TMED10 mediates the trafficking of insulin-like growth factor 2 along the secretory pathway for myoblast differentiation.

The insulin-like growth factor 2 (IGF2) plays critical roles in cell proliferation, migration, differentiation, and survival. Despite its importance, the molecular mechanisms mediating the trafficking of IGF2 along the secretory pathway remain unclear. Here, we utilized a Retention Using Selective Hook system to analyze molecular mechanisms that regulate the secretion of IGF2. We found that a type I transmembrane protein, TMED10, is essential for the secretion of IGF2 and for differentiation of mouse myoblast C2C12 cells. Further analyses indicate that the residues 112-140 in IGF2 are important for the secretion of IGF2 and these residues directly interact with the GOLD domain of TMED10. We then reconstituted the release of IGF2 into COPII vesicles. This assay suggests that TMED10 mediates the packaging of IGF2 into COPII vesicles to be efficiently delivered to the Golgi. Moreover, TMED10 also mediates ER export of TGN-localized cargo receptor, sortilin, which subsequently mediates TGN export of IGF2. These analyses indicate that TMED10 is critical for IGF2 secretion by directly regulating ER export and indirectly regulating TGN export of IGF2, providing insights into trafficking of IGF2 for myoblast differentiation.

Identifications of novel host cell factors that interact with the receptor-binding domain of the SARS-CoV-2 spike protein.

SARS-CoV-2 entry into host cells is facilitated by the interaction between the receptor-binding domain of its spike protein (CoV2-RBD) and host cell receptor, ACE2, promoting viral membrane fusion. The virus also uses endocytic pathways for entry, but the mediating host factors remain largely unknown. It is also unknown whether mutations in the RBD of SARS-CoV-2 variants promote interactions with additional host factors to promote viral entry. Here, we used the GST pull-down approach to identify novel surface-located host factors that bind to CoV2-RBD. One of these factors, SH3BP4, regulates internalization of CoV2-RBD in an ACE2-independent but integrin- and clathrin-dependent manner and mediates SARS-CoV-2 pseudovirus entry, suggesting that SH3BP4 promotes viral entry via the endocytic route. Many of the identified factors, including SH3BP4, ADAM9, and TMEM2, show stronger affinity to CoV2-RBD than to RBD of the less infective SARS-CoV, suggesting SARS-CoV-2-specific utilization. We also found factors preferentially binding to the RBD of the SARS-CoV-2 Delta variant, potentially enhancing its entry. These data identify the repertoire of host cell surface factors that function in the events leading to the entry of SARS-CoV-2.

Targeted Quantification of Glutathione/Arginine Redox Metabolism Based on a Novel Paired Mass Spectrometry Probe Approach for the Functional Assessment of Redox Status.

Glutathione (GSH) redox control and arginine metabolism are critical in regulating the physiological response to injury and oxidative stress. Quantification assessment of the GSH/arginine redox metabolism supports monitoring metabolic pathway shifts during pathological processes and their linkages to redox regulation. However, assessing the redox status of organisms with complex matrices is challenging, and single redox molecule analysis may not be accurate for interrogating the redox status in cells and in vivo. Herein, guided by a paired derivatization strategy, we present a new ultraperformance liquid chromatography tandem mass spectrometry (UPLC-MS/MS)-based approach for the functional assessment of biological redox status. Two structurally analogous probes, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) and newly synthesized 2-methyl-6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (MeAQC), were set for paired derivatization. The developed approach was successfully applied to LPS-stimulated RAW 264.7 cells and HDM-induced asthma mice to obtain quantitative information on GSH/arginine redox metabolism. The results suggest that the redox status was remarkably altered upon LPS and HDM stimulation. We expect that this approach will be of good use in a clinical biomarker assay and potential drug screening associated with redox metabolism, oxidative damage, and redox signaling.

An in vitro vesicle formation assay reveals cargo clients and factors that mediate vesicular trafficking.

