Protein Analysis

• Protein Quantification - Direct Method (UV-VIS spectroscopy)

  1. Shine a UV light on protein mixture, calibrate the curves, make a spectra

  2. Measure protein absorbance at roughly 280nm (this is the wavelength aromatic amino acids absorb UV light)

  3. UV-VIS absorption is proportional to their aromatic amino acid content and total conc (more absorption = more aromatic amino acids)
• Protein Quantification - Biuret assay

  Take protein, add in copper ions (blue), when it complexes with protein it generates deep purple colour which you can measure. More intense colour = more protein
• Pros of Biuret assay

  • Compatibility with most surfactants (detergents) – good for impure protein mixes
  • Linear response curve (R2 > 0.95)
  • Less protein-protein variation than the Coomassie dye-based assays
• Cons of Biuret assay

  • Incompatibility with substances that reduce copper
  • Incompatibility with common reducing agents such as DTT
• Nuclear magnetic resonance (NMR)

  NMR provides a real-life image of the protein structure in solution

  Final step is to do additional input whether this is determining hydrogen bonds, composition of amino acids, etc. to construct a fairly accurate representation of the protein in solution
• X-ray crystallography

  Snapshot of the protein at a certain point in time

  Take solution of protein and crytallise protein in soluble

  Technically freezing protein in a particular state
• Some amino acids are prone to degradation by 6 molar HCl used in amino acid analysis (e.g. serine, threonine, tyrosine, tryptophan, glutamine, cysteine) and this can throw off analysis
• Amino Acid Analysis Process - Determination of amino acid sequence

  1. Use ion-exchange and/or HPLC

  2. Run free amino acids through the chromatography to create a standard

  3. This generates a reference sheet – we know what amino acids come out of the elusion column at what time and in what order

  4. When we run our sample through the same column, we know what we have based on the timing's things come out of the column

  5. We map that against our standards
• Mass Spectrometry - Proteomics

  • Technique used to detect, identify, and quantitate proteins based on the mass to charge ratio (m/z)
  • High sensitivity – analytes detected at attomolar range (10-18)
  • Two ion sources – electrospray and MALDI
  • Neutrally charged compounds cannot be analysed by a mass spectroscopy – compound has to be ionised
  • Ion source sprayed into analyser which are in a vacuum (vacuum maintains the ions – ions would bump into each other and neutralise without it and not be analysed)
  • You can isolate in 2 ways: HPLC chromatography before the mass spec, or put the mixture into the mass spec and use the mass analyser to separate them out before sending them to the detector
• Pros of Mass Spectrometry - Proteomics

  • Used to detect and quantify proteins, allowing you to determine how much of a chosen analyte there is in a mixed sample
  • Very sensitive technique (attomolar range)
• Usage of mass spec

  • Proteomics – analysis of proteins within mass spec (different from cell biology proteomics)
  • Determine protein structure, function, folding, interactions
  • Identify a protein from the mass of its peptide fragments
  • Detect specific post-translational modifications throughout complex biological mixtures using workflows for phosphoproteomics and protein glycosylation
  • Quantitate proteins (relative or absolute) in a given sample
  • Monitor enzyme reactions, chemical modifications, and protein digestion
• Bottom up proteomics workflow

  Start from single amino acid and build the protein, Use enzyme called trypsin to digest protein down into single peptide fragments, Then do mass spec analysis and end up with picture of all the different fragments the protein is made of, This method will also detect glycosylation (will tell what the sugars are and where they are located)

  
• Top down proteomics workflow

  Starts with intact protein, Stick it straight into mass spec, From that you get entire molecular weight of protein, If you have a complexed protein (e.g. dimer, trimer) the mass will correspond with the complexed form not the individual protein, so correction is needed to get the right weight, Once you have this weight you can then fragment proteins into ions and analyse them individually to allow you to identify any modifications the protein has gone through

  
• Protein glycosylation

  Plays a vital role in a variety of biochemical processes as many glycans undergo disease-related expression, Allows us to identify every sugar on a protein, where they are, their nature, and give us a clear indication of what the protein consists of as a whole, This is especially important in the analysis of monoclonal antibodies as these have a lot of sugars on their surface which are vital to function

  
• Protein glycosylation analysis workflow

  Probably began top down: Sugars digested, either chemically or enzymatically, releasing them from the protein, Then chemical modifications, Then put into a mass spec to analyse the simple sugars

  The intact, whole protein is analysed through mass spec: This allows you to have your protein molecular weight, identification of post-translational modifications, and the mass of sugars within there

  You can also digest the proteins into fragments which will each contain certain sugars in different positions, You can then separate the different pieces, do a mass spec, and software will allow you to construct the fragments back into the original protein: This will give you things like sequence confirmation, glycan confirmation, etc.

  
• Monoclonal Antibody Analysis

  Understanding all sources of variation that may impact critical quality attributes (CQAs) such as glycosylation pattern, charger variants, aggregates, and fragments or low molecular weight species to enable identification of potential deleterious variants and allow assessment of product heterogeneity

  Analysis of McA needs to assess: Physiochemical properties (MW, UV-VIS and circular dichroism spectra), Structural properties (amino acid sequence, assessment of PTM glycosylation with identification of glycosylation sites/linkage), Bioactivity

  
• Isoelectric Point

  The pH at which a particular molecule carries no net electrical charge (the positive ions balance the negative ions)

  For proteins, it is the sum of the charges of ALL amino acids in the sequence that determine the overall protein pI

  pI is a critical parameter for many analytical biochemistry and proteomic techniques (2D gel electrophoresis, capillary isoelectric focusing, x-ray crystallography, and LC-MS)
• Determining protein isoelectric point

  It's the amount of balance between protonation and deprotonation of the amino and carboxylic acid groups that will determine the overall charge

  In acidic medium, the carboxylic acid group becomes protonated leading to a net positive charge

  In basic medium, the NH3 group becomes deprotonated leaving a net negative charge from the carboxylic acid

  For proteins, it's the sum of all the positive and negative charges from the amino acids that determines the overall net charge and isoelectric point
• When dealing with proteins, we have huge sequences of amino acids that contain both positive and negative charge residues
• Affinity chromatography


  Technique to purify proteins by exploiting specific interactions between the protein and a solid support

  
• Capillary electrophoresis-mass spectrometry (CE-MS)


  Technique that combines capillary electrophoresis separation with mass spectrometry detection