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Cited by 511Mass Spectrometry Techniques and ApplicationsAdvanced Proteomics Techniques and ApplicationsMetabolomics and Mass Spectrometry Studies

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High-Resolution Native Mass Spectrometry

Open access
Tamara, Sem, den Boer, Maurits A., Heck, Albert J. R.·2022·Chemical Reviews
DOI 10.1021/acs.chemrev.1c00212PMC PMC9052423
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Abstract

Native mass spectrometry (MS) involves the analysis and characterization of macromolecules, predominantly intact proteins and protein complexes, whereby as much as possible the native structural features of the analytes are retained. As such, native MS enables the study of secondary, tertiary, and even quaternary structure of proteins and other biomolecules. Native MS represents a relatively recent addition to the analytical toolbox of mass spectrometry and has over the past decade experienced immense growth, especially in enhancing sensitivity and resolving power but also in ease of use. With the advent of dedicated mass analyzers, sample preparation and separation approaches, targeted fragmentation techniques, and software solutions, the number of practitioners and novel applications has risen in both academia and industry. This review focuses on recent developments, particularly in high-resolution native MS, describing applications in the structural analysis of protein assemblies, proteoform profiling of─among others─biopharmaceuticals and plasma proteins, and quantitative and qualitative analysis of protein-ligand interactions, with the latter covering lipid, drug, and carbohydrate molecules, to name a few.

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Figures

Figure 1 Native MS has over the past decade experienced immense advances, primarily in enhancement of sensitivity and resolving power of mass analyzers but also in its ease of use. These advances have enabled the use of high-resolution native MS to analyze and characterize a wide range of macromolecules, including protein and ribonucleoprotein assemblies (section 7) and proteoforms of intact biopharmaceuticals and plasma proteins (section 8), and binding between proteins and small-molecule ligands such as lipids, drugs, and carbohydrates (section 9).
Figure 1 Native MS has over the past decade experienced immense advances, primarily in enhancement of sensitivity and resolving power of mass analyzers but also in its ease of use. These advances have enabled the use of high-resolution native MS to analyze and characterize a wide range of macromolecules, including protein and ribonucleoprotein assemblies (section 7) and proteoforms of intact biopharmaceuticals and plasma proteins (section 8), and binding between proteins and small-molecule ligands such as lipids, drugs, and carbohydrates (section 9).CC BY-NC-ND · PubMed Central
Figure 2 Challenges for resolving isotopologues with high-resolution native MS. (A) Adduct ions affect the mass resolving power. Baseline isotope mass resolution does not permit bare, sodium-bound, and ammonium-bound ions of a 150 kDa averagine protein to be distinguished using native ESI-MS. According to the empirical charging behavior of globular proteins in native MS, 26+ is the most abundant charge state for a molecular weight of 150 kDa. Therefore, the peaks of 26-fold-charged cations of a 150 kDa averagine protein (1350 averagine residues) were generated with MassLynx ver. 4.1, assuming baseline isotope mass resolution (R = 500 000). The isotope distributions of unmodified (green), ammonium-bound (orange), and sodium-bound (red) protein ions were simulated individually and subsequently summed to produce their combined mass spectrum (black). (B) Experimental peaks of globular protein complexes are substantially broader than simulated peaks of their molecular ions. The apparent mass resolution depends on the preset instrumental resolution and the efficiency of adduct removal. Shown are mass spectra of a GroEL ion with a charge of 71+. These were measured on Orbitrap Exactive Plus (blue) and a QToF (cyan) instruments, both operating at an instrument mass resolution of 5000, or simulated with MassLynx ver. 4.1 at mass resolutions of 5000 (red), 10 000 (orange), 20 000 (yellow), and 40 000 (green). The black curve represents the natural isotope envelope of GroEL. Numbers in parentheses correspond to the apparent mass resolutions Rnat determined by measuring the experimental peak widths. Reproduced from ref (32). Copyright 2014 American Society for Mass Spectrometry.
Figure 2 Challenges for resolving isotopologues with high-resolution native MS. (A) Adduct ions affect the mass resolving power. Baseline isotope mass resolution does not permit bare, sodium-bound, and ammonium-bound ions of a 150 kDa averagine protein to be distinguished using native ESI-MS. According to the empirical charging behavior of globular proteins in native MS, 26+ is the most abundant charge state for a molecular weight of 150 kDa. Therefore, the peaks of 26-fold-charged cations of a 150 kDa averagine protein (1350 averagine residues) were generated with MassLynx ver. 4.1, assuming baseline isotope mass resolution (R = 500 000). The isotope distributions of unmodified (green), ammonium-bound (orange), and sodium-bound (red) protein ions were simulated individually and subsequently summed to produce their combined mass spectrum (black). (B) Experimental peaks of globular protein complexes are substantially broader than simulated peaks of their molecular ions. The apparent mass resolution depends on the preset instrumental resolution and the efficiency of adduct removal. Shown are mass spectra of a GroEL ion with a charge of 71+. These were measured on Orbitrap Exactive Plus (blue) and a QToF (cyan) instruments, both operating at an instrument mass resolution of 5000, or simulated with MassLynx ver. 4.1 at mass resolutions of 5000 (red), 10 000 (orange), 20 000 (yellow), and 40 000 (green). The black curve represents the natural isotope envelope of GroEL. Numbers in parentheses correspond to the apparent mass resolutions Rnat determined by measuring the experimental peak widths. Reproduced from ref (32). Copyright 2014 American Society for Mass Spectrometry.CC BY-NC-ND · PubMed Central
Figure 3 Schematics of mass spectrometers commonly used for native MS: (A) 15 T FT-ICR. Adapted from ref (76). Copyright 2017 American Society for Mass Spectrometry. (B) Synapt G2 instrument featuring a Q-ToF mass analyzer. Reproduced with permission from ref (77). Copyright 2013 Nature Publishing Group. (C) Orbitrap Q-Exactive UHMR instrument with extended mass range. Reproduced with permission from ref (66). Copyright 2017 Nature Publishing Group.
