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N -glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N -glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N -glycosylation at the intact N -glycopeptide level, providing comprehensive information of peptide backbones, N -glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N -glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N -glycopeptides were identified, where 422 site- and structure-specific differential N -glycosylation on 145 N -glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N -glycoproteomes at large scale. [Display omitted] • One-hour N -glycoproteomics with 1-h gradient is developed for fast analysis. • 85 % more IDs was achieved per elution time than previous 4-h gradient. • Fast N -glycoproteomics may find wide application of large-cohort study. [ABSTRACT FROM AUTHOR]

When fluid flows through a pipe that is packed with sand particles, the fluid will bear the resistance from the sand-pack, as well as the viscous shear from the pipe wall. If the viscous shear from the pipe wall can be neglected, the fluid flow will obey Darcy's law, and one can think the equivalent permeability of the packed-pipe equals the permeability of the sand-pack. However, if the viscous shear from the pipe wall cannot be neglected, the fluid flow will obey the Brinkman equation, and the permeability of the packed-pipe will be less than that of the sand-pack due to the additional viscous drag. In this work, on the basis of the Brinkman equation, we derived a series of analytical solutions for characterizing the fluid flow in packed-pipes. These solutions can be used to depict the velocity profiles, estimate the flux rate, and calculate the equivalent permeability of a packed-pipe. On the basis of these analytical solutions, we found that Poiseuille's law is a special form of the derived equivalent permeability solution. We further divided the fluid flow in a packed-pipe into three regimes, including N-S flow, Brinkman flow, and Darcy flow. During the regime of Brinkman flow, the dimensionless flow velocity at the pipe center is 1, and the dimensionless flow velocity is gradually decreased to 0 at the pipe wall. We also investigated the effects of sorting, sand particle size, and sand-pack porosity on the packed-pipe permeability. The calculated results show that a more uniform size of the sand particles or a smaller mean particle diameter can lead to lower packed-pipe permeability. Compared to the sorting and mean particle diameter, the sand-pack porosity exerts a more significant effect on the packed-pipe permeability. [ABSTRACT FROM AUTHOR]

In this study, a facile synthesis of the titanium(IV) ion‐immobilized poly‐glycidyl methacrylate microparticles functionalized with polyethylenimine and adenosine triphosphate was developed for efficient enrichment of intact phosphoproteins. The titanium(IV) ion‐immobilized microparticles had higher saturated adsorption capacity for phosphoproteins (1217.6 mg/g for β‐casein) than nonphosphoproteins (97.1 mg/g for bovine serum albumin) and the average particle diameter was about 1.4 μm with good dispersibility. In application, as demonstrated by SDS‐PAGE, titanium(IV) ion‐immobilized microparticles exhibited good performance in enriching intact phosphoproteins from standard protein mixtures of β‐casein and bovine serum albumin with high specificity and selectivity. In addition, titanium(IV) ion‐immobilized microparticles were also successfully applied in intact phosphoprotein enrichment from complex biological samples including nonfat milk, chicken egg white, and mouse heart tissue extract. [ABSTRACT FROM AUTHOR]

Analysis of phosphoproteins always faces the challenge of low stoichiometry, which demands highly selective and efficient enrichment in the initial sample preparation. Here we report our synthesis of the novel titanium (IV) ion immobilized adenosine triphosphate functionalized silica nanoparticles (Ti 4+ -ATP-NPs) for efficient enrichment of intact phosphoproteins. The average diameter of Ti 4+ -ATP-NPs was about 128 nm with good dispersibility and the saturated adsorption capacity for β-casein was 1046.5 mg/g. In addition, Ti 4+ -ATP-NPs exhibited high specificity and selectivity in enriching phosphoproteins from both standard protein mixtures and complex biological samples (non-fat milk, chicken egg white and mouse heart tissue extract) as demonstrated by SDS-PAGE. [ABSTRACT FROM AUTHOR]

N-linked glycosylation (N-glycosylation) is a common protein post-translational modification, occurring on more than half of mammalian proteins; in striking contract with small molecule modifications (such as methylation, phosphorylation) with only single structures, N-glycosylation has multiple dimensional structural features (monosaccharide composition, sequence, linkage, anomer), which generates enormous N-glycan structures; and these structures widely regulate protein structure and functions. For the modification site, N-glycosylation occurs on the Asn residue among the consensus N-X-S/T/C (X≠P) motif; mutation-originated amino acid change may lead to loss of such an original motif and thus loss-of-glycosylation (LoG) or gain of such a new motif and thus gain-of-glycosylation (GoG). Both LoG and GoG generates new structures and functions of glycoproteins, which has been observed in the S protein of SARS-Cov-2 as well as malignant diseases. Here we report our glycoproteome-wide qualitative N-glycoproteomics characterization of GoGs in breast cancer Adriamycin drug resistance (ADR) cells (MCF-7/ADR) and cancer stem cells (MCF-7/ADR CSCs); comprehensive N-glycosite and N-glycan structure information at the intact N-glycopeptide level were reported. [Display omitted] • The method of building the putative gain-of-glycosylation (GoG) protein database is established • The glycoproteome level characterization of GoGs is benchmarked with two cell lines • GoGs from common mutations including MUTAGEN, VARIANT and VAR_SEQ are demonstrated. [ABSTRACT FROM AUTHOR]