The fidelity of protein transport in the secretory pathway relies on the accurate sorting of proteins to their correct destinations. To deepen our understanding of the underlying molecular mechanisms, it is important to develop a robust approach to systematically reveal cargo proteins that depend on specific sorting machinery to be enriched into transport vesicles. Here, we used an in vitro assay that reconstitutes packaging of human cargo proteins into vesicles to quantify cargo capture. Quantitative mass spectrometry (MS) analyses of the isolated vesicles revealed cytosolic proteins that are associated with vesicle membranes in a GTP-dependent manner. We found that two of them, FAM84B (also known as LRAT domain containing 2 or LRATD2) and PRRC1, contain proline-rich domains and regulate anterograde trafficking. Further analyses revealed that PRRC1 is recruited to endoplasmic reticulum (ER) exit sites, interacts with the inner COPII coat, and its absence increases membrane association of COPII. In addition, we uncovered cargo proteins that depend on GTP hydrolysis to be captured into vesicles. Comparing control cells with cells depleted of the cargo receptors, SURF4 or ERGIC53, we revealed specific clients of each of these two export adaptors. Our results indicate that the vesicle formation assay in combination with quantitative MS analysis is a robust and powerful tool to uncover novel factors that mediate vesicular trafficking and to uncover cargo clients of specific cellular factors.

Lysyl hydroxylase LH1 promotes confined migration and metastasis of cancer cells by stabilizing Septin2 to enhance actin network.

BACKGROUND: Excessive extracellular matrix deposition and increased stiffness are typical features of solid tumors such as hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma (PDAC). These conditions create confined spaces for tumor cell migration and metastasis. The regulatory mechanism of confined migration remains unclear. METHODS: LC-MS was applied to determine the differentially expressed proteins between HCC tissues and corresponding adjacent tissue. Collective migration and single cell migration microfluidic devices with 6 µm-high confined channels were designed and fabricated to mimic the in vivo confined space. 3D invasion assay was created by Matrigel and Collagen I mixture treat to adherent cells. 3D spheroid formation under various stiffness environment was developed by different substitution percentage GelMA. Immunoprecipitation was performed to pull down the LH1-binding proteins, which were identified by LC-MS. Immunofluorescent staining, FRET, RT-PCR, Western blotting, FRAP, CCK-8, transwell cell migration, wound healing, orthotopic liver injection mouse model and in vivo imaging were used to evaluate the target expression and cellular phenotype. RESULTS: Lysyl hydroxylase 1 (LH1) promoted the confined migration of cancer cells at both collective and single cell levels. In addition, LH1 enhanced cell invasion in a 3D biomimetic model and spheroid formation in stiffer environments. High LH1 expression correlated with poor prognosis of both HCC and PDAC patients, while it also promoted in vivo metastasis. Mechanistically, LH1 bound and stabilized Septin2 (SEPT2) to enhance actin polymerization, depending on the hydroxylase domain. Finally, the subpopulation with high expression of both LH1 and SEPT2 had the poorest prognosis. CONCLUSIONS: LH1 promotes the confined migration and metastasis of cancer cells by stabilizing SEPT2 and thus facilitating actin polymerization.

Interdomain flexibility and interfacial integrity of ß-lactamase inhibitory protein (BLIP) modulate its binding to class A ß-lactamases.