Figure 3 Schematics of mass spectrometers commonly used for native MS: (A) 15 T FT-ICR. Adapted from ref (76). Copyright 2017 American Society for Mass Spectrometry. (B) Synapt G2 instrument featuring a Q-ToF mass analyzer. Reproduced with permission from ref (77). Copyright 2013 Nature Publishing Group. (C) Orbitrap Q-Exactive UHMR instrument with extended mass range. Reproduced with permission from ref (66). Copyright 2017 Nature Publishing Group.CC BY-NC-ND · PubMed Central
Figure 4 Theoretical and experimental mass resolutions (m/Δm) in the m/z range from 1000 to 50 000 Th for the common mass analyzers used in native MS. Shown are plots of R vs m/z for the three mass analyzers (FT-ICR, Orbitrap, and ToF) based on their respective Rmax values reported at m/z = 5000–6000 Th (FTICR, 40 000; Orbitrap; 25 000; ToF, 7000) and the theoretically achievable relations between analyzer mass resolution and m/z (FT-ICR (black line), R ∼ z/m; Orbitrap (blue line), R ∼ (z/m)0.5; ToF (cyan line), R = constant). Dotted lines in the FT-ICR and Orbitrap graphs represent theoretical values for m/z ranges that were not accessible with these mass analyzers at the time of the publication. Reproduced from ref (32). Copyright 2014 American Society for Mass Spectrometry.
Figure 4 Theoretical and experimental mass resolutions (m/Δm) in the m/z range from 1000 to 50 000 Th for the common mass analyzers used in native MS. Shown are plots of R vs m/z for the three mass analyzers (FT-ICR, Orbitrap, and ToF) based on their respective Rmax values reported at m/z = 5000–6000 Th (FTICR, 40 000; Orbitrap; 25 000; ToF, 7000) and the theoretically achievable relations between analyzer mass resolution and m/z (FT-ICR (black line), R ∼ z/m; Orbitrap (blue line), R ∼ (z/m)0.5; ToF (cyan line), R = constant). Dotted lines in the FT-ICR and Orbitrap graphs represent theoretical values for m/z ranges that were not accessible with these mass analyzers at the time of the publication. Reproduced from ref (32). Copyright 2014 American Society for Mass Spectrometry.CC BY-NC-ND · PubMed Central
Figure 5 High-resolution native MS analyses of antibody–drug conjugates using a 15 T FT-ICR instrument. Shown are the native MS spectra of (a) 5 equiv (biotin) lysine conjugate, (b) 10 equiv (biotin) lysine conjugate, (c) 5 equiv tris(2-carboxyethyl)phosphine (TCEP) and 10 equiv (biotin) cysteine conjugate, and (d) 10 equiv TCEP and 10 equiv (biotin) cysteine conjugate. Drug-to-antibody ratio (DAR) values are annotated on selected charge states and all of the deconvoluted spectra. Asterisks (*) represent a +162 Da glycation mass increase. Data are displayed in magnitude mode using a symmetric Hann apodization function. The annotated DAR values in insets III and IV of (c) and (d) are consistent with the numbers of biotin moieties covalently attached to the specific species and not the full intact initial monoclonal antibody conjugate molecule. Peak widths at half-height for z = 28+ are annotated. The peak widths at half-height for the equivalent deconvoluted data range from 39.2 to 41.1 Th. Reproduced from ref (108). Copyright 2017 American Chemical Society.
Figure 5 High-resolution native MS analyses of antibody–drug conjugates using a 15 T FT-ICR instrument. Shown are the native MS spectra of (a) 5 equiv (biotin) lysine conjugate, (b) 10 equiv (biotin) lysine conjugate, (c) 5 equiv tris(2-carboxyethyl)phosphine (TCEP) and 10 equiv (biotin) cysteine conjugate, and (d) 10 equiv TCEP and 10 equiv (biotin) cysteine conjugate. Drug-to-antibody ratio (DAR) values are annotated on selected charge states and all of the deconvoluted spectra. Asterisks (*) represent a +162 Da glycation mass increase. Data are displayed in magnitude mode using a symmetric Hann apodization function. The annotated DAR values in insets III and IV of (c) and (d) are consistent with the numbers of biotin moieties covalently attached to the specific species and not the full intact initial monoclonal antibody conjugate molecule. Peak widths at half-height for z = 28+ are annotated. The peak widths at half-height for the equivalent deconvoluted data range from 39.2 to 41.1 Th. Reproduced from ref (108). Copyright 2017 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 6 Spectral deconvolution of native MS spectra using UniDec software. The native mass spectra of AqpZ with bound POPC (a) and HSP17.7 (d) show satellite artifacts (red) when deconvolved (b, e) that are removed by addition of the SoftMax function (c, f). POPC nanodiscs with mixed heavy and light belts (g) show harmonic artifacts (orange) after deconvolution (h) that are removed by the SoftMax function (i). The triplet peaks from the mixed belts are preserved by the SoftMax function, as shown in the inset. Reproduced from ref (164). Copyright 2019 American Society for Mass Spectrometry.
Figure 6 Spectral deconvolution of native MS spectra using UniDec software. The native mass spectra of AqpZ with bound POPC (a) and HSP17.7 (d) show satellite artifacts (red) when deconvolved (b, e) that are removed by addition of the SoftMax function (c, f). POPC nanodiscs with mixed heavy and light belts (g) show harmonic artifacts (orange) after deconvolution (h) that are removed by the SoftMax function (i). The triplet peaks from the mixed belts are preserved by the SoftMax function, as shown in the inset. Reproduced from ref (164). Copyright 2019 American Society for Mass Spectrometry.CC BY-NC-ND · PubMed Central
Figure 7 Spectral deconvolution by PMI Intact software. Shown is the deconvolution of the highly polydisperse native mass spectrum of an empty MSP1D1 nanodisc containing the phospholipid DMPC analyzed on an Orbitrap-EMR. The inset displays the initial native mass spectrum. The Comb filter, available in PMI Intact, results in the successful deconvolution of this complex spectrum. Reproduced from ref (166). Copyright 2019 American Chemical Society.
Figure 7 Spectral deconvolution by PMI Intact software. Shown is the deconvolution of the highly polydisperse native mass spectrum of an empty MSP1D1 nanodisc containing the phospholipid DMPC analyzed on an Orbitrap-EMR. The inset displays the initial native mass spectrum. The Comb filter, available in PMI Intact, results in the successful deconvolution of this complex spectrum. Reproduced from ref (166). Copyright 2019 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 8 Native mass spectra of DPPC-MSP1E3D1 nanodiscs acquired on a Q-ToF mass analyzer and corresponding Fourier spectra for (a) the fundamentals and (b) the second harmonics. Inverse Fourier transforms of the charge-state-specific peaks in Fourier spectra (insets) are shown as overlaid envelope functions of the same color in the mass spectra. (c) Harmonic-averaged reconstruction of envelope functions. (d) Zero-charge spectrum (black) calculated from harmonic-averaged spectra for all charge states. Reproduced from ref (169). Copyright 2018 American Society for Mass Spectrometry.
