MultiSig: a new high-precision approach to the analysis of complex biomolecular systems

Abstract

MultiSig is a newly developed mode of analysis of sedimentation equilibrium (SE) experiments in the analytical ultracentrifuge, having the capability of taking advantage of the remarkable precision (~0.1 % of signal) of the principal optical (fringe) system employed, thus supplanting existing methods of analysis through reducing the ‘noise’ level of certain important parameter estimates by up to orders of magnitude. Long-known limitations of the SE method, arising from lack of knowledge of the true fringe number in fringe optics and from the use of unstable numerical algorithms such as numerical differentiation, have been transcended. An approach to data analysis, akin to ‘spatial filtering’, has been developed, and shown by both simulation and practical application to be a powerful aid to the precision with which near-monodisperse systems can be analysed, potentially yielding information on protein-solvent interaction. For oligo- and poly-disperse systems the information returned includes precise average mass distributions over both cell radial and concentration ranges and mass-frequency histograms at fixed radial positions. The application of MultiSig analysis to various complex heterogenous systems and potentially multiply-interacting carbohydrate oligomers is described.

Glargine and degludec: Solution behaviour of higher dose synthetic insulins

“…Sedimentation equilibrium profiles were analysed according to two relevant analysis routines: INVEQ 16 and MULTISIG 17 . INVEQ is able to provide estimates of the second virial coefficient; and MULTISIG provides low-resolution distributions of molar masses.…”

“…Data were analysed using two algorithms. The MULTISIG-RADIUS algorithm 17 was used to fit distributions of molar mass over the radius of the cell using the fitting software ProFit (QuantumSoft, Switzerland). An adaptation of the INVEQ algorithm 16 was used to yield the second virial coefficient, a measure of non-ideality of a macromolecular solution, fitted using OriginLab (Northampton, MA, USA).…”

“…Charge effects ( B Z ) can be reduced to insignificant levels through an excess of ionic strength ( I ). Analysis was also carried out using the MULTISIG algorithm 17 : Where j is the fringe increment (the concentration as measured using Rayleigh Interference optics from AUC), uncorrected for baseline E , at radial position r or reference radial position r ref . c i and σ i represent relative concentration and sigma for iteration i .…”

SEDFIT–MSTAR: molecular weight and molecular weight distribution analysis of polymers by sedimentation equilibrium in the ultracentrifuge

“…In recently published work 41 a complementary approach to sedimentation equilibrium analysis of polydisperse systems has been presented, with a focus on point average molecular weights at specific radial positions. The ‘MultiSig’ algorithm – based on a multi-exponential approach – (a) yields profiles of (reduced flotational) molecular weights (i.e.…”

Variable-Field Analytical Ultracentrifugation: I. Time-Optimized Sedimentation Equilibrium

“…This is true in partic-ular for systems in which rapid chemical interconversion, on the timescale of sedimentation in SV, causes sedimentation boundary patterns that do not directly resolve the interacting species (12), or in which intermediate reaction kinetics, thermodynamic and hydrodynamic nonideality, or microheterogeneity complicate the computational analysis of sedimentation velocity boundary shapes. Unfortunately, although important progress has been made in many aspects of SE data analysis of noninteracting and interacting systems (9,(13)(14)(15)(16)(17), SE remains limited by the sometimes prohibitively long experimental times required to reach equilibrium.…”