Deconvolving Native and Intact Protein Mass Spectra with UniDec

Intact Protein ◽

Top Down ◽

Protein Mass ◽

Isotopic Resolution ◽

Charge States ◽

User Friendly

Intact protein, top-down, and native mass spectrometry (MS) generally require the deconvolution of electrospray ionization (ESI) mass spectra to assign the mass of components from their charge state distribution. For small, well-resolved proteins, the charge can usually be assigned based on the isotope distribution. However, it can be challenging to determine charge states with larger proteins that lack isotopic resolution, in complex mass spectra with overlapping charge states, and in native spectra that show adduction. To overcome these challenges, UniDec uses Bayesian deconvolution to assign charge states and to create a zero-charge mass distribution. UniDec is fast, user-friendly, and includes a range of advanced tools to assist in intact protein, top-down, and native MS data analysis. This chapter provides a step-by-step protocol, an in-depth explanation of the UniDec algorithm, and highlights the parameters that affect the deconvolution. It also covers advanced data analysis tools, such as macromolecular mass defect analysis and tools for assigning potential PTMs and bound ligands. Overall, the chapter provides users with a deeper understanding of UniDec, which will enhance the quality of deconvolutions and allow for more intricate MS experiments.<br>

A Universal Score for Deconvolution of Intact Protein and Native Electrospray Mass Spectra

10.26434/chemrxiv.10278041.v1 ◽

2019 ◽

Author(s):

Michael Marty

Keyword(s):

Data Analysis ◽

Mass Spectra ◽

Weighted Average ◽

Simulated Data ◽

Intact Protein ◽

Protein Mass ◽

Charge States

The growing use of intact protein mass analysis, top-down proteomics, and native mass spectrometry have created a need for improved data analysis pipelines for deconvolution of electrospray (ESI) mass spectra containing multiple charge states and potentially without isotopic resolution. Although there are multiple deconvolution algorithms, there is no consensus for how to judge the quality of the deconvolution, and many scoring schemes are not published. Here, an intuitive universal score (UniScore) for ESI deconvolution is presented. The UniScore is the weighted average of deconvolution scores (DScores) for each peak. Each DScore is composed of separate components to score 1) the uniqueness and fit of the deconvolution to the data, 2) the consistency of the peak shape across different charge states, 3) the smoothness of the charge state distribution, and 4) symmetry and separation of the peak. Example scores are provided for a range of experimental and simulated data. By providing a means of judging the quality of the overall deconvolution as well as individual mass peaks, the UniScore scheme provides a foundation for standardizing ESI data analysis of larger molecules and enabling the use of ESI deconvolution in automated data analysis pipelines.

A Universal Score for Deconvolution of Intact Protein and Native Electrospray Mass Spectra

10.26434/chemrxiv.10278041 ◽

2019 ◽

Author(s):

Michael Marty

Keyword(s):

Data Analysis ◽

Mass Spectra ◽

Weighted Average ◽

Simulated Data ◽

Intact Protein ◽

Protein Mass ◽

Charge States

The growing use of intact protein mass analysis, top-down proteomics, and native mass spectrometry have created a need for improved data analysis pipelines for deconvolution of electrospray (ESI) mass spectra containing multiple charge states and potentially without isotopic resolution. Although there are multiple deconvolution algorithms, there is no consensus for how to judge the quality of the deconvolution, and many scoring schemes are not published. Here, an intuitive universal score (UniScore) for ESI deconvolution is presented. The UniScore is the weighted average of deconvolution scores (DScores) for each peak. Each DScore is composed of separate components to score 1) the uniqueness and fit of the deconvolution to the data, 2) the consistency of the peak shape across different charge states, 3) the smoothness of the charge state distribution, and 4) symmetry and separation of the peak. Example scores are provided for a range of experimental and simulated data. By providing a means of judging the quality of the overall deconvolution as well as individual mass peaks, the UniScore scheme provides a foundation for standardizing ESI data analysis of larger molecules and enabling the use of ESI deconvolution in automated data analysis pipelines.

Top-Down and Intact Protein Mass Spectrometry Data Visualization for Proteoform Analysis Using VisioProt-MS

Bioinformatics and Biology Insights ◽

10.1177/1177932219868223 ◽

2019 ◽

Vol 13 ◽

pp. 117793221986822 ◽

Cited By ~ 3

Author(s):

Jean Lesne ◽

Marie-Pierre Bousquet ◽

Julien Marcoux ◽

Marie Locard-Paulet

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Data Visualization ◽

Web Application ◽

Three Dimensional ◽

Mass Spectrometry Data ◽

Intact Protein ◽

Top Down ◽

Molecular Weights ◽

Protein Mass

The rise of intact protein analysis by mass spectrometry (MS) was accompanied by an increasing need for flexible tools allowing data visualization and analysis. These include inspection of the deconvoluted molecular weights of the proteoforms eluted alongside liquid chromatography (LC) through their representation in three-dimensional (3D) liquid chromatography coupled to mass spectrometry (LC-MS) maps (plots of deconvoluted molecular weights, retention times, and intensity of the MS signal). With this aim, we developed a free and open-source web application named VisioProt-MS ( https://masstools.ipbs.fr/mstools/visioprot-ms/ ). VisioProt-MS is highly compatible with many algorithms and software developed by the community to integrate and deconvolute top-down and intact protein MS data. Its dynamic and user-friendly features greatly facilitate analysis through several graphical representations dedicated to MS and tandem mass spectrometry (MS/MS) analysis of proteoforms in complex samples. Here, we will illustrate the importance of LC-MS map visualization to optimize top-down acquisition/search parameters and analyze intact protein MS data. We will go through the main features of VisioProt-MS using the human proteasomal 20S core particle as a user-case.