Figure 8 Native mass spectra of DPPC-MSP1E3D1 nanodiscs acquired on a Q-ToF mass analyzer and corresponding Fourier spectra for (a) the fundamentals and (b) the second harmonics. Inverse Fourier transforms of the charge-state-specific peaks in Fourier spectra (insets) are shown as overlaid envelope functions of the same color in the mass spectra. (c) Harmonic-averaged reconstruction of envelope functions. (d) Zero-charge spectrum (black) calculated from harmonic-averaged spectra for all charge states. Reproduced from ref (169). Copyright 2018 American Society for Mass Spectrometry.CC BY-NC-ND · PubMed Central
Figure 9 Example of a higher-order native top-down MS approach used for the characterization of membrane proteins such as pMMO. Nanodiscs embedded with pMMO were subjected to CID in the source region for ejection of the protein complex from the nanodisc, resulting in the stabilization of the pMMO protomer species (MS1). Increasing collisional activation breaks up the protomer into individual pMMO subunits (MS2). Further collisional activation in the HCD cell of the Orbitrap-UHMR enables backbone fragmentation of each subunit (MS3). From ref (235). CC BY 4.0.
Figure 9 Example of a higher-order native top-down MS approach used for the characterization of membrane proteins such as pMMO. Nanodiscs embedded with pMMO were subjected to CID in the source region for ejection of the protein complex from the nanodisc, resulting in the stabilization of the pMMO protomer species (MS1). Increasing collisional activation breaks up the protomer into individual pMMO subunits (MS2). Further collisional activation in the HCD cell of the Orbitrap-UHMR enables backbone fragmentation of each subunit (MS3). From ref (235). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 10 Identification of unknown ligands bound to TSPO and subsequent fitting of PE 16:0/18:1 lipids into unresolved electron density in the X-ray structure. (a) Schematic showing the release of the TSPO dimer from detergent micelles (the subunits are shown as red and blue cartoons and the detergent micelle as orange spheres). (b) Native mass spectrum of the TSPO dimer (pseudo-MS2 step (pMS2)) and isolation of the 7– charge state (red box) (inset). (c) Collisional activation (pseudo-MS3 step (pMS3)) yields dissociated monomer, apo dimer produced from neutral ligand loss, and multiple ligands at low m/z (blue box). (d) Zoom of the low-m/z region showing two peak series corresponding to multiple lipids: PE (red) and PG (orange). (e–g) Isolation and subsequent fragmentation of released lipids (pseudo-MS4 step (pMS4)) define the hydrocarbon chain length and extent of unsaturation. (h) Position of the most abundant PE lipid identified, PE (16:0/18:1) (red sticks), in the TSPO A139T structure (PDB entry 4UC1). (i) Critical protein–lipid interactions (zoom box). Reproduced with permission from ref (74). Copyright 2020 Gault et al.
Figure 10 Identification of unknown ligands bound to TSPO and subsequent fitting of PE 16:0/18:1 lipids into unresolved electron density in the X-ray structure. (a) Schematic showing the release of the TSPO dimer from detergent micelles (the subunits are shown as red and blue cartoons and the detergent micelle as orange spheres). (b) Native mass spectrum of the TSPO dimer (pseudo-MS2 step (pMS2)) and isolation of the 7– charge state (red box) (inset). (c) Collisional activation (pseudo-MS3 step (pMS3)) yields dissociated monomer, apo dimer produced from neutral ligand loss, and multiple ligands at low m/z (blue box). (d) Zoom of the low-m/z region showing two peak series corresponding to multiple lipids: PE (red) and PG (orange). (e–g) Isolation and subsequent fragmentation of released lipids (pseudo-MS4 step (pMS4)) define the hydrocarbon chain length and extent of unsaturation. (h) Position of the most abundant PE lipid identified, PE (16:0/18:1) (red sticks), in the TSPO A139T structure (PDB entry 4UC1). (i) Critical protein–lipid interactions (zoom box). Reproduced with permission from ref (74). Copyright 2020 Gault et al.CC BY-NC-ND · PubMed Central
Figure 11 High-resolution native MS of ribosomal particles. (a) (left) Structures of the E. coli 70S ribosome consisting of the 50S (proteins shown in blue and rRNA in green) and 30S (proteins shown in red and rRNA in orange) particles. (right) High-resolution native mass spectra of the 70S, 30S, and 50S ribosomes. (b) Distinct particles of the 30S ribosome, with annotations provided at the top. Masses are shown as mean ± SD. (c) Distinct particles of the 50S ribosome, with annotations provided at the top. In most of the particles, the pentameric stalk complex is absent. Reproduced with permission from ref (66). Copyright 2017 Nature Publishing Group.
Figure 11 High-resolution native MS of ribosomal particles. (a) (left) Structures of the E. coli 70S ribosome consisting of the 50S (proteins shown in blue and rRNA in green) and 30S (proteins shown in red and rRNA in orange) particles. (right) High-resolution native mass spectra of the 70S, 30S, and 50S ribosomes. (b) Distinct particles of the 30S ribosome, with annotations provided at the top. Masses are shown as mean ± SD. (c) Distinct particles of the 50S ribosome, with annotations provided at the top. In most of the particles, the pentameric stalk complex is absent. Reproduced with permission from ref (66). Copyright 2017 Nature Publishing Group.CC BY-NC-ND · PubMed Central
Figure 12 High-resolution native mass spectra of free and internal ribosome entry site (IRES) RNA-bound human 40S subunits. (a) Native mass spectrum of the human 40S subunit acquired with an Orbitrap-UHMR mass analyzer. The well-resolved charge states of three distinct forms of the ribosomal subunit could be detected. The most abundant fully assembled 1.2 MDa 40S particles are labeled in blue, while lower-abundance particles lacking either the S25 or S10 protein are labeled in magenta and red, respectively. (b) Monitoring of the formation of a complex containing human 40S ribosomes and IRES RNA fragments of cricket paralysis virus (CrPV) and hepatits C virus (HCV). The mass spectra of the RNA fragments alone (insets) provide the accurate masses of the IRES elements. Structures of the free 40S ribosomes (PDB entry 5A2Q) and particles bound by CrPV (PDB entry 4V91) and HCV IRES (PDB entry 5A2Q) are shown, with the ribosomal proteins in blue, the rRNA in gray, and the IRES elements in yellow and green, respectively. From ref (53). CC BY 4.0.
