Characterization of protein iv-glycosylation by reversed-phase microbore liquid chromatography / electrospray mass spectrometry,complementary mobile phases,and sequential exoglycosidase digestion

A strategy for the identification of the site occupancy and glycoform heterogeneity, including sialylation occurring at specific sites of N-linked giycoproteins is presented using the asparagine-linked glycosylation on bovine fetuin for illustration. This is achieved by microbore high-performance liquid chromatography/electrospray ionization mass analysis (LC/ESIMS) of the tryptic glycopeptide mixtures with an acetonitrile-based mobile phase followed by sequential steps of residue (and linkage) specific glycoform degradation and LC/ESIMS analysis at each stage. In addition, chromatographic separation of the site-specific glycoforms of tryptic glycopeptides is accomplished by the use of an alternative, mass spectrometrically compatible mobile phase-water/ethanol/propanol/formic acid. By employing this nontraditional mobile phase for characterizing the complete tryptic digest, and using highly specific exoglycosidases in combination with LC/ESIMS analysis, a previously uncharacterized carbohydrate (a disialo biantennary complex oligosaccharide) was identified as a novel structure at Asn⁸¹ of bovine fetuin. (J Am Sot Mass Spectrom 1994, 5, 350-358)     

Reliable Determination of Site-Specific In Vivo Protein N-Glycosylation Based on Collision-Induced MS/MS and Chromatographic Retention Time

Site-specific glycopeptide mapping for simultaneous glycan and peptide characterization by MS is difficult because of the heterogeneity and diversity of glycosylation in proteins and the lack of complete fragmentation information for either peptides or glycans with current fragmentation technologies. Indeed, multiple peptide and glycan combinations can readily match the same mass of glycopeptides even with mass errors less than 5 ppm providing considerably ambiguity and analysis of complex mixtures of glycopeptides becomes quite challenging in the case of large proteins. Here we report a novel strategy to reliably determine site-specific N-glycosylation mapping by combining collision-induced dissociation (CID)-only fragmentation with chromatographic retention times of glycopeptides. This approach leverages an experimental pipeline with parallel analysis of glyco- and deglycopeptides. As the test case we chose ABCA4, a large integral membrane protein with 16 predicted sites for N-glycosylation. Taking advantage of CID features such as high scan speed and high intensity of fragment ions together combined with the retention times of glycopeptides to conclusively identify the non-glycolytic peptide from which the glycopeptide was derived, we obtained virtually complete information about glycan compositions and peptide sequences, as well as the N-glycosylation site occupancy and relative abundances of each glycoform at specific sites for ABCA4. The challenges provided by this example provide guidance in analyzing complex relatively pure glycoproteins and potentially even more complex glycoprotein mixtures.

质谱法定量测定高密度脂蛋白结合蛋白的糖基化水平

采用质谱法对4种高密度脂蛋白(HDL)的结合蛋白重组人载脂蛋白血清淀粉样蛋白A(SAA)、 α1-抗胰蛋白酶(A1AT)、 α2-人体血清糖蛋白(A2HSG)和A载脂蛋白C3(Apo C3)从蛋白质含量(蛋白的绝对定量)、 位点特异性糖基化(糖肽的相对定量)及聚糖位点占有率等方面进行了研究. 利用四极杆-飞行时间质谱仪(Q-TOF)测量糖蛋白标样酶解产物的二级质谱碎片离子, 用Byonic软件发现了新的糖基化位点信息, 即增加了原位点处聚糖糖型的种类. 对于A2HSG, 新增了N-糖基化156位点上的4种糖型, N-糖基化176位点上的6种糖型, O-糖基化319位点的4种O-聚糖和O-糖基化346位点上的1种糖型. 对于Apo C3, 只有O-糖基化94一个位点, 在此位点上新增了9种糖型. 同时, 调整了用于定量蛋白的多肽, 使得定量更加准确. 采用三重四极杆串级质谱仪(UPLC-ESI-QQQ)研究了4种结合蛋白中多肽和糖肽的多反应监测(MRM)行为, 并重新计算了每种聚糖的位点占有率, 优化了现有的定量方法.     