Protein sialylation participates many biological processes in a linkage-specific manner, and aberrant sialylation has been associated with many malignant diseases. Mass spectrometry-based quantitative N-glycoproteomics has been widely adopted for quantitative analysis of aberrant sialylation, yet multiplexing method at intact N-glycopeptides level is still lacking. Here we report our study of sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex tandem mass tags (TMT)-labeling. With lung cancer as a model system, differential sialylation in cancer tissues relative to adjacent non-tumor tissues was characterized at the intact N-glycopeptide level with N-glycosite information. TMT-labeled intact N-glycopeptides with and without sialic acid alkylamidation were subject to reversed-phase liquid chromatography-nano-electron spray ionization-tandem mass spectrometry (RPLC-nanoESI-MS/MS) analysis to provide comprehensive characterization of N-glycosylation with and without sialic acid at the intact N-glycopeptide level with structure and N-glycosite. In this study, 6384 intact N-glycopeptides without sialylation were identified and 521 differentially expressed intact N-glycopeptides from 254 intact N-glycoproteins were quantified. Eight intact N-glycoproteins responsible for N-glycan biosynthesis were identified as glycosyltransferases. In total, 307 sialylated intact N-glycopeptides with linkage-specific sialic acid residues were identified together with 29 N-glycans with α2,6-linked sialic acids and 55 N-glycans with α2,3-linked sialic acids. Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-(COVID)-19. Additionally, many types of N-glycosylation including terminal N-galactosylation, core and/or branch fucosylation, α2,6-sialylation and terminal bisecting N-acetylglucosamine were identified and quantified in intact N-glycoproteins from immunoglobulin family. [Display omitted] • Sialic acid linkage-specific quantitative N-glycoproteomics with selective alkylamidation and multiplex TMT-labeling. • Eight N-glycoproteins about N-glycan biosynthesis were identified as glycosyltransferases. • 307 sialylated intact N-glycopeptides identified with 29 α2,6-sialylated N-glycans and 55 α2,3-sialylated N-glycans. • Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-COVID-19. • Many N-glycosylation especially α2,6-sialylation quantified in intact N-glycopeptides from immunoglobulin family. [ABSTRACT FROM AUTHOR]

Hydrogen–deuterium exchange reactions of 16 small rigid anions were examined using D 2 O, CH 3 OD, CH 3 CO 2 D, and (CF 3 ) 2 C(CH 3 )OD. Bimolecular rate constants and extensive computations are reported. These deuterated reagents were used because D 2 O and CH 3 OD are weak acids that cannot react via sequential deuteron/proton transfers (i.e., the conventional H/D exchange mechanism of DePuy et al.) with the selected anions, and they provide a means to probe alternative pathways. The relative acidity of CH 3 CO 2 D and (CF 3 ) 2 C(CH 3 )OD was measured (ΔΔ H acid ° = 1.1 ± 0.8 kcal mol −1 ) and their thermodynamic similarity but structural differences (i.e., the presence of a C O in the carboxylic acid but not the alcohol) were exploited to systematically investigate flip-flop and relay mechanisms. This has led to a number of generalizations relating to the occurrence of these pathways. [ABSTRACT FROM AUTHOR]

RATIONALE Soft ionization, high-resolution mass spectrometry is widely used to characterize large biological molecules, such as proteins. Deconvolution ('deisotoping') of isotopic envelopes (iEs) in biological mass spectra into monoisotopic or average masses is challenging due to low signals and heavily overlapped iEs, resulting in many wrong interpretations. METHODS Isotopic envelopes (iEs) are directly used without deisotoping to identify biological molecules. An algorithm, isotopic mass-to-charge ratio ( m/z) and envelope fingerprinting (iMEF), was implemented in the ProteinGoggle search engine for top-down intact protein database searching. iMEF combines isotopic m/z fingerprinting (iMF) and isotopic envelop fingerprinting (iEF), where 'Isotopic mass-to-charge ratio' means the m/z value of the most abundant isotopic peak within the iE of a precursor or product ion. iMF is used to 'fish' precursor or product ion candidates from the database, which is pre-built and contains all iE information (precursor and product ions) of all proteoforms of the studied system. iEF identifies matching precursor or product ions. A protein is finally identified with user-specified total number of matching product ions and post-translational modification scores. RESULTS The working principles of iMEF and ProteinGoggle, and the definition of a set of related parameters and scoring metrics, are illustrated with high-resolution tandem mass spectrometric analysis of a mixture of ubiquitin and the HUMAN histone H4 proteoforms. Ubiquitin was confidently identified from its CID, ETD, and HCD spectra with 57, 91, and 66 matching product ions, respectively; 125 proteoforms were confidently found from the H4 dataset. The locations of PTMs in 54 and 6 isoforms were partially and fully identified. CONCLUSIONS Database search with iMEF bypasses 'deisotoping' avoiding associated errors, and also provides full quality control of matching precursor and product ions and finally protein IDs. Overlapped iEs of different product ions could also be confidently unwrapped in situ. Improvement and addition of more functionalities and utilities of ProteinGoggle are underway. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Hydrogen bonds are the dominant motif for organizing the three-dimensional structures of biomolecules such as carbohydrates, nucleic acids, and proteins, and serve as templates for proton transfer reactions. Computations, gas-phase acidity measurements, and pKa determinations in dimethyl sulfoxide on a series of polyols indicate that multiple hydrogen bonds to a single charged center lead to greatly enhanced acidities. A new class of Brønsted acids, consequently, is proposed. [ABSTRACT FROM AUTHOR]