Figure 12 High-resolution native mass spectra of free and internal ribosome entry site (IRES) RNA-bound human 40S subunits. (a) Native mass spectrum of the human 40S subunit acquired with an Orbitrap-UHMR mass analyzer. The well-resolved charge states of three distinct forms of the ribosomal subunit could be detected. The most abundant fully assembled 1.2 MDa 40S particles are labeled in blue, while lower-abundance particles lacking either the S25 or S10 protein are labeled in magenta and red, respectively. (b) Monitoring of the formation of a complex containing human 40S ribosomes and IRES RNA fragments of cricket paralysis virus (CrPV) and hepatits C virus (HCV). The mass spectra of the RNA fragments alone (insets) provide the accurate masses of the IRES elements. Structures of the free 40S ribosomes (PDB entry 5A2Q) and particles bound by CrPV (PDB entry 4V91) and HCV IRES (PDB entry 5A2Q) are shown, with the ribosomal proteins in blue, the rRNA in gray, and the IRES elements in yellow and green, respectively. From ref (53). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 13 20S proteasomal particles analyzed by surface-induced dissociation (SID) combined with ion mobility mass spectrometry (IM-MS). SID-IM-MS spectra of the rabbit 20S proteasome complex reflect the cylindrical topology of the complex. (A) Charge-reduced rabbit 20S proteasome precursor ions were measured on a Synapt G2 instrument equipped with an SID cell (top), and the 43+ and 44+ charge states were isolated (bottom) and accelerated into the surface at 150 V. (B) IM-MS plot of the SID spectra of the rabbit 20S proteasome. The separation in drift time (vertical axis) assists in the discrimination of species that overlap in m/z (horizontal axis), as shown in (C). The major populations of the dissociation products are designated by dashed lines in (B) and labeled with symbols that are graphically depicted in (D–J). The extracted m/z spectra from the underlined regions in the IM-MS plot (D–J) show the identified dissociation products. Reproduced from ref (216). Copyright 2020 American Chemical Society.
Figure 13 20S proteasomal particles analyzed by surface-induced dissociation (SID) combined with ion mobility mass spectrometry (IM-MS). SID-IM-MS spectra of the rabbit 20S proteasome complex reflect the cylindrical topology of the complex. (A) Charge-reduced rabbit 20S proteasome precursor ions were measured on a Synapt G2 instrument equipped with an SID cell (top), and the 43+ and 44+ charge states were isolated (bottom) and accelerated into the surface at 150 V. (B) IM-MS plot of the SID spectra of the rabbit 20S proteasome. The separation in drift time (vertical axis) assists in the discrimination of species that overlap in m/z (horizontal axis), as shown in (C). The major populations of the dissociation products are designated by dashed lines in (B) and labeled with symbols that are graphically depicted in (D–J). The extracted m/z spectra from the underlined regions in the IM-MS plot (D–J) show the identified dissociation products. Reproduced from ref (216). Copyright 2020 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 14 Analysis of adeno-associated virus (AAV) by high-resolution native MS: (A) AAV (structure from PDB entry 3NG9) is composed of 60 copies of a combination of VP1, VP2, and VP3, encoded within the same cap open-reading frame, sharing a common C-terminal sequence. AAV capsid assembly follows a stochastic model, where VP proteins are taken randomly from the pool of expressed VP proteins. The probability for each stoichiometry is determined by the ratio of the expressed VP proteins. (B) Simulated and experimental high-resolution native mass spectra of AAV at mass resolutions corresponding to transient times of 32 and 128 ms. Each plot shows (top to bottom) the simulated mass spectra before and after baseline correction and their experimental counterparts. On the right are depicted zoomed-in parts of these spectra. Adapted from ref (272). CC BY 4.0.
Figure 14 Analysis of adeno-associated virus (AAV) by high-resolution native MS: (A) AAV (structure from PDB entry 3NG9) is composed of 60 copies of a combination of VP1, VP2, and VP3, encoded within the same cap open-reading frame, sharing a common C-terminal sequence. AAV capsid assembly follows a stochastic model, where VP proteins are taken randomly from the pool of expressed VP proteins. The probability for each stoichiometry is determined by the ratio of the expressed VP proteins. (B) Simulated and experimental high-resolution native mass spectra of AAV at mass resolutions corresponding to transient times of 32 and 128 ms. Each plot shows (top to bottom) the simulated mass spectra before and after baseline correction and their experimental counterparts. On the right are depicted zoomed-in parts of these spectra. Adapted from ref (272). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 15 High-resolution native tandem mass spectra of the designed charge-reduced AaLS-neg particles. (top) The charge-reduced intact Cp180 shows a well-resolved charge-state envelope centered at m/z ≈ 30 000 Th. (middle, bottom) The charge-state envelope was subjected to increasing HCD collision energies of 250 and 300 V. At maximal HCD the mass spectrum revealed the consecutive elimination of monomers from the Cp180-mer with fragment ions up to m/z = 70 000 Th. The obtained mass resolution enabled baseline resolution of these different dissociation products. At the highest m/z, the mass resolution was still greater than 500. The figure is adapted from ref (30). Copyright 2017 Royal Society of Chemistry. The inset structure in the top panel is from ref (282). CC BY 4.0.
Figure 15 High-resolution native tandem mass spectra of the designed charge-reduced AaLS-neg particles. (top) The charge-reduced intact Cp180 shows a well-resolved charge-state envelope centered at m/z ≈ 30 000 Th. (middle, bottom) The charge-state envelope was subjected to increasing HCD collision energies of 250 and 300 V. At maximal HCD the mass spectrum revealed the consecutive elimination of monomers from the Cp180-mer with fragment ions up to m/z = 70 000 Th. The obtained mass resolution enabled baseline resolution of these different dissociation products. At the highest m/z, the mass resolution was still greater than 500. The figure is adapted from ref (30). Copyright 2017 Royal Society of Chemistry. The inset structure in the top panel is from ref (282). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 16 Native-MS-assisted analysis of the initial steps in complement activation: stepwise reconstitution of the 2.1 MDa C1:IgG:Ag complexes, comprising 40 protein subunits, monitored by native MS. (A) Native MS analysis of reconstituted C1, C1:IgG, and C1:IgG:Ag complexes. The signals shown in dashed boxes are color-coded according to the stoichiometries of C1 assembly, as specified to the left of the corresponding spectra. The bottom spectrum demonstrates the successful reconstitution of C1, containing C1q, two copies of C1r, and two copies of C1s. The middle and top spectra show that the latter assembly successfully binds to (IgG)6 and subsequently to 12 copies of the antigen (Ag). (B) Model summarizing molecular determinants contributing to IgG-mediated activation of the classical complement pathway. The assembly of the complement initiation complex is proposed to be dependent on (1) the availability of specific antigens recognized by IgG antibodies, (2) Ag compatibility with clustering at the cell surface or in solution, (3) the Fc-domain conformation, (4) the high-avidity binding site for hexavalent C1q, (5) the composition of the IgG glycan chains, (6) the presence of Fab arms, and (7) Ag binding by the Fab arms. Reproduced with permission from ref (293). Copyright 2016 Elsevier Inc.
