PDI stands for polydispersity index. It is a numerical measure of the distribution of molecular sizes, such as mass or radius, in a sample. In the context of proteins and polymers, PDI tells you how uniform or varied the molecules are. A low PDI means the sample is monodisperse, with molecules nearly all the same size. A high PDI means the sample is polydisperse, containing a wide range of different sized molecules.

What does polydispersity mean?

Polydispersity refers to the presence of a mixture of species that differ in size, shape, mass or conformation within a single sample. Instead of being a uniform collection of identical molecules, which would be a monodisperse sample, a polydisperse sample contains a range of different molecular forms.

What do PDI values mean?

In FIDA terms, a sample with a PDI below 0.05 is highly monodisperse. Between 0.05 and 0.15 the sample is monodisperse. Above 0.15 it is polydisperse.

What makes a sample polydisperse?

Polydispersity can result from several different oligomerisation states in the same sample, for example monomers, dimers and trimers. It can come from post-translational modifications such as glycosylation or phosphorylation. It can come from degradation products, for instance partial proteolysis. It can come from aggregation, whether small or large protein aggregates. And it can come from conformational heterogeneity, where the same molecule is present in different folding states.

Most proteins have a single polypeptide length and size, so they should be monodisperse. A polydisperse protein is indicative of aggregation.

Why measure polydispersity?

Knowing the polydispersity of a sample tells you how uniform or heterogeneous the molecules in it are, which directly affects the quality, reliability and interpretability of downstream experiments. It serves three purposes: to examine sample quality, to predict downstream assay challenges, and to detect sample loss.

Where is polydispersity relevant?

Protein quality after purification

During purification the aim is to isolate one specific, uniform form of the protein, correctly folded, monomeric and properly assembled. PDI measures how homogeneous the purified sample actually is in terms of size or molecular weight distribution. A low PDI indicates good purification. A high PDI points to problems: aggregates, oligomers or fragments. The same measurement can also be used to study protein quality in different storage buffers, or after freeze and thaw cycles.

Membrane protein solubilisation

PDI is an important criterion for selecting a solubilising detergent during membrane protein solubilisation. A low PDI means uniform protein and detergent complexes, indicating good solubilisation and producing monodisperse complexes, which are ideal for work such as crystallography, cryo-electron microscopy or functional assays. A high PDI means heterogeneous complexes, some small, some large aggregates, or mixed oligomeric states, which suggests poor solubilisation, aggregation or instability.

Vesicle preparation

In the production of liposomes, exosomes, lipid nanoparticles and other nanoparticles, PDI is a critical measure of how uniform the vesicles are in size. Uniform vesicle size matters for drug delivery, because it gives consistent drug release rates, biodistribution and targeting. It matters in synthetic biology, where stable and predictable systems require vesicles of similar size. And it matters for analytical reproducibility, since heterogeneous vesicles make experimental results noisy or unreliable.

Biomolecular condensate formation

When proteins or RNAs undergo phase separation they form droplets, called condensates, of various sizes. PDI measures the spread of droplet sizes within a population of condensates. A low PDI suggests well regulated growth and fusion of condensates, which may itself be important for biological function. Over time condensates may coarsen, as small droplets fuse into larger ones, or solidify, and PDI increases as the size range broadens.

Agglutination

In agglutination, where particles clump together, often as a result of antibody binding, particles can vary in size after they clump. As agglutination progresses the size distribution of the clumps becomes broader, because some are small aggregates and others are large, irregular clusters. A low PDI suggests the clumps formed are similar in size, meaning the agglutination is relatively uniform. A high PDI indicates a wide range of clump sizes, meaning it is heterogeneous.

How is polydispersity measured in FIDA?

Polydispersity is derived from the same in-solution measurement that gives molecular size, so it does not require a separate experiment. Because of its resolution and precision, FIDA can detect subtle size heterogeneity.

PDI fitting can be applied to a single data point, or to a whole batch of data at once using the automatic analysis option. The processed batch is then sent to the QC dashboard, which compiles all sample parameters including PDI, so polydispersity arrives alongside size, aggregation and the other quality parameters from the same run.

Frequently asked questions

What is the difference between monodisperse and polydisperse?

A monodisperse sample is a uniform collection of identical molecules, all of a similar size. A polydisperse sample contains a distribution of different species, differing in size, shape, mass or conformation.

Does a high PDI mean my protein is aggregating?

Not necessarily on its own, but it is a strong indicator. A polydisperse protein is indicative of aggregation, and aggregation is one of five listed causes of polydispersity, alongside oligomerisation states, post-translational modifications, degradation products and conformational heterogeneity.

Related resources

The poster Characterising Lipid Nanoparticles with FIDA covers PDI in vesicle work, and the poster High Throughput Detergent Screening for Membrane Proteins uses it to choose a solubilising detergent. For oligomerisation as a cause of polydispersity, see the application note Reversible Oligomerization and FIDA, and for gene therapy work 360 degree automated characterization of AAVs.

On this site: the Polydispersity Index readout, What is protein aggregation? and the Molecular Size readout.