Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis - electrospray - time-of-flight mass spectrometry

Glycosylation of recombinant human erythropoietin (rHuEPO) is a post-translational process that alters biological activity, solubility and lifetime of the glycoprotein in blood, and strongly depends on the type of cell and the cell culture conditions. A fast and simple method providing extensive carbohydrate information about the glycans present in rHuEPO and other glycoproteins is needed in order to improve current methods in drug development or product quality control. Here, an improved method for intact rHuEPO glycoform characterization by CZE-ESI-TOF MS has been developed using a novel capillary coating and compared to a previous study. Both methods allow a fast separation in combination with accurate mass characterization of the single protein isoforms. The novel dynamic coating provides a separation at an EOF close to zero, enabling better separation. This results in an improved mass spectrometric resolution and the detection of minor isoforms. In order to assign an unequivocal carbohydrate composition to every intact glycoform, a CZE-ESI-MS separation method for enzymatically released underivatized N-glycans has been developed. The TOF MS allows the correct identification of the glycans due to its high mass accuracy and resolution. Therefore, glycan modifications such as acetylation, oxidation, sulfation and even the exchange of OH by NH(2) are successfully characterized. Information of the protein-backbone molecular mass has been combined with results from peptide analysis (revealing information about O-glycosylation) and from the glycan analysis, including the detection of as yet undescribed glycans containing four antennae and five sialic acids. This allows an unequivocal assignment of an overall glycosylation composition to the molecular masses obtained for the intact rHuEPO glycoforms.     

Collection of alpha1-acid glycoprotein molecular species by capillary electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms

A highly heterogeneous glycoprotein, alpha1-acid glycoprotein, was resolved into their glycoforms by capillary electrophoresis using a surface-modified capillary in 20 mM acetate buffer (pH 4.2) containing 0.5% (w/v) hydroxypropylmethylcellulose. We collected the fractions containing each glycoform as nearly pure state by capillary electrophoresis, and examined the molecular masses of these glycoforms by matrix assisted laser desorption time-of-flight mass spectrometry. We also analyzed carbohydrate chains after releasing them with N-glycosidase F followed by fluorescent labeling with 8-aminopyrene-1,3,6-trisulfonate. We found that the separation of glycoforms was mostly due to the presence of multiantennary carbohydrate chains. We propose that the present technique is useful for the analysis of post translational modification of proteins with carbohydrate chains.     

Characterization of transferrin glycoforms in human serum by CE-UV and CE-ESI-MS

Human transferrin (Tf) is a model glycoprotein for congenital disorders of glycosylation (CDG) diagnosis. In the last few years, new CE-UV methods for intact Tf glycoforms analysis have been developed using nonvolatile BGEs and organic modifiers. However, the use of these BGEs does not allow the coupling of these procedures with electrospray MS (ESI-MS). In this study, a new CE-UV separation method of Tf glycoforms is developed, using a double-layer stable coating and a volatile BGE based on ammonium acetate. The separation method is optimized using standard Tf and their potential is demonstrated applying the method to the analysis of sera Tf from healthy individuals and CDG patients. The CE-UV separation method has been coupled to ESI-MS detection. Main parameters such as sheath liquid composition are optimized in order to obtain a good sensitivity. The CE-ESI-MS method has also been used in serum samples obtaining the separation of the different proteins present in serum and partial separation of Tf glycoforms. Different mass spectra and deconvoluted molecular masses were obtained for each sialoform, allowing unequivocal glycoform identification.     

Capillary electrophoresis and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry for analysis of the novel erythropoiesis-stimulating protein (NESP)

NESP (novel erythropoiesis-stimulating protein) is a recently approved hyperglycosylated analogue of human erythropoietin (EPO) with a long-lasting effect. In this work, the capillary electrophoresis (CE) methodology proposed by the European Pharmacopoeia for the separation of EPO glycoforms has been modified for the separation of NESP glycoforms. Optimization of pH of the separation electrolyte has been fundamental in order to achieve baseline resolution of seven peaks corresponding to NESP glycoforms. Intact NESP has also been characterized by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). An accurate approximation to an average molecular mass of the NESP molecule has been obtained, taking into account the strong influence of laser intensity upon the MALDI-TOF mass spectra found.     