Reversed-phase depletion coupled with hydrophilic affinity enrichment for the selective isolation of N-linked glycopeptides by using Click OEG-CD matrix

Selective enrichment of glycopeptides is of great importance for protein glycosylation analysis using mass spectrometry since the signals of glycopeptides could be severely suppressed by the coexisting non-glycosylated peptides in the protein digest. In the present work, a strategy for N-linked glycopeptide enrichment through reversed-phase depletion coupled with hydrophilic affinity enrichment by applying the customized matrix named Click OEG-CD is developed. Compared with single hydrophilic interaction liquid chromatography (HILIC) mode, the strategy exhibited remarkably higher selectivity for N-linked glycopeptides. As many as 22, 18, and eight glycopeptides were detected in the glycopeptide fraction enriched with the strategy from the digests of human immunoglobulin G, horseradish peroxidase and bovine ribonuclease B, respectively. In addition, the strategy also showed high glycosylation microheterogeneity coverage for the enrichment of human α₁-acid glycoprotein glycopeptides. More than 170 glycopeptides covering all the glycosylation sites were detected in the enriched fraction. The revered-phase liquid chromatography depletion coupled with HILIC enrichment strategy by using Click OEG-CD matrix is expected to show more potential in further applications in glycosylation analysis.     

Selective enrichment of N‐linked glycopeptides and glycans by using a dextran‐modified hydrophilic material

Glycosylation analysis of proteins from biological sources utilizing mass spectrometry based approaches is challenging due to the relatively low abundance of glycopeptides, the structural diversity of glycans, and the coexisting matrices. In this study, a customized dextran‐bonded silica‐based stationary phase was introduced for selective enrichment of glycopeptides and glycans from complex biological samples. This material has exhibited superior selectivity and broader glycosylation site coverage over commercial Sepharose in glycoproteomic evaluation. Additionally, the glycomic analysis of fetuin, α₁‐acid glycoprotein, and human serum N‐glycome also indicated the relatively higher sensitivity, selectivity, and glycoform coverage of dextran‐bonded silica than that of Sepharose and porous graphitized carbon. Therefore, the dextran‐bonded silica is expected to make contributions in the fields of glycoproteomics and glycomics.     

Inherently hydrophilic mesoporous channel coupled with metal oxide for fishing endogenous salivary glycopeptides and phosphopeptides

Both glycosylation and phosphorylation exert crucial rule in multitudinous biological processes. For in-depth profiling of glycosylation and phosphorylation, a magnetic metal oxide is effectively coupled with inherently hydrophilic mesoporous channels (denoted as Fe₃O₄@TiO₂@mSiO₂-TSG). Based on the mechanism of hydrophilic interaction liquid chromatography (HILIC) and metal oxide affinity chromatography (MOAC), the Fe₃O₄@TiO₂@mSiO₂-TSG nanomaterial shows high capacity for simultaneously enriching glycopeptides and phosphopeptides. With human saliva collected in successive four days as practical biological sample, endogenous glycopeptides and phosphopeptides are efficiently enriched. Further gene ontology analysis reveals that the identified endogenous glycopeptides and phosphopeptides participate in diverse molecular functions and biological processes. This strategy is anticipated to promote variation analysis of salivary post-translational modifications.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Absolute Quantitation of Glycosylation Site Occupancy Using Isotopically Labeled Standards and LC-MS

