Game-theory-based search engine to automate the mass assignment in complex native electrospray mass spectra.

Electrospray ionization coupled to native mass spectrometry (MS) has evolved into an important tool in structural biology to decipher the composition of protein complexes. However, the mass analysis of heterogeneous protein assemblies is hampered because of their overlapping charge state distributions, fine structure, and peak broadening. To facilitate the mass analysis, it is of importance to automate preprocessing raw mass spectra, assigning ion series to peaks and deciphering the subunit compositions. So far, the automation of preprocessing raw mass spectra has not been accomplished; Massign was introduced to simplify data analysis and decipher the subunit compositions. In this study, we develop a search engine, AutoMass, to automatically assign ion series to peaks without any additional user input, for example, limited ranges of charge states or ion mass. AutoMass includes an ion intensity-dependent method to check for Gaussian distributions of ion series and an ion intensity-independent method to address highly overlapping and non-Gaussian distributions. The minimax theorem from game theory is adopted to define the boundaries. With AutoMass, the boundaries of ion series in the well-resolved tandem mass spectra of the hepatitis B virus (HBV) capsids and those of the mass spectrum from CRISPR-related cascade protein complex are accurately assigned. Theoretical and experimental HBV ion masses are shown in agreement up to ~0.03%. The analysis is finished within a minute on a regular workstation. Moreover, less well-resolved mass spectra, for example, complicated multimer mass spectra and norovirus capsid mass spectra at different levels of desolvation, are analyzed. In sum, this first-ever fully automatic program reveals the boundaries of overlapping ion peak series and can further aid developing high-throughput native MS and top-down proteomics.

Interpreting the charge state assignment in electrospray mass spectra of bioparticles.

In electrospray ionization mass spectra of heterogeneous protein complexes and other bioparticles, accurate mass determination is often hampered by the inaccuracy in determination of the charge states for individual signals. Here, we describe an algorithm that automatically minimizes the standard deviation in a series of related ion peaks with varying numbers of charges. The algorithm assumes that the mass is invariant and allows the determination of the correct charge state in a peak series. The analysis results in a periodic pattern, which can be interpreted as a harmonic oscillator, when the minimum standard deviation of a charge state series is found. We observed that a mass resolution of much less than 1000 in the acquired mass spectra is sufficient to achieve a correct charge state assignment. Moreover, the boundaries of mixed species can be identified by examining the loss of periodicity in the pattern of the analysis. We tested our algorithm successfully on novel spectra and on spectra reported in the literature with sample masses up to several million Dalton, e.g., viral particles, polyethylene glycol polymers, and polystyrene nanoparticles.

Potential distribution and transmission characteristics in a curved quadrupole ion guide.

The potential distribution in the curved quadrupole is exactly characterized by the Laplace equation, and an approximate solution to the Laplace equation is calculated. We represent the Laplace equation under the coordinates named minimal rotation frame (MRF) and derive an expression on the hexapole and octopole superposition. Our conclusion is in agreement with the results by the numerical (SIMION) method. Based on the Poincare-Lighthill-Kuo (PLK) method reported in our previous work, the nonlinear effects of ion motion are investigated in detail. The frequency shift of ion motion can be well eliminated by coupling the hexapole component with a positive octopole component, and the transmission efficiency of ions is found to decrease dramatically with the increase of the ionic kinetic energy in the z-direction. Furthermore, the transmission characteristics of ions are discussed with regards to the phase-space theory. The results show that the centrifugally introduced axis shift is mainly responsible for the ion losses. A modified direct current (dc) voltage supply pattern is hence proposed to compensate for this effect.