Methyl chitosan coating for glycoform analysis of glycoproteins by capillary electrophoresis

CE is a promising technique for the analysis of glycosylated proteins, especially at the intact level. In the present study, the utility of CE for the separation of protein glycoforms is developed by using methyl chitosan as capillary coating. Methyl chitosan, in contrast to the polymers commonly used for coating, bears different types of amine groups, allowing for tunable charge states for various applications. The addition of methyl chitosan in background electrolyte can modulate the EOF and improve the separation performance. The methyl chitosan-coated capillary provided good separation of acidic or basic glycosylated proteins. Five ribonuclease B glycoforms were resolved by CE in less than 18 min, and the profile was essentially in agreement with that obtained by MALDI-TOF MS. The recombinant human erythropoietin glycoforms were well separated within 9 min. The developed method shows a great potential in protein glycoform analysis.     

Characterization of oligosaccharide moieties of intact glycoproteins by microwave-assisted partial acid hydrolysis and mass spectrometry

A method, which utilizes microwave-assisted partial acid hydrolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), to elucidate oligosaccharide composition of intact glycoproteins is presented here. Glycoproteins, such as ribonuclease B, avidin, alpha1-acid glycoprotein, and fetuin, are used as model systems to demonstrate this technique. Partial cleavage of oligosaccharides from whole intact glycoproteins with trifluoroacetic acid was observed after a short exposure to microwaves. Due to the high-resolution mass spectra obtained by MALDI-TOFMS from glycoproteins with molecular weights less than 20 kDa, the compositions of oligosaccharides are readily derived for ribonuclease B and avidin. The data agree with the proposed oligosaccharide structures of ribonuclease B (five glycoforms) and avidin (eight glycoforms). Larger glycoproteins such as alpha1-acid glycoprotein (many glycoforms) and fetuin (many glycoforms) exhibited only broad peaks with no glycoform resolution. Nevertheless, this method can be used successfully for analysis of glycoproteins with molecular weights greater than 20 kDa to determine the presence or absence of glycosylation.     

Identification of bifucosylated glycoforms using low‐energy CID spectra

We have used tandem mass spectrometry (MS/MS)‐based analysis of glycopeptides in order to identify the composition and structure of rare glycoforms. The results illustrate utility of low‐energy MS/MS for structure identification. We have shown the presence of bifucosylated and trifucosylated glycoforms in human α‐1‐acid glycoprotein (AGP), a major plasma glycoprotein. Fucosylation in the case of AGP always occurs on the antennae; core fucosylation was not observed.     

Profiling of erythropoietin products by capillary electrophoresis with native fluorescence detection

The potential of CE with native fluorescence detection (Flu) for the profiling of the therapeutic protein erythropoietin (EPO) was studied. EPO is a highly heterogeneous glycoprotein comprising a large number of isoforms. CE was applied to induce separation among the various glycoforms. Native Flu of EPO provided high detection selectivity yielding good signal‐to‐noise ratios and stable baselines, particularly when compared to conventional UV absorbance detection. In order to enhance EPO isoform resolution, CE was performed using a capillary with a neutral coating in combination with a simple BGE of 2.0 M acetic acid (pH 2.1). CE‐Flu analysis of the EPO biological reference preparation of the European Pharmacopeia resulted in a highly detailed glycoform profile. Migration time RSDs for selected EPO isoforms were less than 0.22% and 0.80% for intraday and interday repeatability, respectively. RSDs for relative peak intensity of the major EPO isoforms were less than 3%. The achieved resolution, migration time stability, and sensitivity allowed discrimination of different EPO products (EPO‐α and EPO‐β) based on the recorded glycoform pattern. The developed CE‐Flu method is relatively straightforward, and shows potential for quality control in biopharmaceutical production.     

Comparative study on the distribution of ovalbumin glycoforms by capillary electrophoresis.

