3 Amino Acids, Peptides, and Proteins — Protein Separation and Analysis Techniques

Vocabulary-style flashcards covering chromatographic separation, electrophoresis, protein structure, labeling, proteolysis, and mass spectrometry concepts from the notes.

Column Chromatography

A protein separation technique where a buffered mobile phase passes through a porous solid stationary phase; separation depends on protein properties affecting migration rates.

Mobile Phase

The liquid buffer that carries proteins through the column in chromatography.

Stationary Phase (Solid Phase)

The porous solid material through which the mobile phase moves and proteins migrate at different rates.

Ion-Exchange Chromatography

Chromatography that separates proteins based on net charge; pH and salt concentration affect binding to charged resin.

Cation Exchanger

An ion-exchange resin that is negatively charged and binds positively charged ions (cations).

Anion Exchanger

An ion-exchange resin that is positively charged and binds negatively charged ions (anions).

Size-Exclusion Chromatography (Gel Filtration)

Chromatography that separates molecules by size; larger proteins elute first than smaller ones.

Affinity Chromatography

Chromatography that separates based on specific binding interactions between a protein and a ligand bound to a matrix.

Affinity Matrix

Beads with immobilized ligand used to capture proteins that bind to the ligand.

Elution (in chromatography)

The process of washing off bound proteins from the chromatography matrix, often by changing salt or ligand conditions.

Electrophoresis

Technique to visualize and characterize purified proteins; estimates number of proteins, purity, isoelectric point, and approximate molecular weight.

Polyacrylamide Gels

Gels used in electrophoresis to separate proteins based on size and charge.

Coomassie Blue

A dye that binds to proteins to visualize them after electrophoresis.

Isoelectric Point (pI)

The pH at which a protein carries no net electric charge.

Isoelectric Focusing (IEF)

Technique that separates proteins along a pH gradient according to their pI values.

Two-Dimensional Electrophoresis (2D-E)",

An electrophoresis method that first separates by pI (isoelectric focusing) and then by size, increasing resolution.

Primary Structure

Covalent bonds linking amino acid residues in a polypeptide chain.

Secondary Structure

Recurring structural patterns in proteins (e.g., alpha helices and beta sheets).

Tertiary Structure

Three-dimensional folding of a single polypeptide chain.

Quaternary Structure

Organization of two or more polypeptide subunits into a multi-subunit complex.

Edman Degradation

A classic method for sequencing amino acids from the N-terminus of a protein.

Polymorphism (Amino Acid Variants)

Most human proteins are polymorphic and exist as sequence variants.

FDNB, Dansyl Chloride, Dabsyl Chloride

Labeling reagents that tag the amino-terminal and lysine residues to study protein structure.

Performic Acid Oxidation

Oxidation method that breaks disulfide bonds to linearize proteins.

Dithiothreitol (DTT)

A reducing agent that cleaves disulfide bonds in proteins.

Disulfide Bond

Covalent link between cysteine residues that can connect polypeptide chains; reducible to separate chains.

Trypsin

Protease that cleaves after Lys or Arg (except when followed by Pro).

Chymotrypsin

Protease that cleaves after Phe, Trp, or Tyr.

Staphylococcus aureus V8 Protease

Protease that cleaves after Asp or Glu.

Asp-N Protease

Protease that cleaves at the N-terminal side of Asp and Glu.

Pepsin

Protease that cleaves after Leu, Phe, Trp, or Tyr (optimally in acidic conditions).

Endoproteinase Lys-C

Protease that cleaves after Lys residues.

Cyanogen Bromide (CNBr)

Chemical reagent that cleaves at methionine residues.

Mass Spectrometry (MS)

Analytical technique that measures molecular mass with high accuracy and can sequence short peptides and analyze entire proteomes.

MALDI-MS

Mass spectrometry using Matrix-Assisted Laser Desorption/Ionization; ionizes proteins without breaking them.

ESI-MS (Electrospray Ionization)

Mass spectrometry technique that transfers ions from solution to gas phase for analysis.

Time-of-Flight (TOF)

Mass analyzer that separates ions by measuring their flight time, which depends on m/z.

Orbitrap

Mass analyzer that traps ions and measures m/z via orbital motion and Fourier transform.

Tandem Mass Spectrometry (MS/MS)

Two mass filters in sequence; first selects ions, second fragments them to yield sequence information.

LC-MS/MS

Combination of liquid chromatography with tandem mass spectrometry to identify proteins and quantify their abundance.

Overlapping Peptides

Peptide fragments whose sequences overlap, enabling reconstruction of the original protein sequence.

Edman Degradation vs Mass Spectrometry Sequencing

Edman sequentially fragments the N-terminus; MS sequencing deduces sequence from mass and fragment patterns.