Lecture 2: Sample Preparation and Extraction 1

Question 1

What problems do we encounter that highlight the need for sample preparation?

Answer 1

Most biological/clinical samples are heterogeneous/mixtures (e.g. blood/ biopsies/ urine/ swabs).

Measurements usually target one analyte at a time (often found in trace amounts).

Difficulties in detection.

Direct analysis is detrimental to analytical instruments.

Question 2

What solutions do we have surrounding the problems highlighting the need for sample preparation?

Answer 2

Reduce sample complexity.

Target the analyte of interest from the mixture using sample preparation methods.

Exploit the molecular characteristics of the analyte (Molecular size, polarity, functional groups, solubility, etc..)

Convert to a (more) detectable form and ensure analytical tool compatibility (e.g. chromatography, imaging, etc).

Question 3

What is dilution?

Answer 3

Reducing concentration so it’s more suitable for analysis

Question 4

What do you need to ensure for dilution and why?

Answer 4

Ensure Instrument compatibility: Limited linear calibration/detection range (when you measure something, it must be in its dynamic range – not too low, not too high).

Question 5

Examples of when dilution is used

Answer 5

Used in calibration curves for quantification or cell counting.

Question 6

What is homogenisation/dissociation used for?

Answer 6

For heterogenous samples (e.g. solid/soft tissues).
- Isolation of intact cells from tissues.
- Lyse cells to recover subcellular components (nucleic acids/proteins).

Question 7

What methods can be used for homogenisation/dissociation?

Answer 7

Mechanical shear or with surfactants/enzyme-assist.

Disrupt membranes by ultrasonication (high-frequency sound) (32-38kHz)

Freeze-thaw cycling (water forms crystals that punch holes in the cells and make cells expand when frozen which breaks them)

Question 8

What is tissue sectioning?

Answer 8

Slices of tissue (biopsies) for microscopy done with microtome or cryostat.

Question 9

When is a microtome used for tissue sectioning?

Answer 9

Microtome: formalin fixed paraffin-embedded tissue (room temp)

Question 10

When is a cryostat used for tissue sectioning?

Answer 10

Cryostat: fresh frozen tissues to monitor real-time biological processes (fewer artefacts from formalin processing and maintains structures e.g. metabolites).

Question 11

What can tissue sectioning preparation lead to downstream?

Answer 11

• Tissue staining – morphological features
• Immunohistochemistry – protein biomarker
• Mass spec imaging – MALDI-MS
• In-situ hybridisation (ISH) – expression analysis

Question 12

What is fixation for?

Answer 12

After cryo-sectioning to preserve samples in ‘life-like state’ at a ‘fixed’ point in time – tissue sample resilience (ease of transport/storage).

Stops degradation/degeneration process (autolysis) and prevents diffusion of soluble substances.

Required for scanning and transmission EM.

Question 13

What are the methods of fixation?

Answer 13

chemical fixation
physical fixation
perfusion

Question 14

How does chemical fixation work?

Answer 14

ethanol/ formaldehyde/ glutaraldehyde (decreases solubility and allows protein cross-linking).

Question 15

How does physical fixation work?

Answer 15

heating to 37-45 degrees C to increase diffusion rates.

Question 16

How does perfusion work in fixation?

Answer 16

Distributes buffers/fixative agents via circulatory system - remove interferences from blood.

Question 17

What is the purpose of antigen retrieval?

Answer 17

Treatment of tissue sections to unmask/retrieve antigens masked/altered during fixation – breakage of cross-links or restoration of accessibility of epitopes (binding sites for antibody receptors).

Crucial before immunohistochemistry (IHC), immunofluorescence (IF) and in situ hybridisation (ISH) techniques.

Question 18

What are the types of antigen retrieval?

Answer 18

Heat-induced antigen retrieval
Proteolytic-induced antigen retrieval

Question 19

Explain proteolytic-induced antigen retrieval

Answer 19

Proteolytic-induced antigen retrieval (enhance antibody access – causes some tissue degradation).
- Pepsin, proteinase K, and trypsin proteases used at neutral pH and 37 degrees C.

Question 20

Explain heat-induced antigen retrieval

Answer 20

Heat-induced antigen retrieval (denature bridges).
- Citrate pH4.5-6 retrieval buffer for general epitopes & Tris-EDTA pH9 buffer for nuclear antigens (transcription factors inside cells).
- High temperature (95 degrees C) water bath or pulsed microwave treatment.