Two commercial turkey egg ovalbumins (TEOs) with different quantities of mannose, were further purified by reversed-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for either of the purified glycoproteins showed one big wide band and one close small band. Capillary electrophoresis was used for the investigation of the separation of glycoforms of both glycoproteins. The best resolution of the glycoforms was obtained, reproducibly, with 100 mM borate, 1.8 mM 1,4-diaminobutane and pH 8.6 electrophoretic buffer. At least 13 glycoform peaks could be separated for either of the two glycoproteins. Their glycoform patterns were highly similar except for the conspicuous decrease in quantity of four glycoforms in the ovalbumin containing less mannose, compared to that of the other with more mannose. Coinjection electrophoresis of the two glycoproteins indicated that almost every glycoform peak of the former exactly overlapped with its corresponding glycoform peak of the latter. These results clearly indicated that the two TEOs possessed the same glycoform patterns but differed in quantity at least four glycoforms. It was found that the glycoform patterns were remarkably different between TEO and chicken egg ovalbumin.     

Characterisation of intact recombinant human erythropoietins applied in doping by means of planar gel electrophoretic techniques and matrix-assisted laser desorption/ionisation linear time-of-flight mass spectrometry

Our experiments show that it is possible to detect different types of recombinant human erythropoietins (rhEPOs), EPO-alpha, EPO-beta and novel erythropoesis stimulating protein (NESP), based on exact molecular weight (MW) determination by matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) applying a high-resolution time-of-flight (TOF) mass analyser in the linear mode. Detection limits for the highly purified, intact glycoproteins were achievable in the low fmol range (25-50 fmol) using a sample preparation method applying a hydrophobic sample support (DropStop) as MALDI target surface. These results are very promising for the development of highly sensitive detection methods for a direct identification of rhEPO after enrichment from human body fluids. During our investigation we were able to differentiate EPO-alpha, EPO-beta and NESP based on distinct molecular substructures at the protein level by specific enzymatic reactions. MW determination of the intact molecules by high resolving one-dimensional sodium dodecyl sulfate /polyacrylamide gel electrophoresis (1D SDS-PAGE) and isoform separation by planar isoelectric focusing (IEF) was compared with MALDI-MS data. Migration differences between the rhEPOs were observed from gel electrophoresis, whereby MWs of 38 kDa in the case of EPO-alpha/beta and 49 kDa for NESP could be estimated. In contrast, an exact MW determination by MALDI-MS based on internal calibration revealed average MWs of 29.8 +/- 0.3 kDa for EPO-alpha/beta and 36.8 +/- 0.4 kDa for NESP. IEF separation of the intact rhEPOs revealed the presence of four to eight distinct isoforms in EPO-alpha and EPO-beta, while four isoforms, which appeared in the more acidic area of the gels, were detected by immunostaining in NESP. A direct detection of the different N- or O-glycoform pattern from rhEPOs using MALDI-MS was possible by de-sialylation of the glycan structures and after de-N-glycosylation of the intact molecules. Thereby, the main glycoforms of EPO-alpha, EPO-beta and NESP could be characterised based on their N-glycan composition. A microheterogeneity of the molecules based on the degree of sialylation of the O-glycan was observable directly from the de-N-glycosylated protein.     

Anti-EPO and anti-NESP antibodies raised against synthetic peptides that reproduce the minimal amino acid sequence differences between EPO and NESP

