Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry.

Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and ß-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high m/z by >2.5-fold and >1.6-fold, respectively.

Multiplexed mass spectrometry of individual ions improves measurement of proteoforms and their complexes.

An Orbitrap-based ion analysis procedure determines the direct charge for numerous individual protein ions to generate true mass spectra. This individual ion mass spectrometry (I2MS) method for charge detection enables the characterization of highly complicated mixtures of proteoforms and their complexes in both denatured and native modes of operation, revealing information not obtainable by typical measurements of ensembles of ions.

STORI Plots Enable Accurate Tracking of Individual Ion Signals.

Charge detection mass spectrometry (CDMS) of low-level signals is currently limited to the analysis of individual ions that generate a persistent signal during the entire observation period. Ions that disintegrate during the observation period produce reduced frequency domain signal amplitudes, which lead to an underestimation of the ion charge state, and thus the ion mass. The charge assignment can only be corrected through an accurate determination of the time of ion disintegration. The traditional mechanisms for temporal signal analysis have severe limitations for temporal resolution, spectral resolution, and signal-to-noise ratios. Selective Temporal Overview of Resonant Ions (STORI) plots provide a new framework to accurately analyze low-level time domain signals of individual ions. STORI plots allow for complete correction of intermittent signals, the differentiation of single and multiple ions at the same frequency, and the association of signals that spontaneously change frequency.

A method for assessing and maintaining the reproducibility of mass spectrometric analyses of complex samples.

Direct injection mass spectrometric analysis of biological samples is potentially an attractive approach to the discovery of diagnostic patterns for specific pathophysiological conditions because of its speed and simplicity. Despite the possible benefits offered by such a method, its extensive application has been limited so far by several factors, including the inadequate reproducibility of the analytical results. We describe a method for monitoring and optimizing the performance of mass spectrometers used for biomarker discovery studies, based on the analysis of patterns of standardized spectral features. The method was successfully applied to maintaining spectral reproducibility during a multi-day analysis of hundreds of serum samples despite an ion source failure, which necessitated minor maintenance. The monitoring method allowed the early detection of that failure and the restoration of the spectral profiles after the system was restarted.

A method for monitoring and controlling reproducibility of intensity data in complex electrospray mass spectra: a thermometer ion-based strategy.

Reproducibility in mass spectral data is important in both biomarker discovery and spectral database searching. We report a strategy, employing a series of substituted benzylpyridinium thermometer ions that can be used to monitor changes in performance of multiple aspects of an electrospray ionization source that impact the intensity axis of a spectrum. Performance attributes, which could confound even isotope-based quantification strategies, are readily assessed using a mixture of thermometer ions. Based on the observed behavior of the ions, a procedure is proposed for monitoring instrument performance and compensating for factors that affect reproducibility across both time and instruments.