Sung Hwan Yoon

Surface Acoustic Wave Nebulization Produces Ions with Lower Internal Energy than Electrospray Ionization

Surface acoustic wave nebulization (SAWN) has recently been reported as a novel method to transfer non-volatile analytes directly from solution to the gas phase for mass spectrometric analysis. Here we present a comparison of the survival yield of SAWN versus electrospray ionization (ESI) produced ions. A series of substituted benzylpyridinium (BzPy) compounds were utilized to measure ion survival yield from which ion energetics were inferred. We also estimated bond dissociation energies using higher level quantum chemical calculations than previously reported for BzPy ions. Additionally, the effects on BzPy precursor ion survival of SAWN operational parameters such as inlet capillary temperature and solution flow-rate were investigated. Under all conditions tested, SAWN-generated BzPy ions displayed a higher tendency for survival and thus have lower internal energies than those formed by ESI.

Surface Acoustic Wave Nebulization Facilitating Lipid Mass Spectrometric Analysis

Surface acoustic wave nebulization (SAWN) is a novel method to transfer nonvolatile analytes directly from the aqueous phase to the gas phase for mass spectrometric analysis. The lower ion energetics of SAWN and its planar nature make it appealing for analytically challenging lipid samples. This challenge is a result of their amphipathic nature, labile nature, and tendency to form aggregates, which readily precipitate clogging capillaries used for electrospray ionization (ESI). Here, we report the use of SAWN to characterize the complex glycolipid, lipid A, which serves as the membrane anchor component of lipopolysaccharide (LPS) and has a pronounced tendency to clog nano-ESI capillaries. We also show that unlike ESI SAWN is capable of ionizing labile phospholipids without fragmentation. Lastly, we compare the ease of use of SAWN to the more conventional infusion-based ESI methods and demonstrate the ability to generate higher order tandem mass spectral data of lipid A for automated structure assignment using our previously reported hierarchical tandem mass spectrometry (HiTMS) algorithm. The ease of generating SAWN-MS(n) data combined with HiTMS interpretation offers the potential for high throughput lipid A structure analysis.

Structural basis of MsbA-mediated lipopolysaccharide transport.

Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.

Comprehensive glycosylation profiling of IgG and IgG-fusion proteins by top-down MS with multiple fragmentation techniques

UNLABELLED We employed top- and middle-down analyses with multiple fragmentation techniques including electron transfer dissociation (ETD), electron capture dissociation (ECD), and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) for characterization of a reference monoclonal antibody (mAb) IgG1 and a fusion IgG protein. Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) on an Orbitrap was employed. These experiments provided a comprehensive view on the protein species; especially for different glycosylation level in these two proteins, which showed good agreement with oligosaccharide profiling. Top- and middle-down MS provided additional information regarding glycosylation sites and different combinational protein species that were not available from oligosaccharide mapping or conventional bottom-up analysis. Finally, incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS) with MALDI-ISD analysis enabled extended sequence coverage of the internal region of protein without pre-fractionation. BIOLOGICAL SIGNIFICANCE Oligosaccharide profiling together with top- and middle-down methods enabled: 1) detection of heterogeneous glycosylated protein species and sites in intact IgG1 and fusion proteins with high mass accuracy, 2) estimation of relative abundance levels of protein species in the sample, 3) confirmation of the protein termini structural information, and 4) improved sequence coverage by MALDI-ISD analysis for the internal regions of the proteins without sample pre-fractionation.

