Summary and overview of the different particle–protein corona isolation methods and their advantages and disadvantages as discussed in the text.

Method	Advantages	Disadvantages
Centrifugation	- Separation according to density and size	- Long centrifugation times can lead to false particle–protein interactions
	- Most frequently used technique	- Several purification steps needed; modifications in the protein–corona system can occur
	- Widely used and easy to use216	- Magnetic nanoparticles can agglomerate196 false-positive highly abundant proteins due to insufficient washing
	- High throughput216	- Changing of centrifugation tubes is necessary to exclude carryover of proteins adsorbed to the tube walls
	- Centrifugal speeds and times can be optimized according to the nanoparticle material and media	- Outcome affected by centrifugation force, washing duration, washing solution and solution volumes; must be adjusted for each particle type
	- Tuning experimental conditions makes the method available for a wide range of nanoparticles	- The smaller and less dense the particles, the higher the centrifugation speeds chosen; thus aggregation30 occurs
	- High resolution results30	- Not suited for very small (5–20 nm) or low density nanoparticles (1 g cm−3), because unbound proteins and protein corona complexes cannot be separated effectively30,217
	- Possibility of separating different populations co-existing in situ30	- Not preparative, so no populations can be recovered for further studies23,29,206
Size exclusion chromatography (SEC)	- Flexible technique, many stationary/mobile phases	- Interaction between analytes and the stationary phase can occur218
	- can be used with standard lab equipment	- SEC selectivity decreases when applied to analytes with a very high molar mass such as nanoparticles218
	- Analyte resolution and recovery in SEC is generally superior to A4F218	- Low throughput
	- Has been developed into a systematic methodology14	- No full recovery of hard corona complexes for further studies30
	- Less perturbing than centrifugation14
Asymmetric flow-field-flow fractionation (A4F)	- Complex, heterogeneous and polydisperse dispersions can be investigated without extensive sample preparation205	- Long establishment process
	- Reduced to no destruction or alteration of the protein corona	- Must be adjusted for every particle type205
	- Prior fractionation by AF4 allows size investigation of complex heterogeneous and polydisperse mixtures205	- Low throughput205
	- Several detection techniques can be coupled to AF4 (online and offline)205	- Expensive
	- Possible automation205	- Not routinely available in many analytical laboratories
	- Short measurement time205	- Separation of particles from a very polydisperse sample leads to peak broadening and loss of resolution; must thus be divided into several experiments
	- Easy collection of fractions205	- Sample loss due to adsorption on the membrane can occur, affecting retention and disturbing quantification of single fractions205
	- Absence of a packaging material or a stationary phase205	- No full recovery of fraction for further experiments30
	- The potential of AF4 increases with increasing molar mass205
	- Once established, AF4 is a multifunctional technique for separation and characterization of nearly all nano-sized205particles
Magnetism	- Low impact on the structure	- Only practicable for small (∼10 nm), magnetic nanoparticles196
	- High throughput	- Degree of separation decreases with decreasing magnetism196