N-linked glycans are required to maintain appropriate biological functions on proteins. Underglycosylation leads to many diseases in plants and animals; therefore, characterizing the extent of glycosylation on proteins is an important step in understanding, diagnosing, and treating diseases. To determine the glycosylation site occupancy, protein N-glycosidase F (PNGase F) is typically used to detach the glycan from the protein, during which the formerly glycosylated asparagine undergoes deamidation to become an aspartic acid. By comparing the abundance of the resulting peptide containing aspartic acid against the one containing non-glycosylated asparagine, the glycosylation site occupancy can be evaluated. However, this approach can give inaccurate results when spontaneous chemical deamidation of the non-glycosylated asparagine occurs. To overcome this limitation, we developed a new method to measure the glycosylation site occupancy that does not rely on converting glycosylated peptides to their deglycosylated forms. Specifically, the overall protein concentration and the non-glycosylated portion of the protein are quantified simultaneously by using heavy isotope-labeled internal standards coupled with LC-MS analysis, and the extent of site occupancy is accurately determined. The efficacy of the method was demonstrated by quantifying the occupancy of a glycosylation site on bovine fetuin. The developed method is the first work that measures the glycosylation site occupancy without using PNGase F, and it can be done in parallel with glycopeptide analysis because the glycan remains intact throughout the workflow.

HPLC-ESI-MS and MS/MS structural characterization of multifucosylated N-glycoforms of the basic proline-rich protein IB-8a CON1+ in human saliva

This study describes the characterization of the glycan moieties and the peptide backbone of six glycoforms of IB-8a CON1(+), a basic proline-rich protein present in human saliva. MS analyses on the intact glycoproteins before and after N-deglycosylation with PNGase F and high-resolution MS/MS sequencing by LTQ Orbitrap XL of peptides and glycopeptides from tryptic digests allowed the structural characterization of the glycan moieties and the polypeptide backbone, as well as to establish the glycosylation site at the asparagine residue at 98th position. Five of the glycoforms carry a biantennary N-linked glycan fucosylated in the innermost N-acetylglucosamine of the core and showing from zero to four additional fucoses in the antennal region. The sixth glycoform carries a monoantennary monofucosylated oligosaccharide. The glycoform cluster was detected on 28 of 71 adult saliva specimens. Level of fucosylation showed interindividual variability with the major relative abundance for the trifucosylated glycoform. Nonglycosylated IB-8a CON1(+) and the variant IB-8a CON1(-), lacking of the glycosylation site, have been also detected in human saliva.     

Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe₃O₄ nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.     

Zirconia layer coated mesoporous silica microspheres as HILIC SPE materials for selective glycopeptide enrichment

Characterization of protein glycosylation requires highly specific methods for the enrichment of glycopeptides because of their sub-stoichiometric glycosylation-site occupancy. The hydrophilic affinity based strategy has attracted more attention, owing to its broad glycan specificity, good reproducibility, and compatibility with mass spectrometric (MS) analysis. Several polar matrices have emerged for hydrophilic interaction chromatography (HILIC) approaches, including sepharose, cellulose, ZIC-HILIC and titania. Here, we present the solid-phase extraction (SPE) utility of zirconia coated mesoporous silica (ZrO(2)/MPS) microspheres for glycopeptide isolation prior to MS analysis. The high specificity of this SPE approach was demonstrated by the enrichment of glycopeptides from the digests of model glycoproteins in HILIC mode. ZrO(2)/MPS microspheres show superior selectivity and glycosylation heterogeneity coverage for glycopeptide enrichment to conventional sepharose. Furthermore, digested mixtures of the phosphoprotein α-casein and IgG were also treated with ZrO(2)/MPS HILIC SPE materials, which exhibited that glycopeptides could be effectively enriched with interference from phosphorylated peptides.     

Integration of High Accuracy N-Terminus Identification in Peptide Sequencing and Comparative Protein Analysis Via Isothiocyanate-Based Isotope Labeling Reagent with ESI Ion-trap TOF MS

