Fixing Proteomics Campaign

Protocols

Alternative methods available

The problem of post-sampling changes can be addressed in a number of ways. The preferred strategy will depend on multiple diverse factors such as: type of sample, level and nature of degradation, and downstream analysis technique. Below is a table of common alternatives for addressing post-sampling issues with some pros and cons for the various techniques.

Methods	Pros	Cons
Freezing	Low cost No additives required	Damage to morphology Puncture of cell membrane provide enzymes access Only temporary inhibition since degradation re-starts during thawing
Enzyme inhibitors	Selective inhibition possible Preserves activity of interest, allows for measurement of enzyme activity	Not complete inactivation, only reduction in activity Addition of chemicals to sample Time consuming for inhibitors to reach target High toxicity Inhibitors are selective, making optimization of cocktails necessary Affecting certain downstream analysis methods
Microwave (MW) radiation	Rapid process No additives required	Risk of local hot and/or cold spots Specific equipment required (focused MW) Ethical issues (focused MW) Only usable on small rodent skulls (focused MW) Risk for micro steam eruption, compromising morphology Difficult to control Only for fresh tissue Not possible to measure enzyme activity Denaturing buffers required for resolubilization
Stabilizor T1	Useful for both fresh and frozen samples Rapid process No additives required	Specific equipment required Denaturing buffers required for resolubilization Not possible to measure enzyme activity
Organic solvents	Low cost Easy accessible	Toxic and flammable Addition of chemicals to sample Risk of loss due to unspecific adhesion to plastic
Drying/freeze drying	Low cost storage	Sample needs to be frozen Risk of degradation during drying if not frozen Damage to morphology Specific equipment required

Protocols

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