ß-Lactamase inhibitory protein (BLIP) consists of a tandem repeat of αß domains conjugated by an interdomain loop and can effectively bind and inactivate class A ß-lactamases, which are responsible for resistance of bacteria to ß-lactam antibiotics. The varied ability of BLIP to bind different ß-lactamases and the structural determinants for significant enhancement of BLIP variants with a point mutation are poorly understood. Here, we investigated the conformational dynamics of BLIP upon binding to three clinically prevalent class A ß-lactamases (TEM1, SHV1, and PC1) with dissociation constants between subnanomolar and micromolar. Hydrogen deuterium exchange mass spectrometry revealed that the flexibility of the interdomain region was significantly suppressed upon strong binding to TEM1, but was not significantly changed upon weak binding to SHV1 or PC1. E73M and K74G mutations in the interdomain region improved binding affinity toward SHV1 and PC1, respectively, showing significantly increased flexibility of the interdomain region compared to the wild-type and favorable conformational changes upon binding. In contrast, more rigidity of the interfacial loop 135-145 was observed in these BLIP mutants in both free and bound states. Consistently, molecular dynamics simulations of BLIP exhibited drastic changes in the flexibility of the loop 135-145 in all complexes. Our results indicated for the first time that higher flexibility of the interdomain linker, as well as more rigidity of the interfacial loop 135-145, could be desirable determinants for enhancing inhibition of BLIP to class A ß-lactamases. Together, these findings provide unique insights into the design of enhanced inhibitors.

High-Resolution Micro-object Separation by Rotating Magnetic Chromatography.

The development of new technologies for the separation, selection, and isolation of microparticles such as rare target cells, circulating tumor cells, cancer stem cells, and immune cells has become increasingly important in the last few years. Microparticle separation technologies are usually applied to the analysis of disease-associated cells, but these procedures often face a cell separation problem that is often insufficient for single specific cell analyses. To overcome these limitations, a highly accurate size-based microparticle separation technique, herein called "rotating magnetic chromatography", is proposed in this work. Magnetic nanoparticles, placed in a microfluidic separation channel, are forced to move in well-defined trajectories by an external magnetic field, colliding with microparticles that are in this way separated on the basis of their dimensions with high accuracy and reproducibility. The method was optimized by using fluorescein isothiocyanate-modified polystyrene particles (chosen as a reference standard) and then applied to the analysis of cancer cells like Hep-3B and SK-Hep-1, allowing their fast and high-resolution chromatographic separation as a function of their dimensions. Due to its unmatched sub-micrometer cell separation capabilities, RMC can be considered a break-through technique that can unlock new perspectives in different scientific fields, that is, in medical oncology.

Advances in rapid detection of SARS-CoV-2 by mass spectrometry.

COVID-19 has already been lasting for more than two years and it has been severely affecting the whole world. Still, detection of SARS-CoV-2 remains the frontline approach to combat the pandemic, and the reverse transcription polymerase chain reaction (RT-PCR)-based method is the well recognized detection method for the enormous analytical demands. However, the RT-PCR method typically takes a relatively long time, and can produce false positive and false negative results. Mass spectrometry (MS) is a very commonly used technique with extraordinary sensitivity, specificity and speed, and can produce qualitative and quantitative information of various analytes, which cannot be achieved by RT-PCR. Since the pandemic outbreak, various mass spectrometric approaches have been developed for rapid detection of SARS-CoV-2, including the LC-MS/MS approaches that could allow analysis of several hundred clinical samples per day with one MS system, MALDI-MS approaches that could directly analyze clinical samples for the detection, and efforts for the on-site detection with portable devices. In this review, these mass spectrometric approaches were summarized, and their pros and cons as well as further development were also discussed.

Least absolute shrinkage and selection operator-based prediction of collision cross section values for ion mobility mass spectrometric analysis of lipids.

Collision cross section (CCS) values generated from ion mobility mass spectrometry (IM-MS) have commonly been employed to facilitate lipid identification. However, this is hindered by the limited available lipid standards. Recently, CCS values were predicted by means of computational calculations, though the prediction precision was generally not good and the predicted CCS values of the lipid isomers were almost identical. To address this challenge, a least absolute shrinkage and selection operator (LASSO)-based prediction method was developed for the prediction of lipids' CCS values in this study. In this method, an array of molecular descriptors were screened and optimized to reflect the subtle differences in structures among the different lipid isomers. The use of molecular descriptors together with a wealth of standard CCS values for the lipids (365 in total) significantly improved the accuracy and precision of the LASSO model. Its accuracy was externally validated with median relative errors (MREs) of <1.1% using an independent data set. This approach was demonstrated to allow differentiation of cis/trans and sn-positional isomers. The results also indicated that the LASSO-based prediction method could practically reduce false-positive identifications in IM-MS-based lipidomics.