Rapid lipid a structure determination via surface acoustic wave nebulization and hierarchical tandem mass spectrometry algorithm

RATIONALE Surface acoustic wave nebulization (SAWN) is an easy to use sample transfer method for rapid mass spectrometric analysis. A new standing wave (SW) SAWN chip, with higher ionization efficiency than our previously reported design, is used for rapid analysis of lipids. METHODS The crude, yet fast, Caroff protocol was used for lipid A extraction from Francisella novicida. SW-SAWN with a Waters Synapt G2S quadrupole time-of-flight (QTOF) mass spectrometer was used to generate lipid A ions. Quadrupole collision-induced dissociation (Q-CID) of lipid A at varying CID energies was used to approximate the ion trap MSn data required for our hierarchical tandem mass spectrometry (HiTMS) algorithm. Structural hypotheses can be obtained directly from the HiTMS algorithm to identify species-specific lipid A molecules. RESULTS SW-SAWN successfully generated ions from lipid A extracted from Francisella novicida using the faster Caroff method. In addition, varying collision energies were used to generate tandem mass spectra similar to MS3 and MS4 spectra from an ion trap. The Q-CID spectra are compatible with our HiTMS algorithm and offer an improvement over lipid A tandem mass spectra acquired in an ion trap. CONCLUSIONS Combining SW-SAWN and Q-CID enabled more structural assignments than previously reported in half the time. The ease of generating spectra by SAWN tandem MS in combination with HiTMS interpretation offers high-throughput lipid A structural analysis and thereby rapid detection of pathogens based on lipid fingerprinting. Copyright

Surface acoustic wave nebulization device with dual interdigitated transducers improves SAWN-MS performance

We compared mass spectrometric (MS) performance of surface acoustic wave nebulization (SAWN) generated by a single interdigitated transducer (IDT) designed to produce a progressive wave (PW) to one with a dual IDT that can in theory generate standing waves (SW). Given that devices using dual IDTs had been shown to produce fewer large size droplets on average, we hypothesized they would improve MS performance by improving the efficiency of desolvation. Indeed, the SW-SAWN chip provided an improved limit of detection of 1 femtomole of peptide placed on chip making it 100× more sensitive than the PW design. However, as measured by high-speed image recording and phase Doppler particle analyzer measurements, there was only a 26% increase in the small diameter (1-10 µm) droplets produced from the new device, precluding a conclusion that the decrease in droplet size was solely responsible for the improvement in MS signal/noise. Given that the dual IDT design produced a more instantaneous plume than the PW design, the more likely contributor to improved MS signal/noise was concluded to be a higher ion flux entering the mass spectrometer for the dual IDT designs. Notably, the dual IDT device allowed production of much higher quality protein mass spectra up to about 20 kDa, compared with the single IDT device. Copyright

Glycosylation characterization of therapeutic mAbs by top- and middle-down mass spectrometry.

A reference monoclonal antibody IgG1 and a fusion IgG protein were analyzed by top- and middle-down mass spectrometry with multiple fragmentation techniques including electron transfer dissociation (ETD) and matrix-assisted laser desorption ionization in-source decay (MALDI-ISD) to investigate heterogeneity of glycosylated protein species. Specifically, glycan structure, sites, relative abundance levels, and termini structural conformation were investigated by use of Fourier transform ion cyclotron resonance (FT-ICR) or high performance liquid chromatography electrospray ionization (HPLC-ESI) linked to an Orbitrap. Incorporating a limited enzymatic digestion by immunoglobulin G-degrading enzyme Streptococcus pyogenes (IdeS) with MALDI-ISD analysis extended sequence coverage of the internal region of the proteins without pre-fractionation. The data in this article is associated with the research article published in Journal of Proteomics (Tran et al., 2015) [1].

Identification and characterization of a drug-sensitive strain enables puromycin-based translational assays in Saccharomyces cerevisiae

