Journal of the American Society for Mass Spectrometry最新文献

In seawater, most dissolved copper (Cu) is complexed by organic ligands, many of which are thought to be produced by phytoplankton. Although very little is known about the composition and structure of these ligands, they play an important role in determining the reactivity and bioavailability of Cu. In this study, Phaeodactylum tricornutum, a marine diatom known to produce Cu ligands (CuLs), was grown in laboratory pure culture, and the CuLs were recovered from the growth media. Using liquid chromatography coupled to ultrahigh resolution tandem mass spectrometry, 11 Cu ligand complexes were identified and assigned molecular formulas. Molecular formulas were confirmed by comparing the expected and observed relative abundances of ¹⁵N, ¹³C, ⁶⁵Cu, and ¹⁸O isotopologues. The CuLs had molecular weights from 520 to 719 Da and molecular formulas of C_26-35H_23-36O_5-9N_3-4Cu with an average assignment error of 56 ppb. High-resolution tandem mass spectrometry of the Cu-bound and metal-free ligands revealed these to be a suite of tri- and tetrapyrroles stabilized through complexation of Cu by N. The ligands share similar parent structures but differ in the number, type, and arrangement of functional groups that decorate the pyrroles. The similarity of CuL structures with known catabolites of chlorophyll suggests these ligands may be widely produced by marine photoautotrophs.

Electrospray ionization mass spectrometry has long been a prevalent ionization method in the analysis of low volatility molecules with biological, environmental, and industrial relevance. To address analytical challenges associated with molecules suffering from low ionization efficiencies (IEs), chemical derivatization (CD) strategies have been developed and are frequently adopted into workflows. However, only a minute number of CD techniques have been developed for negative polarity. To address this disparity, we evaluated 27 anions based on three criteria: (1) IE relative to a sodium dodecyl sulfate (11 + Na⁺) internal standard; (2) stability to collision induced dissociation; (3) diagnostic tandem mass spectrometry behavior. Highly fluorous ions exhibiting weakly coordinating and hydrophobic properties displayed enhanced IE. Trifluoromethanesulfonyl-containing ions proved to be unexpectedly labile, while tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (23) and bis(nonafluoro-1-butane)sulfonimidate (25) were determined to be of optimal IE of 332% ± 25% and 939% ± 92%, respectively, and in tandem MS exhibited survival yields of 100% ± 0% and 72.6% ± 0.8% at -50 eV. 23 and 25 were also comparable in IE across several solvents, and combinations thereof, that are ubiquitous in liquid chromatography. Various salts of 25 were evaluated for cation effects, where the IE of 25 ranged from 939% ± 92% to 3195% ± 145% across K⁺, NH₄⁺, Na⁺, and H⁺. Compared to tetra-n-butylammonium, tetra-n-butylphosphonium, and (4-methylphenyl)diphenylsulfonium cations, 25 displayed signal enhancements ranging from 136% ± 6% to 181% ± 14%, thereby making it an optimal candidate for CD development.

Glycoproteomics has recently increased in popularity due to instrumental and methodological advances. That said, O-glycoproteomic analysis is still challenging for various reasons, including signal suppression, search algorithm limitations, and co-occupancy of N- and O-glycopeptides. To decrease sample complexity and simplify analysis, most O-glycoproteomic workflows include PNGaseF digestion, which is an endoglycosidase that removes most N-glycan structures. Here, we report that N-glycans released from PNGaseF digestion were identified during data acquisition and hampered detection of O-glycopeptides. Importantly, we noted instances where free glycans adducted to unmodified peptides in the gas phase and were misidentified by search algorithms as O-glycopeptides. We confirmed the presence of free glycans in other experiments performed in our laboratory, as well as from data generated by other groups. To overcome this limitation, we demonstrated that released N-glycans can be removed using a molecular weight cut off filter prior to (glyco)protease digestion, which improves O-glycoproteomic coverage.