Question 21

What is the purpose of precipitation?

Answer 21

To reduce analyte solubility (crashing/come out of solution).

Question 22

What are the methods of precipitation?

Answer 22

salts
trichloroacetic acid (pH change)
solvents
immunoprecipitation
temperature

Question 23

Explain the use of salts in precipitation

Answer 23

Salts: protein aggregation in high-conc. ammonium sulphate (removes water – salt draws the water out).

Question 24

Explain the use of Trichloroacetic acid (pH change) in precipitation

Answer 24

Trichloroacetic acid (pH change): denaturing of tissues/proteins, disrupts hydrogen bonding but leaves peptide bonds intact.

Question 25

Explain the use of solvents in precipitation

Answer 25

Solvents: ethanol, acetone, methanol/chloroform lowers dielectric constant (decrease solubility – lowers water’s ability to solubilise or selective dissolution).

Question 26

Explain the use of immunoprecipitation in precipitation

Answer 26

Immunoprecipitation: antibodies to ‘fish out’ target analytes (antibodies bind to analytes - makes them insoluble).

Question 27

Explain the use of temperature in precipitation

Answer 27

Temperature: cold lowers solubility (degradation hampered in lower temperatures).

Question 28

Explain filtration/dialysis as a sample preparation technique

Answer 28

Size inclusion/exclusion: - Reverse osmosis (0.0001μm – 0.001μm) - Ultrafiltration (0.01μm – 0.1μm) - Microfiltration (0.1μm - 1μm) - Clarification (10μm - 100μm) Filters: - Paper discs (remove large/insoluble particles), membranes (sterile fixation – filters out pathogens), glass fibres - Gravity, vacuum, high pressure - Centrifuge filtration (spin columns – no adhesion, just a filter).

Question 29

What is the purpose of Size-exclusion chromatography (SEC)?

Answer 29

Sort/separate analytes (filtration) by size, aggregation, conformation, and hydrodynamic radius (e.g. proteins, polymers and nanoparticles).

Question 30

Describe the columns in SEC and how this separates analytes.

Answer 30

Columns: - Porous non-adsorbent stationary phase resin (different fractionation range based on pore size). - Elution by isocratic mobile phase - Detection by UV-Vis (light scattering) o 280nm – proteins o 260nm – nucleic acids - Fraction collection Smaller analytes take longer to pass through the column (smaller particles have a longer path unlike the bigger ones which go straight through) - good for purification.

Question 31

What is the purpose of centrifugation?

Answer 31

Separation by differences in density, buoyancy, sedimentation rate (denser goes to bottom). - (non) Cellular components of blood (Ficoll-paque, histopaque, lymphoprep, percoll). - Isolating sub-cellular components of lysed cells (Ultracentrifugation (gravity 20000<)

Question 32

What is the purpose of quenching?

Answer 32

Halt/freeze chemical reaction at specific timepoint (Enzyme kinetics, chemical synthesis, analysis of intermediate product, monitoring reaction states). By changing temperature or reaction mixture (with acids, bases, solvents) rapidly to preserve the substrates and products (by stopping the enzyme). ‘stop solutions’ in ELISAs

Question 33

Why is derivatisation an important technique?

Answer 33

mass spec most commonly used to analyse so analytes must be ionised and derivatisation helps with this

Question 34

Explain derivatisation

Answer 34

Labelling/tagging – alter chemical structure etc. to increase detectability, identification, ionisation efficiency, sensitivity, selectivity, stability (gives constant charge (ionised) therefore always polar). Enable high-throughput analysis: - Multiplexing – derivatives of reagent taken (short time/lots of samples) with different hydrazine tags that have different masses. - Isobaric mass-tags in proteomics (<16 plex). in GC-MS analysis – increase volatility, thermal stability, and aid separation (needs to be non-polar to decrease the boiling point to become gas easily – to be volatile) - Trimethylsilylation (polar tagging) of oestrogens (estrone and estradiol)

Question 35

What is the use for perfusion in medical imaging?

Answer 35

In nuclear/radiation medicine to assess blood flow, organ function, metabolic activity, etc.