Figure 16 Native-MS-assisted analysis of the initial steps in complement activation: stepwise reconstitution of the 2.1 MDa C1:IgG:Ag complexes, comprising 40 protein subunits, monitored by native MS. (A) Native MS analysis of reconstituted C1, C1:IgG, and C1:IgG:Ag complexes. The signals shown in dashed boxes are color-coded according to the stoichiometries of C1 assembly, as specified to the left of the corresponding spectra. The bottom spectrum demonstrates the successful reconstitution of C1, containing C1q, two copies of C1r, and two copies of C1s. The middle and top spectra show that the latter assembly successfully binds to (IgG)6 and subsequently to 12 copies of the antigen (Ag). (B) Model summarizing molecular determinants contributing to IgG-mediated activation of the classical complement pathway. The assembly of the complement initiation complex is proposed to be dependent on (1) the availability of specific antigens recognized by IgG antibodies, (2) Ag compatibility with clustering at the cell surface or in solution, (3) the Fc-domain conformation, (4) the high-avidity binding site for hexavalent C1q, (5) the composition of the IgG glycan chains, (6) the presence of Fab arms, and (7) Ag binding by the Fab arms. Reproduced with permission from ref (293). Copyright 2016 Elsevier Inc.CC BY-NC-ND · PubMed Central
Figure 17 Native MS analysis of a membrane-embedded photosynthetic reaction center: release of the photosynthetic reaction center complex from detergent micelles by collisional activation. At low collisional energy (bottom spectrum), the broad peak represents ions from the micelles. The detergent micelles and extra bound detergent can be removed by increasing the collision energy at the trap region of a Synapt G2 mass spectrometer. Reproduced from ref (299). Copyright 2017 American Chemical Society.
Figure 17 Native MS analysis of a membrane-embedded photosynthetic reaction center: release of the photosynthetic reaction center complex from detergent micelles by collisional activation. At low collisional energy (bottom spectrum), the broad peak represents ions from the micelles. The detergent micelles and extra bound detergent can be removed by increasing the collision energy at the trap region of a Synapt G2 mass spectrometer. Reproduced from ref (299). Copyright 2017 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 18 Native MS and IM-MS of the UVR8 photoreceptor core domain (UVR812–381). (A) Native mass spectrum of the UVR812–381 dimer (pale-orange peaks) in the absence of UV-B light. (B) Monodisperse drift tube collision cross section in helium (DTCCSHe) of the UVR8 dimer. The inset shows the energy-minimized structure of the UVR8 dimer and associated theoretical CCS (ThCCSHe). (C) Mass spectra of UVR812–381 as a function of illumination time (280 nm, 25 mW, 350 mA). Mass spectra were combined over a period of 1.2 s after the light-emitting diode (LED) was switched on at t0. Dimer signal, pale orange; monomer signals, pale green. (D) Ion chromatograms extracted for the UVR812–381 dimer (orange) and monomer (green) as functions of acquisition time. Data to the left of the purple dashed line are from when the ion source tip was in the dark, and the data to the right are from when the tip was illuminated. (E) Native mass spectrum of UVR812–381 following illumination in the source tip with the 280 nm LED for 10 s (to ensure maximum conversion). The spectrum is now dominated by the UVR812–381 monomer (pale-green peaks). (F) Monodisperse DTCCSHe of the UVR812–381 monomer. The inset shows the energy-minimized structure of the UVR812–381 monomer and associated ThCCSHe. From ref (313). CC BY 4.0.
Figure 18 Native MS and IM-MS of the UVR8 photoreceptor core domain (UVR812–381). (A) Native mass spectrum of the UVR812–381 dimer (pale-orange peaks) in the absence of UV-B light. (B) Monodisperse drift tube collision cross section in helium (DTCCSHe) of the UVR8 dimer. The inset shows the energy-minimized structure of the UVR8 dimer and associated theoretical CCS (ThCCSHe). (C) Mass spectra of UVR812–381 as a function of illumination time (280 nm, 25 mW, 350 mA). Mass spectra were combined over a period of 1.2 s after the light-emitting diode (LED) was switched on at t0. Dimer signal, pale orange; monomer signals, pale green. (D) Ion chromatograms extracted for the UVR812–381 dimer (orange) and monomer (green) as functions of acquisition time. Data to the left of the purple dashed line are from when the ion source tip was in the dark, and the data to the right are from when the tip was illuminated. (E) Native mass spectrum of UVR812–381 following illumination in the source tip with the 280 nm LED for 10 s (to ensure maximum conversion). The spectrum is now dominated by the UVR812–381 monomer (pale-green peaks). (F) Monodisperse DTCCSHe of the UVR812–381 monomer. The inset shows the energy-minimized structure of the UVR812–381 monomer and associated ThCCSHe. From ref (313). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 19 Native MS of membrane-embedded protein complexes analyzed directly from their native environment. Regions of the mass spectrum recorded for inner membranes from E. coli yield cytochromes, the Ton complex multidrug transporters, and the intact ATP synthase in complex with the SecYEG translocon. (A, B) Expanded regions of the spectrum assigned to cytochrome bo3 and cytochrome bd oxidase, showing peak splitting due to binding of quinol and heme groups. The pentameric ExbB complex (with one copy of ExbD in the center of the pore) that forms part of the TonB complex is also observed (yellow). (C) High-m/z region of the mass spectrum assigned to the multidrug efflux pumps AcrAB and MdtAB and the intact ATP synthase. Expansion of peaks assigned to the ATPase reveals binding of the SecY (blue), SecYG (green), and SecYEG (orange) charge states 52+, 53+, and 54+. Complexes observed in mass spectra are shown schematically, with subunits that have dissociated shown in gray. Reproduced with permission from ref (323). Copyright 2018 Chorev et al.