The benefits of native mass spectrometry have led to the extensive study of proteins inside mass spectrometers in the gas phase. The expansion of native mass spectrometry requires novel tools for gaining greater insights into protein structures. Herein, we introduce a new approach utilizing gas phase ion/ion reactions, where cross-linking reagents link unprotonated lysine residues, arginine residues, and N-termini with their protonated forms. We used three lengths of linkers, determining that different length cross-linkers resulted in different residues being cross-linked, as we have previously observed for electrostatic-to-electrostatic cross-linkers. However, this new method allows for the probing of both protonated and neutral lysine and arginine residues. Native mass spectrometry often produces fewer charges than protonatable sites, allowing access to a greater number of sites on proteins using an electrostatic-to-covalent cross-linking approach. In this report, we describe the reaction phenomenology and trends at reaction sites. We envision electrostatic-to-covalent cross-linking as a useful structural tool to provide complementary information to other native MS-based measurements such as collision cross section.

Reactions with mobile protons occur under electrospray ionization (ESI) in many applications of mass spectrometry. Understanding how protonation isomers (protomers) form and how molecular structure influences protomer interconversion provides fundamental insight into ESI mechanisms, which can then be exploited to rationalize ion mobility and ion activation processes for robust analyte detection. Using ten arylamine protomer systems, this paper establishes the key substrate properties that influence protomer isomerism. Protomers from ten arylamines are separated by differential ion mobility spectrometry (DMS) mass spectrometry and identified by characteristic collision-induced dissociation mass spectra. These assignments are further rationalized using quantum chemical calculations (M06-2X/6-31G(2df,p)). Based on these assignments, mobility-selected protomers are then allowed to react with methanol vapor under atmospheric and reduced pressure conditions (2.5 mTorr, 300 K). The latter enabled measurements of the second-order rate coefficients for methanol-catalyzed protomer isomerization, which span 3.9 × 10^-11-2 × 10^-13 cm³ molecule^-1 s^-1. Double-hybrid quantum chemical calculations (DSD-PBEP86-D3(BJ)/aug-cc-pVDZ) show that the direction of proton transfer is controlled by protomer relative stability, whereas reaction rates are controlled by a key transition state that separates the protonation sites. Computational exploration of a larger substituted-arylamine test-set shows that the protomer proton affinity generally correlates with energy of the key transition state. Applying the Bell-Evans-Polanyi principle to this reaction set highlights that outliers in the predictive model correspond to transition states with significant displacements along the reaction coordinate. This archetype system of derivatized arylamines provides a foundation to understand how substrate functionalization influences protomer isomerism for ions during ESI and predicts protonation isomer distributions.

Ion mobility mass spectrometry (IM-MS) can assist in the identification of isobaric chemical analytes by exploiting the difference in their gas phase collision cross-section (CCS) property. In glycomics, reliable glycan characterization remains challenging, even with IM-MS, because of closely related isomeric species and the available binding arrangements of substituted monosaccharides, allowing for the formation of complex structures. Here, we present a computational procedure to obtain gas-phase structural information from the experimental IM-MS CCS data of carbohydrates. The workflow proceeds with high throughput charge modeling of glycan seed structures to determine the precise protonation or deprotonation site. The charge models were then screened by using density functional theory (DFT) to produce candidate charge states for conformation generation. An extensive conformational scoring of the glycan ions was performed quantum mechanically at the DFT D3-B3LYP/6-31G+(d,p) level for the negative mode, [M - H]^-, and at the D3-B3LYP/6-31G(d,p) level for the positive mode, [M + H]⁺. For most of our test set, the computed CCS values from the final geometry optimized structures showed good agreement with experiment. We also demonstrated the capability of characterizing configurational and constitutional isomeric species. Altogether, we believe that the method we used in this work can be used to build a reliable theoretical reference database for glycans that can be used for experimental quality control and for assigning molecular structure to experimental IM-MS CCS information.