Puromycin is an aminonucleoside antibiotic with structural similarity to aminoacyl tRNA. This structure allows the drug to bind the ribosomal A site and incorporate into nascent polypeptides, causing chain termination, ribosomal subunit dissociation and widespread translational arrest at high concentrations. In contrast, at sufficiently low concentrations, puromycin incorporates primarily at the C‐terminus of proteins. While a number of techniques utilize puromycin incorporation as a tool for probing translational activity in vivo, these methods cannot be applied in yeasts that are insensitive to puromycin. Here, we describe a mutant strain of the yeast Saccharomyces cerevisiae that is sensitive to puromycin and characterize the cellular response to the drug. Puromycin inhibits the growth of yeast cells mutant for erg6∆, pdr1∆ and pdr3∆ (EPP) on both solid and liquid media. Puromycin also induces the aggregation of the cytoplasmic processing body component Edc3 in the mutant strain. We establish that puromycin is rapidly incorporated into yeast proteins and test the effects of puromycin on translation in vivo. This study establishes the EPP strain as a valuable tool for implementing puromycin‐based assays in yeast, which will enable new avenues of inquiry into protein production and maturation. Copyright

Comprehensive analysis of clinical Burkholderia pseudomallei isolates demonstrates conservation of unique lipid A structure and TLR4-dependent innate immune activation.

Burkholderia pseudomallei is an environmental bacterium that causes melioidosis, a major community-acquired infection in tropical regions. Melioidosis presents with a range of clinical symptoms, is often characterized by a robust inflammatory response, may relapse after treatment, and results in high mortality rates. Lipopolysaccharide (LPS) of B. pseudomallei is a potent immunostimulatory molecule comprised of lipid A, core, and O-polysaccharide (OPS) components. Four B. pseudomallei LPS types have been described based on SDS-PAGE patterns that represent the difference of OPS–type A, type B, type B2 and rough LPS. The majority of B. pseudomallei isolates are type A. We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) followed by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QqTOF MS) and gas chromatography to characterize the lipid A of B. pseudomallei within LPS type A isolates. We determined that B. pseudomallei lipid A is represented by penta- and tetra-acylated species modified with 4-amino-4-deoxy-arabinose (Ara4N). The MALDI-TOF profiles from 171 clinical B. pseudomallei isolates, including 68 paired primary and relapse isolates and 35 within-host isolates were similar. We did not observe lipid A structural changes when the bacteria were cultured in different growth conditions. Dose-dependent NF-κB activation in HEK cells expressing TLR4 was observed using multiple heat-killed B. pseudomallei isolates and corresponding purified LPS. We demonstrated that TLR4-dependent NF-κB activation induced by heat-killed bacteria or LPS prepared from OPS deficient mutant was significantly greater than those induced by wild type B. pseudomallei. These findings suggest that the structure of B. pseudomallei lipid A is highly conserved in a wide variety of clinical and environmental circumstances but that the presence of OPS may modulate LPS-driven innate immune responses in melioidosis.

Structural modification of LPS in colistin-resistant, KPC-producing Klebsiella pneumoniae

Background Colistin resistance in Klebsiella pneumoniae typically involves inactivation or mutations of chromosomal genes mgrB, pmrAB or phoPQ, but data regarding consequent modifications of LPS are limited. Objectives To examine the sequences of chromosomal loci implicated in colistin resistance and the respective LPS-derived lipid A profiles using 11 pairs of colistin-susceptible and -resistant KPC-producing K. pneumoniae clinical strains. Methods The strains were subjected to high-throughput sequencing with Illumina HiSeq. The mgrB gene was amplified by PCR and sequenced. Lipid profiles were determined using MALDI-TOF MS. Results All patients were treated with colistimethate prior to the isolation of colistin-resistant strains (MIC >2 mg/L). Seven of 11 colistin-resistant strains had deletion or insertional inactivation of mgrB. Three strains, including one with an mgrB deletion, had non-synonymous pmrB mutations associated with colistin resistance. When analysed by MALDI-TOF MS, all colistin-resistant strains generated mass spectra containing ions at m/z 1955 and 1971, consistent with addition of 4-amino-4-deoxy-l-arabinose (Ara4N) to lipid A, whereas only one of the susceptible strains displayed this lipid A phenotype. Conclusions The pathway to colistin resistance in K. pneumoniae primarily involves lipid A modification with Ara4N in clinical settings.