Figure 19 Native MS of membrane-embedded protein complexes analyzed directly from their native environment. Regions of the mass spectrum recorded for inner membranes from E. coli yield cytochromes, the Ton complex multidrug transporters, and the intact ATP synthase in complex with the SecYEG translocon. (A, B) Expanded regions of the spectrum assigned to cytochrome bo3 and cytochrome bd oxidase, showing peak splitting due to binding of quinol and heme groups. The pentameric ExbB complex (with one copy of ExbD in the center of the pore) that forms part of the TonB complex is also observed (yellow). (C) High-m/z region of the mass spectrum assigned to the multidrug efflux pumps AcrAB and MdtAB and the intact ATP synthase. Expansion of peaks assigned to the ATPase reveals binding of the SecY (blue), SecYG (green), and SecYEG (orange) charge states 52+, 53+, and 54+. Complexes observed in mass spectra are shown schematically, with subunits that have dissociated shown in gray. Reproduced with permission from ref (323). Copyright 2018 Chorev et al.CC BY-NC-ND · PubMed Central
Figure 20 Eliminating hexosylation bias in glycoproteoform profiling by high-resolution native MS. Glycation obscures differences between the glycoform profiles of two batches of Prolia (old vs new). (a, b) Raw mass spectra of the (a) intact and (b) de-N-glycosylated mAb, corresponding to 2 kDa sections of the respective zero-charge spectra (the secondary x axes indicate the respective masses). (c) Fractional glycoform abundances before and after correction for the effects of glycation. (d) Interbatch differences of glycoform abundances as derived from (c). Lines connect points denoting identical glycoforms. The three most common glycoforms are labeled. pp, percentage points. (e) Absolute deviations of observed and actual glycoform abundances from simulated abundances based on released N-glycan data. In each batch, those five glycoforms for which correction leads to the largest decrease in deviation are highlighted (thick line: most pronounced decrease). Point areas are proportional to observed/actual glycoform abundance. Error bars represent (propagated) 95% confidence intervals from five technical replicates. RMSD, root-mean-square deviation. From ref (356). CC BY 4.0.
Figure 20 Eliminating hexosylation bias in glycoproteoform profiling by high-resolution native MS. Glycation obscures differences between the glycoform profiles of two batches of Prolia (old vs new). (a, b) Raw mass spectra of the (a) intact and (b) de-N-glycosylated mAb, corresponding to 2 kDa sections of the respective zero-charge spectra (the secondary x axes indicate the respective masses). (c) Fractional glycoform abundances before and after correction for the effects of glycation. (d) Interbatch differences of glycoform abundances as derived from (c). Lines connect points denoting identical glycoforms. The three most common glycoforms are labeled. pp, percentage points. (e) Absolute deviations of observed and actual glycoform abundances from simulated abundances based on released N-glycan data. In each batch, those five glycoforms for which correction leads to the largest decrease in deviation are highlighted (thick line: most pronounced decrease). Point areas are proportional to observed/actual glycoform abundance. Error bars represent (propagated) 95% confidence intervals from five technical replicates. RMSD, root-mean-square deviation. From ref (356). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 21 Comparison between cation-exchange chromatography MS (CEX-MS) and capillary electrophoresis MS (CE-MS) in the analysis of cetuximab charge variants. (A) Cetuximab chromatogram and electropherogram, with peaks annotated in red and the corresponding net charges in blue. (B) Deconvoluted native mass spectra corresponding to the peaks in (A). Spectra from the two separation approaches containing the same charge variant species are aligned horizontally. Modifications that cause differences in the net charge are indicated in blue, whereas only the most probable combination of modifications is given. The most abundant species corresponding to biantennary Fab glycan pairs are shown in orange, whereas the most abundant forms carrying triantennary Fab glycans are labeled in gray. (C) Deconvoluted spectra of the peak front, center, and tail for peaks 5 and 3 of the CEX and CE separations, respectively. Associated spectra are aligned horizontally and are shown in the same color. Reproduced from ref (369). Copyright 2020 American Chemical Society.
Figure 21 Comparison between cation-exchange chromatography MS (CEX-MS) and capillary electrophoresis MS (CE-MS) in the analysis of cetuximab charge variants. (A) Cetuximab chromatogram and electropherogram, with peaks annotated in red and the corresponding net charges in blue. (B) Deconvoluted native mass spectra corresponding to the peaks in (A). Spectra from the two separation approaches containing the same charge variant species are aligned horizontally. Modifications that cause differences in the net charge are indicated in blue, whereas only the most probable combination of modifications is given. The most abundant species corresponding to biantennary Fab glycan pairs are shown in orange, whereas the most abundant forms carrying triantennary Fab glycans are labeled in gray. (C) Deconvoluted spectra of the peak front, center, and tail for peaks 5 and 3 of the CEX and CE separations, respectively. Associated spectra are aligned horizontally and are shown in the same color. Reproduced from ref (369). Copyright 2020 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 22 Characterization of wild-type EPO and 23 glycoengineered EPO variants by high-resolution native MS. (A) Illustrative deconvoluted native mass spectra of WT EPO (top) and the knockout-based glycoengineered clones C23 (middle) and C21 (bottom). (B) Comparison of native mass spectra of EPO from two biological replicate clones C22 (black) and C24 (orange) by correlation scoring. (C) Clustering of the glycoengineered EPO clones based on the correlation between their native MS spectra. From ref (381). CC BY 4.0.
Figure 22 Characterization of wild-type EPO and 23 glycoengineered EPO variants by high-resolution native MS. (A) Illustrative deconvoluted native mass spectra of WT EPO (top) and the knockout-based glycoengineered clones C23 (middle) and C21 (bottom). (B) Comparison of native mass spectra of EPO from two biological replicate clones C22 (black) and C24 (orange) by correlation scoring. (C) Clustering of the glycoengineered EPO clones based on the correlation between their native MS spectra. From ref (381). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 23 Molecular structure and high-resolution native mass spectra of etanercept. (a) Structure of dimeric etanercept consisting of a TNFR and an Fc domain. Some of the cleavage sites of IdeS, PNGase F, and sialidase are indicated. (b–d) Native mass spectra of (b) intact etanercept and etanercept treated with (c) sialidase, (d) PNGase F, or (f) a combination of PNGase F and sialidase. (f, g) Annotation of the etanercept O-glycoforms after treatment with (f) PNGase F and sialidase or (g) PNGase F alone. Adapted from ref (383). CC BY 4.0.