Abstract: The reactions of CH3 +, CH2Cl+ and CHCl2 + with standard reference bases were examined in a dual cell Fourier transform mass spectrometer. Deprotonation of methyl cation occurs rapidly via a spin forbidden process to afford triplet methylene even when the reaction is nearly thermoneutral. Bracketing results enable us to assign PA(CHCl)=209.7±2.2kcalmol−1 and PA(CCl2)=205.2±1.9kcalmol−1, the latter value of which is considerably larger than previous determinations. The resulting heats of formation, however, are in good accord with other measurements and lead to recommended values of (CHCl)=74.6±2.4kcalmol−1 and (CCl2)=53.0±2.6kcalmol−1. These values correspond to a 2–3kcalmol−1 lowering of these quantities and are within 1–2kcalmol−1 of high level G3 and W1 predictions based upon the atomization energies and a series of four isodesmic reactions. [Copyright &y& Elsevier]

Abstract: Using a very weak electric field to accelerate the photofragment ions, we have constructed a very simple photofragment translational spectrometer with only a 50 mm total flight path, but we can get photofragment translational spectra with high resolution. On this apparatus, we have performed the photodissociation of CF3I at 281.73 nm, and the same laser will ionize I*(2P1/2) via REMPI. Seven well-resolved vibrational peaks were obtained. Six of them are assigned to the umbrella vibrational mode v2=0–5 states of photofragment CF3. The average internal energy is about 21% of the available energy. [Copyright &y& Elsevier]

The photodissociation of CF3I at 248 nm was performed on our high-resolution photofragment translational spectrometer. For further improvement of the resolution, we have taken the following procedures: (1) Kr was used as carrier gas, (2) optimum molecular beam angle θopt (when VMB⊥VCM) was selected, (3) the longitudinal molecular beam angle spread was reduced, and (4) the ionization region for photofragments was diminished. The photofragment translational spectra of I* (2P1/2) reveal eight well-resolved vibrational peaks. The highest peak was assigned to be v2=5 of the ν2 vibrational mode, and then the dissociation energy would be D0(C–I)=18 810±200cm−1=53.8±0.6 kcal/mol. [Copyright &y& Elsevier]

• HA degradation promotes odontoblastic differentiation and cell migration of mouse dental papilla cells. • During odontoblastic differentiation, HYAL2 is highly localized in odontoblasts in vivo. • Hyal2 knockdown leads to the accumulation of HA, which impedes dentinogenesis and cell migration. • Hyal2 silencing significantly affects the cytoskeletal networks and focal adhesion, and inhibits the activation of PI3K/Akt signaling pathway. The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs. [ABSTRACT FROM AUTHOR]

T cell function is determined by transcriptional networks that are regulated by epigenetic programming via posttranslational modifications (PTMs) to histone proteins and DNA. Bottom-up mass spectrometry (MS) can identify histone PTMs, whereas intact protein analysis by MS can detect species missed by bottom-up approaches. We used a novel approach of online two-dimensional liquid chromatography-tandem MS with high-resolution reversed-phase liquid chromatography (RPLC), alternating electron transfer dissociation (ETD) and collision-induced dissociation (CID) on precursor ions to maximize fragmentation of uniquely modified species. The first online RPLC separation sorted histone families, then RPLC or weak cation exchange hydrophilic interaction liquid chromatography (WCX-HILIC) separated species heavily clad in PTMs. Tentative identifications were assigned by matching proteoform masses to predicted theoretical masses that were verified with tandem MS. We used this innovative approach for histone-intact protein PTM mapping (HiPTMap) to identify and quantify proteoforms purified from CD8 T cells after in vivo influenza infection. Activation significantly altered PTMs following influenza infection, histone maps changed as T cells migrated to the site of infection, and T cells responding to secondary infections had significantly more transcription enhancing modifications. Thus, HiPTMap identified and quantified proteoforms and determined changes in CD8 T cell histone PTMs over the course of infection. [ABSTRACT FROM AUTHOR]

Histone post‐translational modifications (PTMs) play various roles in chromatin‐related cellular processes, and comprehensive analysis of these combinatorial PTMs at the intact protein level by top‐down proteomics is the method of choice to reveal their crosstalk and biological functions. Here, we report our top‐down characterization of the core histones from mouse fibroblasts cells NIH/3T3, which is a classic model used in many kinds of research. With nanoRPLC‐MS/MS analysis and ProteinGoggle database search, 547 protein species were identified with spectrum‐level FDR ≤ 1%, where PTMs in 51 protein species were unambiguously localized with PTM scores ≥1. High‐resolution MS/MS data also allowed the unambiguous identification of acetylation instead of trimethylation. This study presents a general picture of combinatorial PTMs of mouse core histones, which serves as a basic reference for all future related biological studies. [ABSTRACT FROM AUTHOR]

Core histones and their PTMs play important roles in regulating gene transcription and other DNA-related processes. The study of core histones PTMs, their cross-talk and functional roles is not only of broad biological significance but also of wide pathological and clinical relevance. Having the strength of comprehensive proteoform identification with 100% amino acid sequence coverage and combinatorial PTMs, top-down proteomics has become the state-of-the-art analytical tool for combinatorial PTM characterization of core histones. In this study, we report our top-down characterization of chicken (Gallus gallus domesticus) core histones, which have been widely used as models for chromosome re-construction among others because of easy availability and not-so-dense PTMs. With nanoRPLC-MS/MS analysis and ProteinGoggle database search, a total of 58 proteoforms were identified for the core histone families of H4, H2B, H2A, and H3. [ABSTRACT FROM AUTHOR]