A multifunctional isothiocyanate-based isotope labeling reagent, [d ₀]-/[d ₆]-4,6-dimethoxy pyrimidine-2-isothiocyanate (DMPITC), has been developed for accurate N-terminus identification in peptide sequencing and comparative protein analysis by ESI Ion-trap TOF mass spectrometry. In contrast with the conventional labeling reagent phenyl isothiocyanate (PITC), DMPITC showed more desirable properties such as rapid labeling, sensitivity enhancement, and facilitating peptide sequencing. More significantly, DMPITC-based labeling strategy possessed the capacity of higher reliable N-terminus identification owning to the high-yield b₁ ion combined with the isotope validation of 6 Da. Meanwhile, it also showed potential in differentiating isomeric residues of leucine and isoleucine at N-terminus on the basis of the relative abundance ratios between the fragment ions of their respective b₁ ions. The strategy not only allows accurate interpretation for peptide but also ensures rapid and sensitive comparative analysis for protein by direct MS analysis. Using trypsin-digested bovine serum albumin (BSA), both peptide N-terminus identification and quantitative analysis were accomplished with high accuracy, efficiency, and reproducibility. The application of DMPITC-based labeling strategy is expected to serve as a promising tool for proteome research.     

Quantitative mapping of glycoprotein micro‐heterogeneity and macro‐heterogeneity: an evaluation of mass spectrometry signal strengths using synthetic peptides and glycopeptides

Mass spectrometry (MS) is used to quantify the relative distribution of glycans attached to particular protein glycosylation sites (micro‐heterogeneity) and evaluate the molar site occupancy (macro‐heterogeneity) in glycoproteomics. However, the accuracy of MS for such quantitative measurements remains to be clarified. As a key step towards this goal, a panel of related tryptic peptides with and without complex, biantennary, disialylated N‐glycans was chemically synthesised by solid‐phase peptide synthesis. Peptides mimicking those resulting from enzymatic deglycosylation using PNGase F/A and endo D/F/H were synthetically produced, carrying aspartic acid and N‐acetylglucosamine‐linked asparagine residues, respectively, at the glycosylation site. The MS ionisation/detection strengths of these pure, well‐defined and quantified compounds were investigated using various MS ionisation techniques and mass analysers (ESI‐IT, ESI‐Q‐TOF, MALDI‐TOF, ESI/MALDI‐FT‐ICR‐MS). Depending on the ion source/mass analyser, glycopeptides carrying complex‐type N‐glycans exhibited clearly lower signal strengths (10–50% of an unglycosylated peptide) when equimolar amounts were analysed. Less ionisation/detection bias was observed when the glycopeptides were analysed by nano‐ESI and medium‐pressure MALDI. The position of the glycosylation site within the tryptic peptides also influenced the signal response, in particular if detected as singly or doubly charged signals. This is the first study to systematically and quantitatively address and determine MS glycopeptide ionisation/detection strengths to evaluate glycoprotein micro‐heterogeneity and macro‐heterogeneity by label‐free approaches. These data form a much needed knowledge base for accurate quantitative glycoproteomics. Copyright © 2013 John Wiley & Sons, Ltd.     

Use of activated graphitized carbon chips for liquid chromatography/mass spectrometric and tandem mass spectrometric analysis of tryptic glycopeptides

Protein glycosylation has a significant medical importance as changes in glycosylation patterns have been associated with a number of diseases. Therefore, monitoring potential changes in glycan profiles, and the microheterogeneities associated with glycosylation sites, are becoming increasingly important in the search for disease biomarkers. Highly efficient separations and sensitive methods must be developed to effectively monitor changes in the glycoproteome. These methods must not discriminate against hydrophobic or hydrophilic analytes. The use of activated graphitized carbon as a desalting media and a stationary phase for the purification and the separation of glycans, and as a stationary phase for the separation of small glycopeptides, has previously been reported. Here, we describe the use of activated graphitized carbon as a stationary phase for the separation of hydrophilic tryptic glycopeptides, employing a chip‐based liquid chromatographic (LC) system. The capabilities of both activated graphitized carbon and C₁₈ LC chips for the characterization of the glycopeptides appeared to be comparable. Adequate retention time reproducibility was achieved for both packing types in the chip format. However, hydrophilic glycopeptides were preferentially retained on the activated graphitized carbon chip, thus allowing the identification of hydrophilic glycopeptides which were not effectively retained on C₁₈ chips. On the other hand, hydrophobic glycopeptides were better retained on C₁₈ chips. Characterization of the glycosylation sites of glycoproteins possessing both hydrophilic and hydrophobic glycopeptides is comprehensively achieved using both media. This is feasible considering the limited amount of sample required per analysis (<1 pmol). The performance of both media also appeared comparable when analyzing a four‐protein mixture. Similar sequence coverage and MASCOT ion scores were observed for all proteins when using either stationary phase. Copyright © 2009 John Wiley & Sons, Ltd.     