Erythropoietin (EPO) is a hormone that regulates red blood cell production. Recombinant human EPO (rHuEPO) and NESP (novel erythropoiesis stimulating protein) have been produced for therapeutic purposes and also to improve sports performance. The primary sequences of rHuEPO and NESP differ by just five amino acids. Due to the high homology, no antibodies that are able to discriminate between both molecules have been obtained until now. The aim of the present work was to design synthetic peptides corresponding to the sequence that differs between EPO and NESP (87–90aa), that can then be used as immunogens to develop specific rabbit polyclonal antibodies for selectively detecting EPO and NESP. Three peptides were synthesized: EPO (81–95), NESP (81–95), and NESP (86–104), and these were coupled to KLH and OVA for immunization and screening purposes, respectively. The sera obtained were tested by ELISA on synthetic peptide–OVA conjugates and purified by immunoaffinity chromatography against the corresponding synthetic peptide. The specific purified antibodies were characterized by ELISA, SDS-PAGE, and isoelectric focusing, followed by western blot. Antisera raised against EPO (81–95) recognized rHuEPO but not NESP. In contrast, anti-NESP (84–106) sera gave a specific anti-NESP response only after immunoaffinity purification on a NESP (86–91) column. An efficient strategy for generating specific antibodies against EPO and NESP can be achieved by selecting suitable synthetic peptides. The antibodies obtained are able to differentiate between rHuEPO and NESP, and may be particularly useful for screening purposes in both therapeutic and antidoping contexts.     

High-throughput glycosylation analysis of intact monoclonal antibodies by mass spectrometry coupled with capillary electrophoresis and liquid chromatography

The analysis of monoclonal antibodies glycosylation is a crucial quality control attribute of biopharmaceutical drugs. High throughput screening approaches for antibody glycoform analysis are required in various stages of process optimization. Here, we present high throughput screening suitable mass spectrometry-based workflows for the analysis of intact antibody glycosylation out of cell supernatants. Capillary electrophoresis and liquid chromatography were coupled with quadrupole time-of-flight mass spectrometry or Orbitrap mass spectrometry. Both separation methods offer fast separation (10–15 min) and the capability to prevent the separated cell supernatant matrix to enter the mass spectrometry by post-separation valving. Both mass spectrometry instruments provide comparable results and both are sufficient to determine the glycosylation pattern of the five major glycoforms of the measured antibodies. However, the Orbitrap yields higher sensitivity of 25 μg/mL (CE-nanoCEasy-Orbitrap mass spectrometry) and 5 μg/mL (liquid chromatography-Orbitrap mass spectrometry). Data processing was optimized for a faster processing and easier detection of low abundant glycoforms based on averaged charge-deconvoluted mass spectra. This approach combines a non-target glycoform analysis while yielding the same glycosylation pattern as the traditional approach based on extracted ion traces. The presented methods enable the high throughput screening of the glycosylation pattern of antibodies down to low μg/mL-range out of cell supernatant without any sample preparation.     

Complementary middle‐down and intact monoclonal antibody proteoform characterization by capillary zone electrophoresis – mass spectrometry

High‐resolution capillary zone electrophoresis – mass spectrometry (CZE‐MS) has been of increasing interest for the analysis of biopharmaceuticals. In this work, a combination of middle‐down and intact CZE‐MS analyses has been implemented for the characterization of a biotherapeutic monoclonal antibody (mAb) with a variety of post‐translational modifications (PTMs) and glycosylation structures. Middle‐down and intact CZE separations were performed in an acidified methanol‐water background electrolyte on a capillary with a positively charged coating (M7C4I) coupled to an Orbitrap mass spectrometer using a commercial sheathless interface (CESI). Middle‐down analysis of the IdeS‐digested mAb provided characterization of PTMs of digestion fragments. High resolution CZE enabled separation of charge variants corresponding to 2X‐deamidated, 1X‐deamidated, and non‐deamidated forms at baseline resolution. In the course of the middle‐down CZE‐MS analysis, separation of glycoforms of the F_C/2 fragment was accomplished due to hydrodynamic volume differences. Several identified PTMs were confirmed by CZE‐MS². Incorporation of TCEP‐HCl reducing agent in the sample solvent resulted in successful analysis of reduced forms without the need for alkylation. CZE‐MS studies on the intact mAb under denaturing conditions enabled baseline separation of the 2X‐glycosylated, 1X‐glycosylated, and aglycosylated populations as a result of hydrodynamic volume differences. The presence of a trace quantity of dissociated light chain was also detected in the intact protein analysis. Characterization of the mAb under native conditions verified identifications achieved via intact analysis and allowed for quantitative confirmation of proteoforms. Analysis of mAbs using CZE‐MS represents a complementary approach to the more conventional liquid‐chromatography – mass spectrometry‐based approaches.     