Aberrant glycosylation has been commonly observed in various physiological and pathological disorders (including cancers), and quite a few glycoproteins have been approved by the US Food and Drug Administration (FDA) as markers for early diagnosis. Each glycoprotein may have multiple glycoforms, and cancer-related ones can be only some specific glycoforms which have much higher sensitivity and specificity; for example, AFP glycoform AFP-L3 with N-glycan of 01Y(61F)41Y41M(31M41Y41L41S61M41Y41L41S is of bigger diagnostic value for hepatocellular carcinoma than total AFP (i.e., combination of all glycoforms). Mass spectrometry-based glycomics is currently the state-of-the-art instrumental analytical pipeline for high-throughput characterization of various glycoforms, where not only monosaccharide composition but also comprehensive structural information (sequence and linkage) of N-glycans are now reported thanks to our recently developed N-glycan database search engine GlySeeker. With this new capability, here, we report our large-scale characterization of human liver N-glycome with primary structures; 214 unique N-glycans with unique primary structures were identified and visualized with spectrum-level false discovery rate ≤ 1% and number of best hits of 1. The LO2 N-glycans reported here serve as a basic reference for future liver N-glycome study, and further quantitative analysis will enable characterization of differentially expressed N-glycans and discovery of more effective markers for liver and other diseases. Data are available via ProteomeXchange with identifier PXD008158. [ABSTRACT FROM AUTHOR]

Review: 193 refs. [ABSTRACT FROM AUTHOR]

Non-imaging radar can achieve the detection and localization of moving targets, but it faces difficulties in the classification of targets. Aiming for the application of moving target classification by low/medium resolution non-imaging radar, this paper proposes a novel feature extraction method, based on the statistical characteristics of echo amplitude with the L-band fully polarimetric radar. A feature plane, composed of the 3rd-order central moment (skewness) and 4th-order central moment (kurtosis) as the statistical characteristics of the echo envelope, is established. In addition, two types of moving targets, pedestrians and non-motorized vehicles, are able to be effectively distinguished according to whether the echo signals of different polarizations have overlapping areas in the feature plane. The L-band fully polarimetric radar has been developed and field experiments have been conducted. The experimental results verify that the kurtosis and skewness of the fully polarimetric echo amplitude of the pedestrians are very close, and there is an overlapping area in the feature plane, while the statistical characteristics of the fully polarimetric echo signal of non-motor vehicles are different, and there is no overlapping area in the feature plane. This proposed feature extraction method has the advantage of being simple and robust, compared to the traditional imaging approach. [ABSTRACT FROM AUTHOR]

For top-down protein database search and identification from tandem mass spectra, our isotopic envelope fingerprinting search algorithm and ProteinGoggle search engine have demonstrated their strength of efficiently resolving heavily overlapping data as well separating non-ideal data with non-ideal isotopic envelopes from ideal ones with ideal isotopic envelopes. Here we report our updated ProteinGoggle 2.0 for intact protein database search with full-capacity. The indispensable updates include users' optional definition of dynamic post-translational modifications and static chemical labeling during database creation, comprehensive dissociation methods and ion series, as well as a Proteoform Score for each proteoform. ProteinGoggle has previously been benchmarked with both collision-based dissociation (CID, HCD) and electron-based dissociation (ETD) data of either intact proteins or intact proteomes. Here we report our further benchmarking of the new version of ProteinGoggle with publically available photon-based dissociation (UVPD) data ( http://hdl.handle.net/2022/17316 ) of intact E. coli ribosomal proteins. Biological significance Protein species (aka proteoforms) function at their molecular level, and diverse structures and biological roles of every proteoform come from often co-occurring proteolysis, amino acid variation and post-translational modifications. Complete and high-throughput capture of this combinatorial information of proteoforms has become possible in evolving top-down proteomics; yet, various methods and technologies, especially database search and bioinformatics identification tools, in the top-down pipeline are still in their infancy stages and demand intensive research and development. [ABSTRACT FROM AUTHOR]

An efficient and unidirectional optical coupling scheme for metal-dielectric-metal (MDM) waveguides with wide-angle and ultra-broadband efficiency is proposed and demonstrated. Based on joint effects of the plasmonic critical angle phenomenon (PCA), the zero-order grating resonance, and the first-order Fabry–Perot (FP) resonance of the MDM waveguide, the illuminated light could be efficiently coupled into MDM plasmonic waveguide with angular-full-width-half-maximum (AFWHM) widen to 50° over a broad wavelength range 750–1000 nm. The operation principle and performance of the structure are clarified and theoretically illustrated. [ABSTRACT FROM AUTHOR]

N-glycosylation is a common post-translational modification (PTM) in plants that possesses important biological functions including protein folding, protein quality control, protein stability and protein-protein interactions. Nowadays, the interest in analysis of glycoproteins using mass spectrometry is increasing because of advances in instrumentation and automatic data analysis tools. Here, we describe the progresses in mass spectrometry-based N-glycomics and N-glycoproteomics in plants. We also provide a comprehensive summary of N-glycan structures in plants according to the biosynthetic rules and up-to-date reports, which serves as a theoretical database for all N-glycan-related search engines. [Display omitted] • Methods and application progresses in mass spectrometry-based N-glycomics was comprehensively reviewed. • Methods and application progresses in mass spectrometry-based N-glycoproteomics was comprehensively reviewed. • Structural characteristics and core structures of plant N-glycans were summarized as a theoretical reference database. [ABSTRACT FROM AUTHOR]

• Site- and structure-specific identification of N-glycosylation of Arabidopsis at the intact N-glycopeptide level. • With spectrum-level FDR ≤ 1%, 5,687 intact N-glycopeptides coming from 3,713 N-glycosites of 3,140 N-glycoproteins were identified. • With structure-diagnostic fragment ions as experimental evidence, monosaccharide sequence structures of N-glycans on 630 intact N-glycopeptides were unambiguously distinguished from the other sequence isomers with the same monosaccharide compositions. The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR ≤ 1%, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge. Wtih the experimental evidence support of structure-diagnostic fragment ions, 81 glycan structures from 54 glcan compostions were unambiguouly distinguished. The comprehensive experimental site- and structure-specific N-glycosylation data reported in this study will serve as a fundamental valuable reference for the coming functional studies of this widely adopted model organism of plant. [ABSTRACT FROM AUTHOR]