Vertical random variability of the distribution coefficient in the soil and its effect on the migration of fallout radionuclides

In the field, the distribution coefficient, K _d, for the sorption of a radionuclide by the soil cannot be expected to be constant. Even in a well defined soil horizon, K _d will vary stochastically in horizontal as well as in vertical direction around a mean value. The horizontal random variability of K _d produce a pronounced tailing effect in the concentration depth profile of a fallout radionuclide, much less is known on the corresponding effect of the vertical random variability. To analyze this effect theoretically, the classical convection-dispersion model in combination with the random-walk particle method was applied. The concentration depth profile of a radionuclide was calculated one year after deposition assuming (1) constant values of the pore water velocity, the diffusion/dispersion coefficient, and the distribution coefficient (K _d = 100 cm^3.g^-1), and (2) exhibiting a vertical variability for K _d according to a log-normal distribution with a geometric mean of 100 cm^3.g^-1 and a coefficient of variation of CV = 0.53. The results show that these two concentration depth profiles are only slightly different, the location of the peak is shifted somewhat upwards, and the dispersion of the concentration depth profile is slightly larger. A substantial tailing effect of the concentration depth profile is not perceivable. Especially with respect to the location of the peak, a very good approximation of the concentration depth profile is obtained if the arithmetic mean of the K _d-values (K _d = 113 cm^3.g^-1) and a slightly increased dispersion coefficient are used in the analytical solution of the classical convection-dispersion equation with constant K _d. The evaluation of the observed concentration depth profile with the analytical solution of the classical convection-dispersion equation with constant parameters will, within the usual experimental limits, hardly reveal the presence of a log-normal random distribution of K _d in the vertical direction in contrast to the horizontal direction.     

Synthesis of and Specific Antibody Generation for Glycopeptides with Arginine N‐GlcNAcylation

As a unique and unappreciated protein posttranslational modification, arginine N‐glycosylation was recently discovered to play an important role in the process that bacteria counteract host defenses. To provide chemical tools for further proteomic and biochemical studies on arginine N‐glycosylation, we report the first general strategy for a rapid and cost‐effective synthesis of glycopeptides carrying single or multiple arginine N‐GlcNAcyl groups. These glycopeptides were successfully utilized to generate the first antibodies that can specifically recognize arginine N‐GlcNAcylated peptides or proteins in a sequence‐independent manner.     

MALDI‐MS profiling of serum O‐glycosylation and N‐glycosylation in COG5‐CDG

Congenital disorders of glycosylation (CDG) are due to defective glycosylation of glycoconjugates. Conserved oligomeric Golgi (COG)‐CDG are genetic diseases due to defects of the COG complex subunits 1–8 causing N‐glycan and O‐glycan processing abnormalities. In COG‐CDG, isoelectric focusing separation of undersialylated glycoforms of serum transferrin and apolipoprotein C‐III (apoC‐III) allows to detect N‐glycosylation and O‐glycosylation defects, respectively. COG5‐CDG (COG5 subunit deficiency) is a multisystem disease with dysmorphic features, intellectual disability of variable degree, seizures, acquired microcephaly, sensory defects and autistic behavior. We applied matrix‐assisted laser desorption/ionization‐MS for a high‐throughput screening of differential serum O‐glycoform and N‐ glycoform in five patients with COG5‐CDG. When compared with age‐matched controls, COG5‐CDG showed a significant increase of apoC‐III_0a (aglycosylated glycoform), whereas apoC‐III₁ (mono‐sialylated glycoform) decreased significantly. Serum N‐glycome of COG5‐CDG patients was characterized by the relative abundance of undersialylated and undergalactosylated biantennary and triantennary glycans as well as slight increase of high‐mannose structures and hybrid glycans. Using advanced and well‐established MS‐based approaches, the present findings reveal novel aspects on O‐glycan and N‐glycan profiling in COG5‐CDG patients, thus providing an increase of current knowledge on glycosylation defects caused by impairment of COG subunits, in support of clinical diagnosis. Copyright © 2017 John Wiley & Sons, Ltd.     