In some types of mass spectrometers, such as time-of-flight mass spectrometers (TOF-MSs), it is necessary to control pulsed beams of ions. This can be easily accomplished by applying a pulsed voltage to the pusher electrode while the ionizer is continuously flowing ions. This method is preferred for its simplicity, although the ion utilization efficiency is not optimized. Here we employed another pulse-control method with a higher ion utilization rate, which is to bunch ions and kick them out instead of letting them stream. The benefit of this method is that higher sensitivity can be achieved; since the start of new ions cannot be allowed during TOF separation, it is highly advantageous to bunch ions that would otherwise be unusable. In this study, we used analytical and numerical methods to design a new bunching ionizer with reduced resources, adopting the principle of the electrostatic ion beam trap. The test model experimentally demonstrated the bunching performance with respect to the sample gas density and ion bunching time using gas samples and electron impact ionization. We also conducted an experiment connecting the newly developed bunching ionizer with a miniature TOF-MS. As a result, the sensitivity was improved by an order of magnitude compared to the case using a nonbunching ionizer. Since the device is capable of bunching ions with low voltage and power consumption, it will be possible to find applications in portable mass spectrometers with reduced resources.

Neuraminidase (NA) is a kind of glycoside hydrolase enzyme, functioning to remove terminal sialic acid (Sia) from glycans which are located on the cell surface. NA plays an essential role in cell interactions with ligands, microbes, and other cells during physiological and pathological processes. Additionally, NA is a major target for developing anti-influenza drugs and influenza vaccines. Herein, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) based method to quantify NA activity is demonstrated for the first time. A reactive matrix 2-hydrazinoquinoline (2-HQ) is used to on-target label the natural substrate (3-sialyllactose, 3'-SL) and its enzymatic product (Sia). The derivatization enhances the ionization efficiency of 3'-SL and Sia, especially in negative ion detection mode. Moreover, the lactose ion signals and noise are significantly suppressed. Consequently, NA activity in influenza vaccines has been successfully quantified by comparing the relative intensity of 2-HQ derivatized Sia and 3'-SL in the absence of an additional internal standard. Moreover, the inhibition efficiencies of NA inhibitors have also been measured. Due to its operating simplicity, high-throughput capacity, and quantification accuracy, the proposed method has potential to be applied for the detection of different kinds of NA activity to reveal the role of NA in immunity, vaccine, and infection processes.

It is demonstrated here that a recently published LabView-based time-of-flight mass spectrometer (TOFMS) simulation program (named TOFSim) can accurately simulate data collected on a commercial Bruker Autoflex III matrix-assisted laser desorption/ionization (MALDI) TOFMS instrument operating in linear mode. Once the instrument distances are determined by matching measured and simulated flight times, it is shown that both overall flight times and peak widths are reproduced for data collected under both focused and slightly defocused conditions. This work confirms that TOFSim can be used not just for training new instrument operators in the principles of TOFMS but, as demonstrated here, to show how changing the voltage applied to grid G1 in the source or the delayed extraction delay time affects the focusing properties of the instrument. In the future we expect that this will also allow users to perform "what-if" experiments to investigate scenarios which may be difficult or impossible to do in a real instrument.

Sample carryover is a common problem in hydrogen-deuterium exchange mass spectrometry, particularly because immobilized protease columns cannot withstand the high organic solvent concentrations typically used in liquid chromatography-mass spectrometry (LC-MS) for cleaning. Conventional cleaning methods using injections of guanidine HCl still suffer from carryover and may require four blanks after each sample run to fully remove carryover. We have implemented an additional LC pump to deliver customized wash solutions to protease and enzyme columns, and the associated LC capillaries to eliminate carryover. Pump-based washing using the protease-safe wash solutions tested herein was able to fully remove carryover with only one blank run. FOS-choline-12 was found to be the most effective component in wash solutions and even performed well alone at 0.1% volume concentration. Since the protease column washing is performed concurrently during the analytical gradient within a sample run, subsequent blank runs could be reduced from four to one and total run time could be reduced by up to 60%. Savings in total run time could more than double the productivity of data acquisition, which is imperative for pandemic preparedness and for acceleration of biotherapeutics development.