Protein N-glycosylation plays an essential role on cancers and other pathological processes. Its structural and functional studies rely on complete qualitative and quantitative information. Thus, it is important to quantify differentially expressed intact N-glycopeptides (DEGPs) at their molecular level. Here we report our application of stable isotopic diethyl labeling (SIDE) of amino groups for relative quantitation of intact N-glycopeptides. The amino groups from both the N-terminal and lysine residues of N-glycopeptides were diethylated with XH 3 XHO (X = 13C or C) and NaBH 3 CN. A linear quantitation dynamic range up to 50-fold was obtained with R2 = 0.9985 with intact N-glycopeptides from standard glycoprotein ribonuclease B (RNase B). In proof-of-principle comparative N-glycoproteomics study of aberrant N-glycosylation of gastric cancer tissues vs. adjacent tissues using SIDE, 644 DEGPs (≥1.5-fold change, p < 0.05, p was calculated using t-test) were discovered. With its accuracy and big dynamic range, SIDE can be applied to quantitative study of any aberrant glycosylation at the intact glycopeptide level. Image 1 • Reductive stable isotopic diethylation (SIDE) was developed for relative quantitation of intact N-glycopeptides. • A linear quantitation dynamic range up to 50-fold was obtained with R2 = 0.9985. • In characterization of differential N-glycosylation in gastric cancer tissues vs. adjacent tissues, 644 DEGPs were discovered. [ABSTRACT FROM AUTHOR]

Rationale: We previously isolated antibodies binding to glycopeptide neoantigen epitopes centering the GSTA sequence of the highly glycosylated tandem repeat region of MUC1. Epitopes centering the GSTA sequence are also predicted by NetMHC programs to bind to MHC molecules. Detecting MUC1 glycopeptide epitopes remains a challenge since antigenic epitopes are often shorter than 10 amino acids. Methods: In this study, we used pronase from Streptomyces griseus, which has no amino acid sequence preference for enzymatic cleavage sites, to digest synthetic glycopeptides RPAPGST (Tn)APPAHG and RPAPGS (Tn)TAPPAHG, and analyzed the digests by liquid chromatography/mass spectrometry (LC/MS) using electron transfer dissociation (ETD) and higher‐energy collisional dissociation (HCD) methods with an Orbitrap Fusion Lumos Tribid mass spectrometer. Results: We found that short glycopeptides containing 8 to 11 amino acids could be efficiently generated by pronase digestion. Such glycopeptides of minimal epitope lengths were clearly distinguished by characteristic MS/MS ion patterns and LC elution profiles. A glycopeptide library was generated which may serve as a standard for measuring neoantigen epitopes centering the GSTA sequence. Conclusions: Our data established the LC/MS/MS identities of a clinically relevant MUC1 glycopeptide neoantigen epitope centering the GSTA motif. A library of short MUC1 glycopeptides centered on the GSTA motif was created, which is a critical step for analysis of such antigen epitopes in real biological samples. [ABSTRACT FROM AUTHOR]

Isotopic-labeling quantitative N-glycoproteomics characterization of cell-surface differentially expressed N-glycosylation in MCF-7/ADR cancer stem cells (CSCs) relative to MCF-7/ADR cells was carried out at the intact N-glycopeptide level with trypsin digestion, ZIC-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS analysis of the 1:1 mixture, and GPSeeker DB search. With a spectrum-level false discovery rate of ≤ 1%, 1,336 intact N-glycopeptides from the combination of 301 unique peptide backbones and 169 putative N-glycan linkages (52 monosaccharide compositions) were identified; the corresponding intact N-glycoproteins and N-glycosites were 289 and 305, respectively, among which 176 N-glycosites were confirmed with GlcNAc-containing site-determining b/y fragment ion pairs. The N-glycan moieties in 546 intact N-glycopeptide IDs were identified with more than one structure-diagnostic fragment ions where multiple linkage structures exist for each of the monosaccharide compositions. With the criteria of ≥ 1.5-fold change and p value < 0.05, 72 cell-surface differentially expressed intact N-glycopeptides (DEGPs) were found in MCF-7/ADR CSCs relative to MCF-7/ADR cells, where 8 and 64 were downregulated and upregulated, respectively. [ABSTRACT FROM AUTHOR]

Background: Cancer stem cells (CSCs) are reported to be responsible for tumor initiation, progression, metastasis, and therapy resistance where P-glycoprotein (P-gp) as well as other glycoproteins are involved. Identification of these glycoprotein markers is critical for understanding the resistance mechanism and developing therapeutics. Methods: In this study, we report our comparative and quantitative site- and structure-specific N-glycoproteomics study of MCF-7/ADR cancer stem cells (CSCs) vs. MCF-7/ADR cells. With zic-HILIC enrichment, isotopic diethyl labeling, RPLC–MS/MS (HCD) analysis and GPSeeker DB search, differentially expressed N-glycosylation was quantitatively characterized at the intact N-glycopeptide level. Results: 4016 intact N-glycopeptides were identified with spectrum-level FDR ≤ 1%. With the criteria of ≥ 1.5 fold change and p value < 0.05, 247 intact N-glycopeptides were found differentially expressed in MCF-7/ADR CSCs as putative markers. Raw data are available via ProteomeXchange with identifier PXD013836. Conclusions: Quantitative site- and structure-specific N-glycoproteomics characterization may help illustrate the cell stemness property. [ABSTRACT FROM AUTHOR]