Atlastin-mediated membrane tethering is critical for cargo mobility and exit from the endoplasmic reticulum.

Endoplasmic reticulum (ER) membrane junctions are formed by the dynamin-like GTPase atlastin (ATL). Deletion of ATL results in long unbranched ER tubules in cells, and mutation of human ATL1 is linked to hereditary spastic paraplegia. Here, we demonstrate that COPII formation is drastically decreased in the periphery of ATL-deleted cells. ER export of cargo proteins becomes defective; ER exit site initiation is not affected, but many of the sites fail to recruit COPII subunits. The efficiency of cargo packaging into COPII vesicles is significantly reduced in cells lacking ATLs, or when the ER is transiently fragmented. Cargo is less mobile in the ER in the absence of ATL, but the cargo mobility and COPII formation can be restored by ATL R77A, which is capable of tethering, but not fusing, ER tubules. These findings suggest that the generation of ER junctions by ATL plays a critical role in maintaining the necessary mobility of ER contents to allow efficient packaging of cargo proteins into COPII vesicles.

Dual-Mode Sensing Platform for Electrochemiluminescence and Colorimetry Detection Based on a Closed Bipolar Electrode.

Development of sensors uniting different sensing principles is in line with the concept of reliable, comprehensive, and diversified equipment construction. However, the current exploration in this field is obstructed by compromise of reaction conditions and inevitable mutual interference arising from different sensing modes. This work reported a closed bipolar electrode (c-BPE) strategy for dual-modality detection or dual-target detection. To this end, a c-BPE sensing platform installed in physically separated anode and cathode compartments was well designed and carefully optimized. If luminol was present in the anode section and Prussian blue (PB) was at the cathode part, single stimulation could realize electrochemiluminescence (ECL) from luminol at the anode and conversion of PB to Prussian white (PW) at the cathode. The latter reaction helped elevate the ECL signal and also prepared for colorimetric detection as color change from PW to PB under the trigger of oxidant (like H2O2) was used to track the content of the oxidant. Thus, dual signals were obtained for dual-modality detection of single target or the detection of different targets was realized at different poles. Detection of glucose was carried out to validate the application for dual-modality detection, while VLDL/AChE and NADH/H2O2 assays illustrated the potential of dual-target detection. The proposed platform possesses outstanding sensing performance including selectivity, repeatability, long-term stability, accuracy, and so forth. This work implements a breakthrough in designing dual-mode sensors and is expected to present a rational basis for development of a diversified sensing platform.

Measurement of Intracellular Nitric Oxide with a Quantitative Mass Spectrometry Probe Approach.

Nitric oxide (NO) is a molecule of physiological importance, and the function of NO depends on its concentration in biological systems, particularly in cells. Concentration-based analysis of intracellular NO can provide insight into its precise role in health and disease. However, current methods for detecting intracellular NO are still inadequate for quantitative analysis. In this study, we report a quantitative mass spectrometry probe approach to measure NO levels in cells. The probe, Amlodipine (AML), comprises a Hantzsch ester group that reacts with NO to form a pyridine, Dehydro Amlodipine (DAM). Quantification of DAM by ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) allows specific measurement of intracellular NO levels. Notably, the AML/NO reaction proceeds rapidly (within 1 s), which is favorable for NO detection considering its large diffusivity and short half-life. Meanwhile, studies under simulated physiological conditions revealed that the AML response to NO is proportional and selective. The presented UPLC-MS/MS method showed high sensitivity (LLOQ = 0.24 nM) and low matrix interference (less than 15%) in DAM quantification. Furthermore, the mass spectrometry probe approach was demonstrated by enabling the measurement of endogenous and exogenous NO in cells. Hence, the quantitative UPLC-MS/MS method developed using AML as a probe is expected to be a new method for intracellular NO analysis.