Application of capillary electrophoresis- electrospray-mass spectrometry to the separation and characterization of isomeric lipopolysaccharides of Neisseria meningitidis

A capillary electrophoresis-electrospray-mass spectrometry technique for the characterization of lipopolysaccharides (LPSs) was developed, permitting the separation of trace-level O-deacylated LPS isoforms for subsequent structural characterization using tandem mass spectrometry (MS/MS). The separation buffer and electrospray interface were optimized first using O-deacylated LPS samples from large-scale preparations. It was found that with microelectrospray or sheath-solution interface, we could separate LPS in anionic forms and detect them using either negative or positive ion mode MS. For negative ion detection mode MS, 30 mM morpholine with addition of 5% v/v methanol was employed as separation buffer. When positive ion detection mode MS was required, 10 mM ammonium acetate with addition of 5% methanol was used as separation buffer. The structural assignments obtained from MS/MS and capillary zone electrophoresis-electrospray-MS (CZE-ESMS) analyses enabled the identification of isomeric glycoforms. Application of this technique to the analysis of LPS from the galE mutants of Neisseria meningitidis strain BZ157 B5+ revealed the presence of isomeric glycoforms, in which the location of a functional group phosphoethanolamine was found to be in either inner core or lipid A-OH regions. The described technique was also applied to the analysis of LPS samples from the galE mutant of N. meningitidis strains F1576 A4+ and A4-. The occurrence of isomeric LPS glycoforms differing by the location or presence of neutral sugar residues, such as hexoses, can also be characterized using MS/MS.     

Synthetic Glycoforms Reveal Carbohydrate‐Dependent Bioactivity of Human Saposin D

The main glycoforms of the hydrophobic lysosomal glycoprotein saposin D (SapD) were synthesized by native chemical ligation. An approach for the challenging solid‐phase synthesis of the fragments was developed. Three SapD glycoforms were obtained following a general and robust refolding and purification protocol. A crystal structure of one glycoform confirmed its native structure and disulfide pattern. Functional assays revealed that the lipid‐binding properties of three SapD glycoforms are highly affected by the single sugar moiety of SapD showing a dependency of the size and the type of N‐glycan.     

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.     

Glycoform Heterogeneity of Human Serum α1-Acid Glycoprotein Determined by CZE in Malignant Diseases

A novel capillary zone electrophoresis method was developed to investigate the glycoform heterogeneity of human serum α1-acid glycoprotein (AGP). The simultaneous application of a dimethyl polysiloxane coated capillary and oligoamine additives, particularly spermidine resulted in a more detailed separation of AGP glycoforms than reported previously. The relative distribution of AGP glycoforms in CZE was determined by baseline integration of peak areas and verified by peak-fitting analysis. Providing high purity of AGP samples suitable for CZE a schedule of isolation and purification steps including sample preparation and an improved technique of ion exchange chromatography was applied. Based on data obtained by CZE and on the serum AGP levels measured the serum concentrations of AGP glycoforms were calculated in cancer patients with Hodgkin and non-Hodgkin lymphoma, ovary carcinoma and melanoma compared to healthy donors. Results presented here demonstrated a significant increase in the serum concentration of the more acidic AGP fractions also indicating the overproduction of these glycoforms in cancer. In conclusion, our observations may raise the clinical diagnostic relevance of changes in the molecular heterogeneity of AGP detected by CZE in the various forms of malignant diseases.     