N-glycosylation is one of the most frequently occurring protein post-translational modifications (PTMs) with broad cellular, physiological and pathological relevance. Mass spectrometry-based N-glycomics has become the state-of-the-art instrumental analytical pipeline for sensitive, high-throughput and comprehensive characterization of N-glycans and N-glycomes. Improvement and new development of methods in N-glycan release, enrichment, derivatization, isotopic labeling, separation, ionization, MS, tandem MS and informatics accompany side-by-side wider and deeper application. This review provides a comprehensive update of mass spectrometry-based qualitative and quantitative N-glycomics in the years of 2017–2018. Image 1 • MS-based N-glycomics is the best tool for high-throughput profiling of N-glycans. • Qualitative information of N-glycans at multi-level are readily available. • Updated progresses in frontier methods and applications in 2017–2018 are reviewed. [ABSTRACT FROM AUTHOR]

High-throughput proteome-level characterization of stemness markers of MCF-7 cancer stem cells was carried out using our recently developed site- and structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. 144 differentially expressed intact N-glycopeptides were quantified all at once yet with good accurate consistency with the individual biochemical measurements of proteins, RNA or DNA. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is one of the most common protein PTMs, and the topological structure (monosaccharide composition and sequence as well as glycosidic linkages) of N-glycans is vital information to understand their biological functions and roles. Tandem mass spectrometry has been widely used for topological structure characterization of N-glycans, where comprehensive understanding of fragmentation pathways and characteristics of product ions are essential to achieve best interpretation of MS/MS data and highest confidence of identification. Here, we report our glycomic study of N-glycome of mouse brain as well as fragmentation pathway analysis of the identified N-glycans. With LC-MS/MS analysis at both the positive and negative ESI modes together with our recently developed N-glycan database search engine GlySeeker, 221 unique N-glycans with putative topological structures were identified with target-decoy searches and number of best hits of 1. Analysis of fragmentation pathways and characteristics of product ions of permethylated N-glycans in the positive mode and native N-glycans in the negative mode were further carried out. The reported N-glycans serve as a basic reference for future glycosylation study of mouse brain; and in general database search of tandem mass spectra of N-glycans, B/Y/Z ions should be preferentially considered for the permethylated form in the positive mode and B/C/Z ions for the native form in the negative mode. [ABSTRACT FROM AUTHOR]

Rationale: The functional study and application of an intact glycoprotein require the structural characterization of both the protein backbone and the glycan moiety; the former has been successfully demonstrated with selective fragmentation of the protein backbone in CID and ExD; whether the latter can be achieved with selective fragmentation of the glycan moiety remains to be explored. Methods: RNase B solution was electrosprayed and its intact glycoforms of GlcNAc2Mann (n = 5–9) with the highest abundance (charge state z = 16) were isolated individually and fragmented using CID, ETD, HCD, ETciD, and EThcD on the Orbitrap Fusion Lumos Tribrid mass spectrometer; the dissociation parameters were optimized for selective fragmentation of the N‐glycan moiety and protein backbone as well as high sequence coverage. The obtained spectra were interpreted using the protein and N‐glycan database search engines ProteinGoggle and GlySeeker, respectively. Results: With exploration of different dissociation parameters for all the five methods, selective fragmentation of the N‐glycan moiety (the protein backbone staying intact) was observed in both HCD and EThcD at low collisional energies, but only a few matched product ions were observed; more comprehensive fragmentation was observed at high collisional energies (the protein backbone lost). Selective protein backbone fragmentation was observed in all the five dissociation methods. Conclusions: For comprehensive structural characterization of intact N‐glycoproteins using tandem mass spectrometry, the composition and topology of the N‐glycan moiety can be identified using HCD and EThcD complementarily at low and high energies; while the amino acid sequence and glycosite can be identified using CID, ETD, HCD, ETciD, and EThcD with their optimal dissociation parameters. [ABSTRACT FROM AUTHOR]

Rationale: Most of the current popular tandem mass spectrometers have the capability of resolving the primary structures (monosaccharide composition, sequence and linkage) of N-glycans; however, compositions or putative structures have mostly been reported so far. Identification and visualization tools of N-glycans are needed. Methods: The isotopic mass-to-charge ratio and envelope fingerprinting algorithm, which has been successfully used for intact protein database search and identification, was adapted for N-glycan database search, and a stand-alone N-glycan database search engine, GlySeeker, for automated N-glycan identification and visualization was developed and successfully benchmarked. Both pseudo 2D graph and one-line text formats with one-letter symbols for monosaccharides were proposed for representing N-glycans. N-glycans were identified with comprehensive interpretation of product ions and false discovery rate (FDR) control. Results: In a database search of reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) datasets of the N-glycome enriched from OVCAR-3 ovarian cancer cells, with FDR ≤1% and number of best hits (NoBHs) = 1-30, 1525 N-glycans with comprehensive primary structural information (composition, sequence and linkage) were identified and visualized; among these 1525 N-glycans, 559 had NoBHs = 1, i.e. their structures were uniquely identified. This represents a large-scale identification and visualization of N-glycans with primary structures from tandem mass spectra. Conclusions: A stand-alone N-glycan database search engine called GlySeeker has been developed for large-scale identification and visualization of N-glycans with comprehensive interpretation of tandem mass spectra and FDR control. [ABSTRACT FROM AUTHOR]