Conformational Dynamics of the Helix 10 Region as an Allosteric Site in Class A ß-Lactamase Inhibitory Binding.

ß-Lactamase inhibitory protein (BLIP) can effectively inactivate class A ß-lactamases, but with very different degrees of potency. Understanding the different roles of BLIP in class A ß-lactamases inhibition can provide insights for inhibitor design. However, this problem was poorly solved on the basis of the static structures obtained by X-ray crystallography. In this work, ion mobility mass spectrometry, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation revealed the conformational dynamics of three class A ß-lactamases with varying inhibition efficiencies by BLIP. A more extended conformation of PC1 was shown compared to those of TEM1 and SHV1. Localized dynamics differed in several important loop regions, that is, the protruding loop, H10 loop, Ω loop, and SDN loop. Upon binding with BLIP, these loops cooperatively rearranged to enhance the binding interface and to inactivate the catalytic sites. In particular, unfavorable changes in conformational dynamics were found in the protruding loop of SHV1 and PC1, showing less effective binding. Intriguingly, the single mutation on BLIP could compensate for the unfavored changes in this region, and thus exhibit enhanced inhibition toward SHV1 and PC1. Additionally, the H10 region was revealed as an important allosteric site that could modulate the inhibition of class A ß-lactamases. It was suggested that the rigid protruding loop and flexible H10 region might be determinants for the effective inhibition of TEM1. Our findings provided unique and explicit insights into the conformational dynamics of ß-lactamases and their bindings with BLIP. This work can be extended to other ß-lactamases of interest and inspire the design of novel inhibitors.

Quantitative Analysis of α-1-Antitrypsin Glycosylation Isoforms in HCC Patients Using LC-HCD-PRM-MS.

The change in glycosylation of serum proteins is often associated with the development of various diseases and thus can be used for diagnosis. In this study, a liquid chromatography-tandem mass spectrometry-based method is used for accurate structural analysis and quantification of site-specific glycoforms of serum α-1-antitrypsin (A1AT) in early-stage HCC and cirrhosis patients. Serum protein A1AT was purified from patient sera by immunoprecipitation with anti-A1AT antibody conjugated agarose beads, and the isolated A1AT protein was digested and analyzed by LC-MS/MS. Two tandem mass spectrometry strategies are integrated in this study: a nontargeted stepped HCD strategy for structural analysis of A1AT glycopeptides and a targeted parallel reaction monitoring (PRM) strategy for quantification of site-specific glycoforms of A1AT in HCC and cirrhosis patient sera. Accordingly, pGlyco2.0 software was used for glycopeptide identification, and Skyline software was used for glycoform quantification using the Y1 ion (peptide+GlcNAc) in MS/MS spectra. Ten site-specific glycopeptides of A1AT were identified with stepped HCD-MS/MS in patient samples, 7 of which were further quantified using HCD-PRM-MS among patient samples. We found that our strategy was able to distinguish isomers of glycopeptides where several isomers showed distinctly different patterns between cirrhosis and HCC patients. We also found that the ratio of different charge states (2+/3+) of one glycopeptide of A1AT can significantly discriminate early-stage HCC from cirrhosis with the area under the receiver operating characteristic curve AUC of 0.9. Further analysis showed that the difference may be related to the sialic acid/galactose linkage of the glycan motif.

n-type polyaniline hole-blocking layer for high-efficiency QDSC by one-pot electropolymerization and selective aprotic cation ([EMIM]+) doping.