Figure 23 Molecular structure and high-resolution native mass spectra of etanercept. (a) Structure of dimeric etanercept consisting of a TNFR and an Fc domain. Some of the cleavage sites of IdeS, PNGase F, and sialidase are indicated. (b–d) Native mass spectra of (b) intact etanercept and etanercept treated with (c) sialidase, (d) PNGase F, or (f) a combination of PNGase F and sialidase. (f, g) Annotation of the etanercept O-glycoforms after treatment with (f) PNGase F and sialidase or (g) PNGase F alone. Adapted from ref (383). CC BY 4.0.CC BY-NC-ND · PubMed Central
Figure 24 Glycan-annotated high-resolution native mass spectra of α1-acid glycoprotein (AGP). (A) High-resolution native mass spectrum and (B) zero-charged and glycan annotated deconvoluted mass spectrum of asialo-AGP. The inset in (A) shows the AGP structure with five highly branched N-glycans at Asn15, Asn38, Asn54, Asn75, and Asn85. From ref (394). CC BY-NC-ND 4.0.
Figure 24 Glycan-annotated high-resolution native mass spectra of α1-acid glycoprotein (AGP). (A) High-resolution native mass spectrum and (B) zero-charged and glycan annotated deconvoluted mass spectrum of asialo-AGP. The inset in (A) shows the AGP structure with five highly branched N-glycans at Asn15, Asn38, Asn54, Asn75, and Asn85. From ref (394). CC BY-NC-ND 4.0.CC BY-NC-ND · PubMed Central
Figure 25 Native-MS-derived proteome profiles of serum SerpinA3 (AACT) from a donor before and after a septic episode revealing an extensive increase in glycosylation.397
Figure 25 Native-MS-derived proteome profiles of serum SerpinA3 (AACT) from a donor before and after a septic episode revealing an extensive increase in glycosylation.397CC BY-NC-ND · PubMed Central
Figure 26 Comparison of deconvoluted high-resolution native mass spectra with simulated mass spectra reconstructed from intact-mass hydrophilic interaction chromatography (HILIC) LC-MS data. (A, C, E) Mirror plots for Hp 2-1 multimers: (A) (α1)2α2β3 trimer, (C) (α1)2(α2)2β4 tetramer, and (E) (α1)2(α2)3β5 pentamer. The zero-charged deconvoluted native MS spectrum is depicted at the top and the in silico-reconstructed spectrum at the bottom. The Pearson correlation coefficient (r) for each pair of native and in silico-reconstructed spectra is given in the top left corner of each panel, and a schematic drawing of each Hp 2-1 multimer is shown in the bottom left corner of each panel. (B, D, F) Annotation of peaks detected in the native deconvoluted MS spectra for the (B) trimer, (D) tetramer, and (F) pentamer of Hp 2-1. From ref (402). CC BY-NC-ND 4.0.
Figure 26 Comparison of deconvoluted high-resolution native mass spectra with simulated mass spectra reconstructed from intact-mass hydrophilic interaction chromatography (HILIC) LC-MS data. (A, C, E) Mirror plots for Hp 2-1 multimers: (A) (α1)2α2β3 trimer, (C) (α1)2(α2)2β4 tetramer, and (E) (α1)2(α2)3β5 pentamer. The zero-charged deconvoluted native MS spectrum is depicted at the top and the in silico-reconstructed spectrum at the bottom. The Pearson correlation coefficient (r) for each pair of native and in silico-reconstructed spectra is given in the top left corner of each panel, and a schematic drawing of each Hp 2-1 multimer is shown in the bottom left corner of each panel. (B, D, F) Annotation of peaks detected in the native deconvoluted MS spectra for the (B) trimer, (D) tetramer, and (F) pentamer of Hp 2-1. From ref (402). CC BY-NC-ND 4.0.CC BY-NC-ND · PubMed Central
Figure 27 Narrow-bore nanoelectrospray ionization emitters with inner tip diameters less than 1000 nm can be used to obtain more accurate Kd values for carbonic anhydrase (P) inhibitors (L). (a–d) nESI mass spectra of aqueous solutions containing 5 μM human carbonic anhydrase I, 2 μM ethoxzolamide, and 70 mM ammonium acetate obtained using emitter tips with inner diameters of (a) ∼2000, (b) ∼850, (c) ∼500, and (d) ∼250 nm. (e) Kd values measured using nESI as a function of the emitter tip diameter for the binding of ethoxzolamide (squares), brinzolamide (circles), furosemide (triangles), and dichlorphenamide (diamonds) to human carbonic anhydrase I. Reproduced from ref (435). Copyright 2019 American Chemical Society.
Figure 27 Narrow-bore nanoelectrospray ionization emitters with inner tip diameters less than 1000 nm can be used to obtain more accurate Kd values for carbonic anhydrase (P) inhibitors (L). (a–d) nESI mass spectra of aqueous solutions containing 5 μM human carbonic anhydrase I, 2 μM ethoxzolamide, and 70 mM ammonium acetate obtained using emitter tips with inner diameters of (a) ∼2000, (b) ∼850, (c) ∼500, and (d) ∼250 nm. (e) Kd values measured using nESI as a function of the emitter tip diameter for the binding of ethoxzolamide (squares), brinzolamide (circles), furosemide (triangles), and dichlorphenamide (diamonds) to human carbonic anhydrase I. Reproduced from ref (435). Copyright 2019 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 28 Automated screening of protein–ligand interactions using a compound library and native SEC-MS. (a–c) Native mass spectra of the enzyme indoleamine-pyrrole 2,3-dioxygenase (IDO1) alone, IDO1 with compound 1, and IDO1 with compound 2, respectively. The left panels show the charge-state distributions, and the right panels focus on the enlargements of the +14 charge state for identification. For all of the spectra shown, the concentration of IDO1 was 5 μM (2 μM for IDO1 w/heme), whereas the concentration of compound 1 was 3.5 μM in (b) and that of compound 2 was 10 μM in (c). (d) Saturation curves from titration experiments. The concentration of the IDO1 sample was 13 μM (9 μM for IDO1 w/heme). The concentrations of compounds 1 and 2 ranged from 0.5 to 100 μM. (e) In-source dissociation profiles for the IDO1–compound 1 and IDO1–compound 2 complexes. Dashed lines indicate the corresponding Vc50 values. The concentration of the IDO1 sample was 12 μM, and the concentrations of compounds 1 and 2 were 89 μM. (f) Plot of Vc50 against initial complex percentage at different compound 1/compound 2 to IDO1 ratios. (g) Competition experiment for evaluation of relative binding affinities. IDO1 (total 13 μM, IDO1 w/heme 9 μM) was incubated with compounds 1 and 2 (each at 10 μM). Each precursor experiences a different lab-frame energy based on its charge state. All of the data points in (a) and (b) were replicated three times, with error bars shown on the graph. Adapted from ref (456). Copyright 2018 American Chemical Society.