Phosphorylation is one of the most important and widely studied protein post-translational modifications. Tandem mass spectrometry using higher-energy collisional dissociation has evolved into a state-of-the-art analytical platform for both phosphorylation identification and site localization. Tens of thousands of phosphopeptides can now be routinely identified from a single shotgun proteomics study; site localization, however, is much more complicated and many challenges still exist. Here, we report our development of P-bracket using direct experimental evidence of phospho-containing site-determining product ions for accurate site localization without the need for additional FLR control. A P-bracket is defined as a complementary product ion pair that forms a bracket to confine a phosphorylation event to a unique site. P-bracket has been successfully benchmarked with a set of six synthetic phosphopeptides with a single phosphorylation event, a set of 96 synthetic peptides and phosphopeptide reference libraries, and two HeLa phosphopeptide LC-MS/MS (HCD) datasets; Accurate phosphosite localization by P-bracket will greatly enhance identification confidence of phosphopeptides and contribute to structural and functional studies of phosphoproteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • 2764 IDs distinguished from sequence isomers by structure-diagnostic fragment ions. • With fold change ≥1.5 and p value < 0.05, 104 DEGPs were determined. • ITGAV, ITGB5 and LAMA1 were associated with ECM-receptor interaction by KEGG analysis. • APC, DVL3 and INVS were associated with Wnt signaling pathway by KEGG analysis. • Down-regulated intact N-glycopeptide with bisecting GlcNAc from SPT20H were found. • Upregulated intact N-glycopeptide with β1,6-branching N-glycan from ITGB5 were found. Breast cancer is responsible for the highest mortality all over the world. Cancer stem cells (CSCs) along with epithelial mesenchymal transition (EMT) are identified as a driver of cancer which are responsible for cancer metastasis and drug resistance. Several signaling pathways are associated with drug resistance. Additionally, glycosyltransferases regulate different types of glycosylation which are involved in drug resistance. To the end, it is urgent to figure out the knowledge on cell-surface altered N-glycosylation and putative markers. Here, differential cell-surface intact N-glycopeptides in adriamycin (ADR)-resistant michigan breast cancer foundation-7 stem cells (MCF-7/ADR CSCs) relative to ADR-sensitive MCF-7 CSCs were analyzed with site- and structure-specific quantitative N-glycoproteomics. The intact N-glycopeptides and differentially expressed intact N-glycopeptides (DEGPs) were determined and quantified via intact N-glycopeptide search engine GPSeeker. Totally, 4777 intact N-glycopeptides were identified and N-glycan sequence structures among 2764 IDs were distinguished from their isomers by structure-diagnostic fragment ions. Among 1717 quantified intact N-glycopeptides, 104 DEGPs were determined (fold change ≥ 1.5 and p value < 0.05). Annotation of protein–protein interaction and biological processes among others of DEGPs were finally carried out; down-regulated intact N-glycopeptide with bisecting GlcNAc from p38-interacting protein and up-regulated intact N-glycopeptide with β1,6-branching N-glycan from integrin beta-5 were found. [ABSTRACT FROM AUTHOR]

Neutral loss and internal product ions have been found to be significant in both peptide and protein tandem mass spectra and they have been proposed to be included in database search and for protein identification. In addition to common canonical b/y ions in collision-based dissociation or c/z ions in electron-based dissociation, inclusion of neutral loss and internal product ions would certainly make better use of tandem mass spectra data; however, their ultimate utility for protein identification with false discovery rate control remains unclear. Here we report our proteome-level utility benchmarking of neutral loss and internal product ions with tandem mass spectra of intact E. coli proteome. Utility of internal product ions was further evaluated at the protein level using selected tandem mass spectra of individual E. coli proteins. We found that both neutral loss and internal products ions do not have direct utility for protein identification when they were used for scoring of P Score; but they do have indirect utility for provision of more canonical b/y ions when they are included in the database search and overlapping ions between different ion types are resolved. Biological significance Tandem mass spectrometry has evolved to be a state-of-the-art method for characterization of protein primary structures (including amino acid sequence, post-translational modifications (PTMs) as well as their site location), where full study and utilization tandem mass spectra and product ions are indispensable. This primary structure information is essential for higher order structure and eventual function study of proteins. [ABSTRACT FROM AUTHOR]