In the field of clean solar-to-current devices, the photoelectron transfer process is essential for photovoltaic conversion in the typical n-i-p solar-cell structure. With regard to the oriented injection and ejection of photoelectrons, the development of hole-blocking layer (HBL) materials with a high electron transfer capability are exceedingly desirable. Profiting from the distortion of the p-π electron cloud attracted by a doped aprotic cation, a modified n-type polyaniline (PANI) as the HBL of a photoanode has been successfully fabricated through a facial one-pot square-wave potentiostatic electropolymerization method. In terms of flat-band potential, charge-carrier concentration and device impedance, the synthesized n-type polyaniline layer doped by aprotic ionic liquid (AIL; [EMIM] [EtSO4]) (AIL-PA layer) for quantum dot solar cells (QDSCs) directly facilitates the high electron carrying capacity as well as the electron transfer driving force. Furthermore, the n-type polyaniline layer doped by AIL ([EMIM] [EtSO4]) (AIL-PA layer) has a widely matching band gap for electron exportation and improved photovoltaic performance of CdSxSe1-x QDSCs: the power conversion efficiency is 10.5% and the J sc is 21.59 mA cm-2 for the device with an AIL-PA HBL. The electron diffusion length L D is 8.07 µm for the photoanode with AIL-PA I and 7.58 µm for the photoanode with AIL-PA II.

Protein dynamics revealed by hydrogen/deuterium exchange mass spectrometry: Correlation between experiments and simulation.

Hydrogen deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for studying protein dynamics, which is an important factor governing protein functions. However, the process of hydrogen/deuterium exchange (HDX) of proteins is highly complex and the underlying mechanism has not yet been fully elucidated. Meanwhile, molecular dynamics (MD) simulation is a computational technique that can be used to elucidate HDX behaviour on proteins and facilitate interpretation of HDX-MS data. This article aims to summarize the current understandings on the mechanism of HDX and its correlation with MD simulation, to discuss the recent developments in the techniques of HDX-MS and MD simulation and to extend the perspectives of these two techniques in protein dynamics study.

Construction of TiO2 Nanotubes/C/MnO2 Composite Films as a Binder-Free Electrode for a High-Performance Supercapacitor.

Although titanium dioxide (TiO2) exhibits excellent promise in electrode materials for supercapacitors, its poor conductivity and low areal specific capacitance hamper further development. In this work, we have designed a clever way to deposit manganese dioxide (MnO2) in order to improve its electrochemical performance via a facile and typical hydrothermal method. In a hydrothermal process, carbon (C), which deposited via new gas thermal penetration, acts as a reducing agent, while a potassium permanganate (KMnO4) solution acts as an oxidant. In this way, MnO2, which has a high theoretical capacity, is generated on TiO2 nanotube arrays (denoted as TNTs) successfully. Remarkably, a TNTs/C/MnO2 film prepared at a hydrothermal temperature of 90 °C and 0.3 g of KMnO4 revealed a superior electrochemical property with 55 mF/cm2 areal capacitance at a scan rate of 5 mV/s, 23 times more enhanced than that of a TNTs/C film. Also, the energy density of a TNTs/C/MnO2 film reached 46.8 Wh/cm2 when the power density was 0.12 mW/cm2, and the energy density still remained at 22.4 Wh/cm2 at a high power density of 0.8 mW/cm2. After 1000 cycle tests, 93.2% capacitance was still retained, indicating excellent reversibility and cycle stability of TNTs/C/MnO2 electrode. This work opens up a facile path for efficient growth of electrode materials with high performance for energy storage devices.

Self-Assembled Binuclear Cu(II)-Histidine Complex for Absolute Configuration and Enantiomeric Excess Determination of Naproxen by Tandem Mass Spectrometry.