Profiling Dopamine-Induced Oxidized Proteoforms of β-Synuclein by Top-Down Mass Spectrometry

Antioxidants ◽

10.3390/antiox10060893 ◽

2021 ◽

Vol 10 (6) ◽

pp. 893

Author(s):

Arianna Luise ◽

Elena De Cecco ◽

Erika Ponzini ◽

Martina Sollazzo ◽

PierLuigi Mauri ◽

...

Keyword(s):

Mass Spectrometry ◽

Electron Transfer Dissociation ◽

Biochemical Properties ◽

Intact Protein ◽

Top Down ◽

Systematic Assessment ◽

Methionine Residues ◽

Stress States ◽

Oxidation Of Methionine

The formation of multiple proteoforms by post-translational modifications (PTMs) enables a single protein to acquire distinct functional roles in its biological context. Oxidation of methionine residues (Met) is a common PTM, involved in physiological (e.g., signaling) and pathological (e.g., oxidative stress) states. This PTM typically maps at multiple protein sites, generating a heterogeneous population of proteoforms with specific biophysical and biochemical properties. The identification and quantitation of the variety of oxidized proteoforms originated under a given condition is required to assess the exact molecular nature of the species responsible for the process under investigation. In this work, the binding and oxidation of human β-synuclein (BS) by dopamine (DA) has been explored. Native mass spectrometry (MS) has been employed to analyze the interaction of BS with DA. In a second step, top-down fragmentation of the intact protein from denaturing conditions has been performed to identify and quantify the distinct proteoforms generated by DA-induced oxidation. The analysis of isobaric proteoforms is approached by a combination of electron-transfer dissociation (ETD) at each extent of modification, quantitation of methionine-containing fragments and combinatorial analysis of the fragmentation products by multiple linear regression. This procedure represents a promising approach to systematic assessment of proteoforms variety and their relative abundance. The method can be adapted, in principle, to any protein containing any number of methionine residues, allowing for a full structural characterization of the protein oxidation states.

Tunable intact protein mass increases (TIPMI) as a simple and affordable method for relative intact protein quantitation and intact protein turnover rate optimized for top-down liquid chromatography-mass spectrometry (LC-MS) approach

10.17760/d20248916 ◽

2017 ◽

Author(s):

Jeniffer del Valle Quijada

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Protein Turnover ◽

Turnover Rate ◽

Intact Protein ◽

Liquid Chromatography Mass Spectrometry ◽

Top Down ◽

Protein Mass ◽

Protein Quantitation ◽

Chromatography Mass Spectrometry

Chapter 8 Intact Protein Mass Measurements and Top-Down Mass Spectrometry: Application to Integral Membrane Proteins

Comprehensive Analytical Chemistry - Protein Mass Spectrometry ◽

10.1016/s0166-526x(08)00208-0 ◽

2008 ◽

pp. 179-196 ◽

Cited By ~ 1

Author(s):

Julian P. Whitelegge

Keyword(s):

Mass Spectrometry ◽

Membrane Proteins ◽

Integral Membrane Proteins ◽

Intact Protein ◽

Mass Measurements ◽

Top Down ◽

Protein Mass ◽

Mass Spectrometry Application ◽

Top Down Mass Spectrometry

Intact protein mass spectrometry and top-down proteomics

Expert Review of Proteomics ◽

10.1586/epr.13.10 ◽

2013 ◽

Vol 10 (2) ◽

pp. 127-129 ◽

Cited By ~ 14

Author(s):

Julian Whitelegge

Keyword(s):

Mass Spectrometry ◽

Intact Protein ◽

Top Down ◽

Protein Mass ◽

Protein Mass Spectrometry

Probing Folded Proteins and Intact Protein Complexes by Desorption Electrospray Ionization Mass Spectrometry

10.26434/chemrxiv.12925295.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Bin Yan ◽

Josephine Bunch

Keyword(s):

Mass Spectrometry ◽

Protein Complexes ◽

Protein Structures ◽

Desorption Electrospray Ionization ◽

Intact Protein ◽

Ionization Mass ◽

Folded Proteins ◽

Folded Structures

Native mass spectrometry (Native MS) enables the study of intact proteins as well as non-covalent protein-protein and protein-ligand complexes in their biological state. In this work we present the application of a prototype Waters DESI source for rapid surface measurements of folded and native protein structures. Ions with narrow charge state distribution (CSD), i.e. folded structures are observed in the spectra of protein samples with the molecular weight ranging from 8.6 kDa up to 66.4 kDa. Intact protein complexes of holo-myoglobin and tetrameric hemoglobin are also successfully detected from a surface. These results reveal that DESI could be gentle enough to detect compact structures and noncovalent bond interactions. We also examine whether unfolded proteins and protein complexes can refold during transient spray solvent-sample interactions during DESI. Our results from ion mobility experiments of standards of ubiquitin, cytochrome c and protein complex myoglobin indicate that such phenomenon may occur, presenting artificial native-like spectra. Nevertheless, the observation of hemoglobin tetramer is promising as it demonstrates the capability of DESI to maintain truly native structures.