Glycoform Heterogeneity of Human Serum α1-Acid Glycoprotein Determined by CZE in Malignant Diseases

A novel capillary zone electrophoresis method was developed to investigate the glycoform heterogeneity of human serum α1-acid glycoprotein (AGP). The simultaneous application of a dimethyl polysiloxane coated capillary and oligoamine additives, particularly spermidine resulted in a more detailed separation of AGP glycoforms than reported previously. The relative distribution of AGP glycoforms in CZE was determined by baseline integration of peak areas and verified by peak-fitting analysis. Providing high purity of AGP samples suitable for CZE a schedule of isolation and purification steps including sample preparation and an improved technique of ion exchange chromatography was applied. Based on data obtained by CZE and on the serum AGP levels measured the serum concentrations of AGP glycoforms were calculated in cancer patients with Hodgkin and non-Hodgkin lymphoma, ovary carcinoma and melanoma compared to healthy donors. Results presented here demonstrated a significant increase in the serum concentration of the more acidic AGP fractions also indicating the overproduction of these glycoforms in cancer. In conclusion, our observations may raise the clinical diagnostic relevance of changes in the molecular heterogeneity of AGP detected by CZE in the various forms of malignant diseases.

     

One-step capillary isoelectric focusing for the separation of the recombinant human immunodeficiency virus envelope glycoprotein glycoforms

One-step capillary isoelectric focusing was investigated as a rapid method to resolve the glycoforms of the heterogeneous recombinant human immunodeficiency virus (HIV) envelope glycoprotein (rgp 160sMN/LAI). The separation was performed in a poly(vinyl alcohol) (PVA) coated capillary using a mixture of ampholyte of narrow and wide pH range. A combination of saccaharose and 3-(cyclohexylamino)-1-propanesulfonic acid was shown to be the most efficient additive to avoid protein precipitation which occurs at a pH close to its pI. Although the calibration curve [isoelectric point (pI) vs. migration times] showed a non-linear relationship, an adequate linearity could be yielded for short pI ranges permitting to exhibit the acidic character of the different glycoforms of the rgp 160s MN/LAI (pI from 4.00 to 4.95). Reproducibility evaluated by comparing the performance of a polyacrylamide and a PVA coated capillary showed that low RSD values were obtained for intra-day (0.5 to 1.9%) and inter-day (1.6 to 7.6%) measurements using the PVA capillary. Moreover, the long term stability of the PVA capillary was demonstrated by measuring the variation of migration times of the protein markers for a long period of use. Finally, this method was able to differentiate the glycoform pattern of two close glycoproteins such as the rgp 160 of two sub-populations of the virus HIV-1.     

Convergent synthesis of homogeneous Glc1Man9GlcNAc2-protein and derivatives as ligands of molecular chaperones in protein quality control

A detailed understanding of the molecular mechanism of chaperone-assisted protein quality control is often hampered by the lack of well-defined homogeneous glycoprotein probes. We describe here a highly convergent chemoenzymatic synthesis of the monoglucosylated glycoforms of bovine ribonuclease (RNase) as specific ligands of lectin-like chaperones calnexin (CNX) and calreticulin (CRT) that are known to recognize the monoglucosylated high-mannose oligosaccharide component of glycoproteins in protein folding. The synthesis of a selectively modified glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase was accomplished by chemical synthesis of a large N-glycan oxazoline and its subsequent enzymatic ligation to GlcNAc-RNase under the catalysis of a glycosynthase. Selective removal of the terminal galactose by a β-galactosidase gave the Glc(1)Man(9)GlcNAc(2)-RNase glycoform in excellent yield. CD spectroscopic analysis and RNA-hydrolyzing assay indicated that the synthetic RNase glycoforms maintained essentially the same global conformations and were fully active as the natural bovine ribonuclease B. SPR binding studies revealed that the Glc(1)Man(9)GlcNAc(2)-RNase had high affinity to lectin CRT, while the synthetic Man(9)GlcNAc(2)-RNase glycoform and natural RNase B did not show CRT-binding activity. These results confirmed the essential role of the glucose moiety in the chaperone molecular recognition. Interestingly, the galactose-masked glycoform Gal(1)Glc(1)Man(9)GlcNAc(2)-RNase also showed significant affinity to lectin CRT, suggesting that a galactose β-1,4-linked to the key glucose moiety does not significantly block the lectin binding. These synthetic homogeneous glycoprotein probes should be valuable for a detailed mechanistic study on how molecular chaperones work in concert to distinguish between misfolded and folded glycoproteins in the protein quality control cycle.