Naproxen is one of the most consumed nonsteroidal anti-inflammatory drugs and marketed as S-naproxen since R-naproxen is hepatotoxic. In this study, chiral recognition of naproxen has been investigated by tandem mass spectrometry (MS/MS). Among all diastereomeric complexes formed between naproxen and the examined chiral selectors, including cyclodextrins (α/ß/γ-CD), modified phenylalanines ( N-acetyl-phenylalanine, N-t-butoxycarbonyl-phenylalanine, N-9-fluorenylmethyloxycarbonyl-phenylalanine), amino acids (Trp, Phe, Tyr, His), glucose, tartaric acid, and vancomycin, a novel binuclear metal bound diastereomeric complexes [(M(II))2( S/ R-naproxen)(l-His)2-3H]+ (M = Cu, Ni, or Co with Cu being the best) could allow effective identification of the absolute configuration of naproxen and determination of its enantiomeric excess ( ee) through MS/MS analysis. The key candidate structure of [(Cu(II))2( S/ R-naproxen)(l-His)2-3H]+ has been revealed by means of collision-induced dissociation, ion mobility mass spectrometry and density functional theory calculations, indicating an interesting and unusual self-assembled compact geometry with the two Cu(II) ions bridged closely together (Cu-Cu distance is 3.04 Å) by the carboxylate groups of the two histidines. It was shown that the difference in dissociation efficiency between the two diastereomers was attributed to the interaction between the NH2 bond of the amino group of one histidine and the naphthyl ring of naproxen. The present report is the first to observe and characterize the complex of (Cu(II))2(His)2 with aromatic acid, which could contribute to the chiral recognition of other chiral aromatic acids, design of catalysts based on binuclear copper bound complex, as well as the better understanding of metal ion complexation by His or His-containing ligands.

Surface-Modified Wooden-Tip Electrospray Ionization Mass Spectrometry for Enhanced Detection of Analytes in Complex Samples.

Replacement of capillary with solid substrates for sample loading and ionization has created many new possibilities for electrospray ionization mass spectrometry (ESI-MS). Surface modification is an attractive strategy to enhance the analytical capability of solid-substrate ESI-MS and allow understanding the relationship between surface activity of solid substrates and analytical properties. In this study, we performed surface modification of wooden tips with hydrophobic (-C18), basic (-NH2), and acidic (-SO3H) functional groups and applied various sampling methods, i.e., extractive sampling and direct loading, to comprehensively investigate the analytical properties of solid-substrate ESI-MS. Our results showed that, for the direct loading method, analytes with weak interactions with solid-substrate surface could be readily sprayed out for detection. While for the extractive sampling method, analytes strongly retained on solid-substrate surface could be selectively enriched and detected, and a washing step after sample loading could effectively remove unbound components for reducing interference. Overall, the insights on the effects of surface-analyte interactions on the analytical features obtained in this study could aid the development of surface-modified strategies for enhancing the analytical capability of solid-substrate ESI-MS.

The analysis of alpha-1-antitrypsin glycosylation with direct LC-MS/MS.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based methodology has been developed to differentiate core- and antennary-fucosylated glycosylation of glycopeptides. Both the glycosylation sites (heterogeneity) and multiple possible glycan occupancy at each site (microheterogeneity) can be resolved via intact glycopeptide analysis. The serum glycoprotein alpha-1-antitrypsin (A1AT) which contains both core- and antennary-fucosylated glycosites was used in this study. Sialidase was used to remove the sialic acids in order to simplify the glycosylation microheterogeneity and to enhance the MS signal of glycopeptides with similar glycan structures. ß1-3,4 galactosidase was used to differentiate core- and antennary-fucosylation. In-source dissociation was found to severely affect the identification and quantification of glycopeptides with low abundance glycan modification. The settings of the mass spectrometer were therefore optimized to minimize the in-source dissociation. A three-step mass spectrometry fragmentation strategy was used for glycopeptide identification, facilitated by pGlyco software annotation and manual checking. The collision energy used for initial glycopeptide fragmentation was found to be crucial for improved detection of oxonium ions and better selection of Y1 ion (peptide+GlcNAc). Structural assignments revealed that all three glycosylation sites of A1AT glycopeptides contain complex N-glycan structures: site Asn70 contains biantennary glycans without fucosylation; site Asn107 contains bi-, tri- and tetra-antennary glycans with both core- and antennary-fucosylation; site Asn271 contains bi- and tri-antennary glycans with both core- and antennary-fucosylation. The relative intensity of core- and antennary-fucosylation on Asn107 was similar to that of the A1AT protein indicating that the glycosylation level of Asn107 is much larger than the other two sites.