Sedimentation velocity analytical ultracentrifugation has become a very popular technique to study size distributions and
interactions of macromolecules. Recently, a method termed two-dimensional spectrum analysis (2DSA) for the determination of
size-and-shape distributions was described by Demeler and colleagues (Eur Biophys J 2009). It is based on novel ideas conceived for fitting the integral equations of the size-and-shape distribution to experimental
data, illustrated with an example but provided without proof of the principle of the algorithm. In the present work, we examine
the 2DSA algorithm by comparison with the mathematical reference frame and simple well-known numerical concepts for solving
Fredholm integral equations, and test the key assumptions underlying the 2DSA method in an example application. While the
2DSA appears computationally excessively wasteful, key elements also appear to be in conflict with mathematical results. This
raises doubts about the correctness of the results from 2DSA analysis.
Keywords Analytical ultracentrifugation - Fredholm integral equations - 2DSA - Sedimentation velocity - Lamm equation - Size distribution