Analysis of recombinant human erythropoietin glycopeptides by capillary electrophoresis electrospray–time of flight-mass spectrometry

Capillary electrophoresis electrospray–mass spectrometry was used to detect and characterize the great variety of O- and N-glycopeptide glycoforms of recombinant human erythropoietin (rhEPO) using an orthogonal accelerating time-of-flight mass spectrometer to obtain their exact molecular masses (CE–TOF-MS). rhEPO was digested with trypsin and Glu-C and analyzed by CE–TOF-MS to detect O₁₂₆, N₈₃, N₂₄–N₃₈ and N₂₄ and N₃₈ glycopeptide glycoforms, respectively. Neuraminidase was first used to enhance the detection of the glycopeptides and detect all possible glycoforms contained in each glycosylation site. O₁₂₆ and N₈₃ glycopeptides were extensively characterized. Twelve sialoforms corresponding to 5 different glycoforms were detected in N₈₃, and for the first time, a sulfated sialoform of this glycopeptide was also detected. In the case of O₁₂₆, different sialoforms with different types of sialic acids (Neu5Gc and Neu5Ac) were detected and an estimation of the relative percentage of Neu5Gc versus Neu5Ac was also carried out for this glycopeptide. N₂₄ and N₃₈ glycosylation sites were also characterized by CE–TOF-MS after Glu-C digestion and these results permitted to rule out some glycan combinations for N₂₄–N₃₈ glycopeptide glycoforms. This study provided a reliable glycopeptide map of rhEPO and may be regarded as an excellent starting point to analyze rhEPO glycopeptides in biological fluids and detect the use of this hormone in sports.     

Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography

Comprehensive glycoprotein characterization based on mass spectrometry (MS) is challenging because of low concentration of glycopeptides and suppression effect of abundant non-glycosylated peptides in MS. Therefore, it is vital to enrich glycopeptides before MS analysis. A new method was developed to selectively enrich glycopeptides from complex sample by coupling C18 fractionation with titanium dioxide (TiO(2)) enrichment. The new method allows to selectively enrich N-linked glycopeptides with various glycan forms and different sequence lengths. Compared with single TiO(2) method, the established method demonstrated higher glycopeptide selectivity and higher glycosylation heterogeneity coverage. Further application of this method to mixture of non-glycosylated protein and glycoprotein digests at different levels reveals the feasibility of enrichment of tryptic glycopeptides from simple proteomics samples.     

Localized Chemical Remodeling for Live Cell Imaging of Protein‐Specific Glycoform

Live cell imaging of protein‐specific glycoforms is important for the elucidation of glycosylation mechanisms and identification of disease states. The currently used metabolic oligosaccharide engineering (MOE) technology permits routinely global chemical remodeling (GCM) for carbohydrate site of interest, but can exert unnecessary whole‐cell scale perturbation and generate unpredictable metabolic efficiency issue. A localized chemical remodeling (LCM) strategy for efficient and reliable access to protein‐specific glycoform information is reported. The proof‐of‐concept protocol developed for MUC1‐specific terminal galactose/N ‐acetylgalactosamine (Gal/GalNAc) combines affinity binding, off‐on switchable catalytic activity, and proximity catalysis to create a reactive handle for bioorthogonal labeling and imaging. Noteworthy assay features associated with LCM as compared with MOE include minimum target cell perturbation, short reaction timeframe, effectiveness as a molecular ruler, and quantitative analysis capability.