Ferritin is a common iron storage protein complex found in both eukaryotic and prokaryotic organisms. Although horse spleen holoferritin (HS-HoloFt) has been widely studied, this is the first report of mass spectrometry (MS) analysis of the intact form, likely because of its high molecular weight ∼850 kDa and broad iron-core mass distribution. The 24-subunit ferritin heteropolymer protein shell consists of light (L) and heavy (H) subunits and a ferrihydrite-like iron core. The H/L heterogeneity ratio of the horse spleen apoferritin (HS-ApoFt) shell was found to be ∼1:10 by liquid chromatography-electrospray ionization mass spectrometry. Superconducting tunneling junction (STJ) cryodetection matrix-assisted laser desorption ionization time-of-flight MS was utilized to determine the masses of intact HS-ApoFt, HS-HoloFt, and the HS-HoloFt dimer to be ∼505 kDa, ∼835 kDa, and ∼1.63 MDa, respectively. The structural integrity of HS-HoloFt and the proposed mineral adducts found for both purified L and H subunits suggest a robust biomacromolecular complex that is internally stabilized by the iron-based core. However, cross-linking experiments of HS-HoloFt with glutaraldehyde, unexpectedly, showed the complete release of the iron-based core in a one-step process revealing a cross-linked HS-ApoFt with a narrow fwhm peak width of 31.4 kTh compared to 295 kTh for HS-HoloFt. The MS analysis of HS-HoloFt revealed a semiquantitative description of the iron content and core dispersity of 3400 ± 1600 (2σ) iron atoms. Commercially prepared HS-ApoFt was estimated to still contain an average of 240 iron atoms. These iron abundance and dispersity results suggest the use of STJ cryodetection MS for the clinical analysis of iron deficient/overload diseases.

The characterization of nanomaterials is critical to understand the size/structure-dependent properties of these particles. In this report, a form of heavy ion mass spectrometry, namely, superconducting tunnel junction (STJ) cryodetection mass spectrometry (MS) is used to characterize quantum dot semiconductor nanocrystals and gold nanoparticles. The nanoparticles studied ranged in mass from 200 kDa to >1.5 MDa and included lead sulfide quantum dots, various cadmium selenide and/or telluride-based core-shell quantum dots coated with different ligands, and gold nanoparticles. Nanoparticles were ionized by both matrix-assisted laser desorption ionization (MALDI) and laser desorption ionization (LDI), shot with an aimed ion gun into a flight tube, mass separated by time-of-flight (TOF), and detected by an energy-sensitive STJ cryodetector. STJ cryodetection MS can be used to analyze intact heterogeneous nanoparticles, allowing determination of average particle mass, dispersity, and ligand loading. Some nanoparticles, however, do undergo fragmentation during the MALDI or LDI-TOF mass analyses. The measurement of the energy deposited into the detector was found to be different for different types of particles. Metastable fragments from these nanoparticles were observed at lower energies. The lower energies deposited for metastable fragments can provide insight into the stability and surface compositions of these materials. Cadmium selenide core-shell quantum dots (655 nm emission) conjugated to biomacromolecules, such as cholera toxin B and human serum transferrin, were also analyzed. When compared to unconjugated particles by mass, it was determined that ∼96 cholera toxin B and ∼14 transferrin proteins were attached to the surface of these nanoparticles.

Zinc oxide (ZnO) nanoparticles coated with either n-octylamine (OA) or α-amino poly(styrene-co-acrylonitrile) (PSAN) ligands (L) have been analyzed using laser desorption/ionization and matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) superconducting tunnel junction (STJ) cryodetection mass spectrometry. STJ cryodetection has the advantage of high m/z detection and allows for the determination of average molecular weights and dispersities for 500-600 kDa ZnO-L nanoparticles. The ability to detect the relative energies deposited into the STJs has allowed for investigation of ZnO-L metastable fragmentation. ZnO-L precursor ions gain enough internal energy during the MALDI process to undergo metastable fragmentation in the flight tube. These fragments produced a lower energy peak, which was assigned as ligand-stripped ZnO cores whereas the individual ligands were at too low of an energy to be observed. From these STJ energy resolved peaks, the average weight percentage of inorganic material making up the nanoparticle was determined, where ZnO-OA and ZnO-PSAN nanoparticles are comprised of ~62% and ~68% wt ZnO, respectively. In one example, grafting densities were calculated based on the metastable fragmentation of ligands from the core to be 16 and 1.1 nm for ZnO-OA and ZnO-PSAN, respectively, and compared with values determined by thermogravimetric analysis (TGA) and transmission electron microscopy (TEM). Graphical Abstract ᅟ.

Cryodetection mass spectrometry (MS) was used to study the Au10(TBBT)10 (TBBT = 4-tert-butylbenzenethiolate) catenane nanocluster. The matrix-assisted laser desorption ionization (MALDI) process generates distinct fragments that can be arranged into two distinct regimes: (i) in-source fragmentation, which occurs rapidly in a relatively short (<170 ns) time frame, and (ii) metastable fragmentation, which occurs postacceleration during a time-of-flight (TOF) mass analysis over a longer time frame (>170 ns–250 μs). Using MALDI-TOF MS with superconducting tunnel junction (STJ) cryodetection, distinct metastable nanocluster fragments were resolved at lower energies deposited into the detector. The results also demonstrated that STJ cryodetection MS can be used to acquire multiple (>10), simultaneous tandem mass spectra in a single experiment. Simulated fragmentation of the Au10 nanocluster using ab initio molecular dynamics (AIMD) revealed the different fragmentation processes and confirmed the MS results. Using both the empirical MS data and AIMD calculations, fragmentation pathways are proposed for Au10(TBBT)10, which terminate with two small, stable ringed species.

resolution, we might be able to obtain information about structural compactness. ■ ASSOCIATED CONTENT * sı Supporting InformationThe Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.1c05074.Figures for STJ energy response calibration, pressure dependent experiments of cytochrome c, and additional mass spectra for carbonic anhydrase and BSA (PDF)