Rationale The mass measurement accuracy (MMA) of Orbitrap mass spectrometers is 1-5 ppm according to the manufacturer's specification; yet, up to 50 ppm has been used as mass tolerance to interpret Orbitrap data in the literature. A systematic evaluation of MMA is thus necessary to find the optimal mass tolerance to be used. Methods Reversed-phase liquid chromatography/tandem mass spectrometry (RPLC/MS/MS) analyses of the intact E. coli proteome were carried out on a Q Exactive Orbitrap mass spectrometer coupled to a Dionex UltiMate 3000 RSLCnano system. The analysis included three technical replicates each day and was repeated for six continuous days right after a mass calibration. The obtained raw datasets were searched using ProteinGoggle 2.0 under four different mass tolerances of 5, 10, 15, and 20 ppm. Results With both forward and random database searches and FDR ≤1% at the spectrum level, the most protein spectrum matches and protein IDs were obtained at a mass tolerance of 15 ppm. The average mass accuracy of both precursor and product ions from three representative high, medium, and low abundance proteins as well as the common proteins identified in all the 18 replicate runs was found to be 0-4 ppm; and no significant drift of measured mass accuracy was observed within the calibration period of 1 week. Conclusions Despite the mass measurement accuracy of 1-5 ppm of the Orbitrap stated by the manufacturer, the optimal mass tolerance for protein identification was found to be 15 ppm for both the precursor and product ions. Weekly mass calibration is appropriate because no significant drift in MMA was found within the 6-day period. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the most malignant diseases among females. N-glycoproteomics studies have shown that N-glycosylation alteration of tumor cells is the key player of cancer progression, multidrug resistance (MDR) and high mortality. Cancer stem cells (CSCs) have the remarkable potential of self-renewing and differentiation which leads to drug resistance and metastasis. To investigate the differentially expressed N-glycosylation in adriamycin-resistant breast cancer stem cells MCF-7/ADR CSCs (relative to MCF-7 CSCs) and find the putative biomarkers, 1:1 paired ZIC-HILIC-enriched and stable isotopic diethyl labelled (SIDE) intact N-glycopeptides from MCF-7/ADR CSCs and MCF-7 CSCs were analyzed with C18-RPLC-ESI-MS/MS (HCD with stepped NCE); differentially expressed intact N-glycopeptides (DEGPs) were identified and quantified via search engine GPSeeker. With control of spectrum-level FDR≤1%, 5515 intact N-glycopeptides were identified (1737 N-glycosites, 1705 peptide backbones and 1516 intact N-glycoproteins; 181 putative N-glycan linkages and 68 monosaccharide compositions). Among 5515 intact N-glycopeptide IDs, 3864 were identified with glycoform score≥1, i.e., one or more structure-diagnostic fragment ions were observed to distinguish sequence isomers. With the three technical replicates and the criteria of fold change≥1.5 and p value<0.05, 380 DEGPs (corresponding to 153 intact N-glycoproteins) were found along with 293 down-regulated and 87 up-regulated. For these 153 intact N-glycoproteins, the molecular functions and biological processes of were comprehensively discussed, and side-to-side comparison of differential expression results with other method were also made. • Among 5515 IDs, 380 DEGPs (fold change≥1.5 and p<0.05) were found as putative N-glycoprotein drug resistance biomarkers. • With control of no less than 1.5-fold change and p < 0.05. 380 DEGPs (corresponding to 153 intact N-glycoproteins) were found along with 293 down-regulated and 87 up-regulated. • These 153 intact N-glycoproteins were found as putative N-glycoprotein drug resistance biomarkers candidates for further biological validation and final understanding of the drug resistance mechanisms. [ABSTRACT FROM AUTHOR]

By fixing the hydrogen bond length (O⋯H⋯N) at a value, the potential energy curves of the [M⋯H⋯NH3]+ were explored at the HF/6-31G(d) and B3LYP/6-31G(d) level of theory. The calculations indicate that as the hydrogen bond length is increased, the proton transfer barrier increases and the single-well potential energy curve tends to become double-welled. To model the proton transfer (PT) in ‘real’ system, the transition state connecting M⋯(H–NH3)+ and (M–H)+⋯NH3 has been optimized and the reaction path gives an estimate of the PT barrier. Correlated electronic structure calculation, coupled with ZPE correction, provides a better understanding of the PT barrier. [Copyright &y& Elsevier]

Glycosylation is a common protein PTM, and its aberrant regulation has been widely linked to various pathological conditions including cancers. Our recent development of intact N-glycopeptide search engine GPSeeker has enabled relatively quantitative structure-specific characterization of differentially expressed intact N-glycopeptides (DEGPs) with isotopic labeling of the peptide backbones and structure-specific fragment ions of the N-glycan moieties. Here we report our site- and structure-specific relatively quantitative N-glycoproteomics study of breast MCF-7 cancer cells (relative to epithelial MCF-10A cells) using RPLC-nanoESI-MS/MS and GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones (amino acid sequences, N-glycosites) and the N-glycan moieties (monosaccharide composition, sequence and linkages) were identified from five technical replicates (TR1, TR2, TR3, TR4 and TR5). With the criteria of quantified at least thrice out of the five technical replicates with no <1.5-fold change, p ≤.05 and RSD ≤ 20%, 56 DEGPs were quantified from 23 N-glycosites on 19 intact N-glycoproteins. For the 19 intact N-glycoproteins observed with differential N-glycosylation expression, 14 (each with one or more DEGPs) were observed with uniform down regulation; 6 (each with one or more DEGPs) were observed with uniform up regulation; whereas one was observed with both up and down regulation. Differential N-glycosylation in breast MCF-7 cancer cells (relative to MCF-10A epithelial cells) were qualitatively and quantitatively characterized with site- and structure-specific N-glycoproteomics using RPLC-nanoESI-MS/MS (HCD with stepped NCEs) and intact N-glycopeptide search engine GPSeeker. With spectrum-level FDR ≤ 1%, 581 intact N-glycopeptides with comprehensive structural information of both the peptide backbones and the N-glycan moieties were identified; For the 248 putative N-glycosites, 248 were confirmed where 125 have not been annotated in UniProt as of July 25, 2019. For the 114 N-glycan putative linkage structures, 44 were confirmed with no less than one structure-diagnostic fragment ions. With the criteria of quantified at least thrice out of the five technical replicates with no < 1.5-fold change and p ≤.05, 56 DEGPs were quantified from 21 intact N-glycoproteins; 13 and 5 intact N-glycoproteins (each with one or more DEGPs) were observed with uniform down and up regulation; whereas one were observed with simultaneous up and down regulation. Unlabelled Image • Site- and structure-specific N-glycoproteomics study of breast cancer MCF-7 cells. • 1612 intact N-glycopeptides were identified with FDR ≤ 1%. • 122 differentially expressed intact N-glycopeptides were quantified. • 51 out of the 216 linkage structures were confirmed with diagnostic fragment ions. • 23 out of the 392 N-glycosites were found new to UniProt. [ABSTRACT FROM AUTHOR]