Figure 28 Automated screening of protein–ligand interactions using a compound library and native SEC-MS. (a–c) Native mass spectra of the enzyme indoleamine-pyrrole 2,3-dioxygenase (IDO1) alone, IDO1 with compound 1, and IDO1 with compound 2, respectively. The left panels show the charge-state distributions, and the right panels focus on the enlargements of the +14 charge state for identification. For all of the spectra shown, the concentration of IDO1 was 5 μM (2 μM for IDO1 w/heme), whereas the concentration of compound 1 was 3.5 μM in (b) and that of compound 2 was 10 μM in (c). (d) Saturation curves from titration experiments. The concentration of the IDO1 sample was 13 μM (9 μM for IDO1 w/heme). The concentrations of compounds 1 and 2 ranged from 0.5 to 100 μM. (e) In-source dissociation profiles for the IDO1–compound 1 and IDO1–compound 2 complexes. Dashed lines indicate the corresponding Vc50 values. The concentration of the IDO1 sample was 12 μM, and the concentrations of compounds 1 and 2 were 89 μM. (f) Plot of Vc50 against initial complex percentage at different compound 1/compound 2 to IDO1 ratios. (g) Competition experiment for evaluation of relative binding affinities. IDO1 (total 13 μM, IDO1 w/heme 9 μM) was incubated with compounds 1 and 2 (each at 10 μM). Each precursor experiences a different lab-frame energy based on its charge state. All of the data points in (a) and (b) were replicated three times, with error bars shown on the graph. Adapted from ref (456). Copyright 2018 American Chemical Society.CC BY-NC-ND · PubMed Central
Figure 29 Native MS spectrum (main panel) of trimeric OmpF bound to an equimolar ratio of DMPG, DPPG, and POPG lipids (cartoon inset). The 22+ charge state is shown in an expanded view (right), with peaks showing up to three bound lipids. Theoretical distributions corresponding to different combinations of lipids are shown by colored lines and correlate with the spectrum. Reproduced with permission from ref (21). Copyright 2016 Nature Publishing Group.
Figure 29 Native MS spectrum (main panel) of trimeric OmpF bound to an equimolar ratio of DMPG, DPPG, and POPG lipids (cartoon inset). The 22+ charge state is shown in an expanded view (right), with peaks showing up to three bound lipids. Theoretical distributions corresponding to different combinations of lipids are shown by colored lines and correlate with the spectrum. Reproduced with permission from ref (21). Copyright 2016 Nature Publishing Group.CC BY-NC-ND · PubMed Central
Figure 30 Determination of the guanosine triphosphate (GTP) hydrolysis rate (khyd,GTP) for K-RAS. (A) Representative native mass spectra recorded at different time points for K-RAS (2 μM) loaded with GTP incubated at 25 °C. The abundance of K-RAS bound to guanosine diphosphate (GDP) increases as GTP is hydrolyzed. Asterisks denote species with bound sodium or magnesium adducts. (B) The concentration of K-RAS bound to GDP and GTP was determined from deconvolution of the native MS data (dots) and fitted to a first-order rate constant model (solid lines). (C) Plot of inorganic phosphate concentration determined for K-RAS·GTP using a malachite green (MG) assay. The inset shows the similar rate constant values determined by native MS and the MG assay. Reproduced from ref (469). Copyright 2019 American Chemical Society.
Figure 30 Determination of the guanosine triphosphate (GTP) hydrolysis rate (khyd,GTP) for K-RAS. (A) Representative native mass spectra recorded at different time points for K-RAS (2 μM) loaded with GTP incubated at 25 °C. The abundance of K-RAS bound to guanosine diphosphate (GDP) increases as GTP is hydrolyzed. Asterisks denote species with bound sodium or magnesium adducts. (B) The concentration of K-RAS bound to GDP and GTP was determined from deconvolution of the native MS data (dots) and fitted to a first-order rate constant model (solid lines). (C) Plot of inorganic phosphate concentration determined for K-RAS·GTP using a malachite green (MG) assay. The inset shows the similar rate constant values determined by native MS and the MG assay. Reproduced from ref (469). Copyright 2019 American Chemical Society.CC BY-NC-ND · PubMed Central

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  1. McNaught A. D., 1997, Compendium of Chemical Terminology: IUPAC Recommendations (The “Gold Book”)
  2. Mamyrin B. A., 1973, Zh. Ehksp. Teor. Fiz., 64, 82
  3. Ye B., 2003, Clin. Cancer Res., 9, 2904

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Authors
Tamara, S., den Boer, M. A., & Heck, A. J. R.
Affiliations
  • Utrecht University (NL)
  • Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands
Corresponding author
Heck, Albert J. R.
ORCID
Venue
Chemical Reviews
Publisher
American Chemical Society (ACS)
Published
2021-08-20
Volume
122
Issue
8
Pages
7269-7326
ISSN
  • 0009-2665 (ISSN-L)
  • 1520-6890
DOI
10.1021/acs.chemrev.1c00212
PMC
PMC9052423
OpenAlex
W3194897413
Cited by
511
References
494
Citations by year
2026: 95 · 2025: 138 · 2024: 123 · 2023: 95 · 2022: 54 · 2021: 6
FWCI
7.75
Type
journal article
Language
en
Open access
Licence
Locations
  • Chemical Reviews (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
Funders
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NACTAR 16442, SPI.2017.028)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Mass Spectrometry Techniques and Applications
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Metabolomics and Mass Spectrometry Studies
Keywords
native mass spectrometry, high-resolution mass spectrometry, protein structure, protein complexes, protein assembly, biopharmaceuticals, plasma proteins, protein-ligand interactions, protein-lipid interactions, drug-protein interactions, carbohydrate-protein interactions, mass analyzers
MeSH terms
Ligands, Proteins, Mass Spectrometry, Macromolecular Substances

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