Page 22)

    • Example sequence fragment: Ala-Gly-Asp-Phe-Arg-Gly.

    • Steps to determine sequence yield:

    • Hydrolyze with 6 M HCl to break peptide bonds.

    • Use ion-exchange chromatography to separate amino acids.

    • Quantify with ninhydrin reagent (or fluorescamine for small quantities).

  • Edman degradation sequencing (page 23)

    • Edman degradation sequence method (Edman degradation): sequence a peptide one amino acid at a time from the N-terminus.

    • Process outline:

    • Label the N-terminal amino acid with phenyl isothiocyanate (PITC).

    • Release the N-terminal residue as a phenylthiohydantoin (PTH) derivative after a reaction step.

    • Repeat for successive rounds to obtain sequence (e.g., first few residues shown in a schematic).

    • Visual notation in figures shows sequential labeling and release steps, with the PTH-AA fragment identified after each cycle.

  • High-Performance analyses: HPLC for Edman products (Page 24)

    • HPLC is used to separate PTH derivatives produced by Edman degradation.

    • Detection often via absorbance at 254 nm (A254).

    • Elution profiles show distinct peaks corresponding to successive PTH amino acids.

  • MALDI-TOF Mass Spectrometry: basic principles (_pages 25–26)

    • MALDI-TOF basics:

    • Sample is mixed with a matrix and ionized by a laser.

    • Ions are accelerated by an electric field into a flight tube.

    • Lighter ions reach the detector first (time-of-flight principle).

    • Typical MALDI-TOF spectrum shows a series of peaks corresponding to the protein/peptide masses (m/z values).

    • Practical notes:

    • Provides accurate molecular weights and proteoform information for relatively large biomolecules.

    • Visual example peaks include insulin, β-lactoglobulin, etc., at characteristic m/z values.

    • Example interpretation: a peak at a particular m/z corresponds to a charge state and the mass of the ionized species; common charge states are considered for deconvolution.

  • Sequencing via Mass Spectrometry (MS/MS) (Page 27)

    • Tandem MS (MS/MS) concept:

    • First mass analyzer selects a parent ion (the intact peptide/protein).

    • The ion is fragmented (e.g., by collision with inert gas like He or Ar).

    • A second mass analyzer measures the mass-to-charge ratios of the resulting product ions.

    • Summary workflow:

    • Obtain parent peak in MS (MS1).

    • Fragment to produce product ions (MS2) for sequencing.

    • Practical resources: JOVE links referenced for tandem MS and peptide identification using MS/MS.

  • MS/MS data interpretation (example fragments) (Page 28)

    • Illustration shows fragment ions corresponding to different cleavages along a peptide sequence (e.g., Glu-Gly-Arg-Glu-Gly-Met-Arg side chains).

    • Reading MS/MS spectra involves matching observed fragment masses to theoretical b- and y-ions (sequence is inferred from mass differences).

    • Visuals provide ordering of fragments and their intensities to deduce amino acid sequence.

  • Tackling larger proteins with cleavage strategies (Page 29)

    • Proteins too large for straightforward sequencing can be digested into smaller peptides.

    • Example cleavages:

    • Trypsin (cleaves after Lys or Arg)

    • CNBr (cyanogen bromide) cleaves at methionine residues

    • Other reagents/enzymes listed for partial digestion strategies to produce manageable fragments.

    • Concept: partial proteolysis aids in sequence determination by generating a library of overlapping peptides for assembly.

  • Partial proteolysis and specific cleavage strategies (Table 3.3, Page 30)

    • Chemical cleavage reagents and their cleavage sites:

    • Cyanogen bromide: carboxyl side of methionine residues

    • O-lodosobenzoate: carboxyl side of tryptophan residues

    • Hydroxylamine: Asn-Gly bonds

    • 2-Nitro-5-thiocyanobenzoate: amino side of cysteine residues

    • Enzymatic cleavage options:

    • Trypsin: carboxyl side of lysine and arginine residues (often yields manageable peptides)

    • Chymotrypsin: carboxyl side of aromatic residues (Phe, Tyr, Trp)

    • Other proteases listed with specific specificities (e.g., clostripain, thrombin, carboxypeptidases)

    • Purpose: create overlapping peptides enabling reconstruction of the full sequence.

  • Building the sequence from peptide clues (Page 31)

    • Example workflow: compile tryptic and chymotryptic peptides, identify overlaps, and assemble into a contiguous sequence.

    • Illustrative peptides:

    • Tryptic peptides: Ala-Ala-Trp-Gly-Lys; Thr-Phe-Val-Lys

    • Chymotryptic peptide: Val-Lys-Ala-Ala-Trp

    • Tryptic peptide: Thr-Phe-Val-Lys-Ala-Ala-Trp-Gly-Lys

    • Overlaps between tryptic and chymotryptic fragments enable sequential assembly of the protein sequence.

  • Next time topics (Page 32 recap)

    • Revisit the six major topics:

    • Protein purification

    • Protein sequencing

    • Immunology (detecting small amounts; in vivo imaging)

    • Artificial protein prep (peptide synthesis)

    • Mass spectrometry

    • 3-D structures

  • Quick connections to foundational principles and real-world relevance

    • Separation science leverages intrinsic properties of biomolecules (size, charge, hydrophobicity, binding affinity).

    • Combining orthogonal methods (e.g., IEF + SDS-PAGE, or Edman degradation + MS) yields high-confidence protein identification.

    • Mass spectrometry-based sequencing has become a central tool for proteomics due to speed, sensitivity, and ability to handle large protein datasets.

    • Purification workflows model the practical balance between purity, yield, and resource use; Table 3.1 illustrates how each step impacts metrics.

  • Ethical, philosophical, and practical implications

    • High-purity protein preparations are crucial for accurate functional studies, therapeutic development, and diagnostic assays.

    • Peptide synthesis and manipulation of protein sequences raise considerations about bioethics, safety, and regulatory oversight when applied to therapeutic contexts.

    • Data interpretation in proteomics (e.g., MS identifications) requires rigorous controls to avoid misassignment and to ensure reproducibility across labs.

  • Key formulas and concepts to remember

    • Electrophoretic mobility relationship (conceptual):

    • extmobility (μ)∝1log⁡(Mw)ext{mobility} \, (\mu) \propto \frac{1}{\log(M_w)}

    • This captures the general idea that larger proteins migrate more slowly in SDS-PAGE due to their larger hydrodynamic radius.

    • Edman degradation yields sequential N-terminal residues with each cycle, provided the N-terminus is free and accessible.

    • Mass spectrometry sequencing relies on accurate mass measurements of parent and fragment ions to deduce amino acid sequences.

  • Practical tips for exam prep

    • Be able to pair a protein question with an appropriate technique based on the protein property to exploit: size, charge, hydrophobicity, or specific binding.

    • Remember the typical order of purification steps and what each step accomplishes (e.g., rough cleanup by salting out, then more selective steps like ion-exchange, then polishing steps like affinity).

    • Know the core MS/MS workflow: select parent ion -> fragment -> detect product ions -> deduce sequence.

    • Be familiar with common proteases and their specific cleavage sites (trypsin after Lys/Arg; chymotrypsin after bulky hydrophobic residues; CNBr after Met).