Profile of native N‐linked glycan structures from human serum using high performance liquid chromatography on a microfluidic chip and time‐of‐flight mass spectrometry

Protein glycosylation involves the addition of monosaccharides in a stepwise process requiring no glycan template. Therefore, identifying the numerous glycoforms, including isomers, can help elucidate the biological function(s) of particular glycans. A method to assess the diversity of the N‐linked oligosaccharides released from human serum without derivatization has been developed using on‐line nanoLC and high resolution TOF MS. The N‐linked oligosaccharides were analyzed with MALDI FT‐ICR MS and microchip LC MS (HPLC–Chip/TOF MS). Two microfluidic chips were employed, the glycan chip (40 nL enrichment column, 43×0.075 mm² i.d. analytical column) and the high capacity chip (160 nL enrichment column, 140×0.075 mm² i.d. analytical column), both with graphitized carbon as the stationary phase. Both chips offered good sensitivity and reproducibility in separating a heterogeneous mixture of neutral and anionic oligosaccharides between injections. Increasing the length and volume of the enrichment and the analytical columns improved resolution of the peaks. Complex type N‐linked oligosaccharides were the most abundant oligosaccharides in human serum accounting for ∼96% of the total glycans identified, while hybrid and high mannose type oligosaccharides comprise the remaining ∼4%.     

An unbiased approach for analysis of protein glycosylation and application to influenza vaccine hemagglutinin

Here a mass spectrometry-based platform for the analysis of glycoproteins is presented. Glycopeptides and released glycans are analyzed, the former by quadrupole orthogonal time-of-flight liquid chromatography/mass spectrometry (QoTOF LC/MS) and the latter by permethylation analysis using matrix-assisted laser desorption/ionization (MALDI)–TOF MS. QoTOF LC/MS analysis reveals the stochastic distribution of glycoforms at occupied sequons, and the latter provides a semiquantitative assessment of overall protein glycosylation. Hydrophilic interaction chromatography (HILIC) was used for unbiased enrichment of glycopeptides and was validated using five model N-glycoproteins bearing a wide array of glycans, including high-mannose, complex, and hybrid subtypes such as sulfo and sialyl forms. Sialyl and especially sulfated glycans are difficult to analyze because these substitutions are labile. The conditions used here allow detection of these compounds quantitatively, intact, and in the context of overall glycosylation. As a test case, we analyzed influenza B/Malaysia/2506/2004 hemagglutinin, a component of the 2006–2007 influenza vaccine. It bears 11 glycosylation sites. Approximately 90% of its glycans are high mannose, and 10% are present as complex and hybrid types, including those with sulfate. The stochastic distribution of glycoforms at glycosylation sites is revealed. This platform should have wide applications to glycoproteins in basic sciences and industry because no apparent bias for any glycoforms is observed.     

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines.     

Enhanced detection of in‐gel released N‐glycans by MALDI‐TOF‐MS

Many biologically relevant glycoproteins need to be separated on 1D‐ or 2D‐gels prior to analysis and are available in picomole amounts. Therefore, it is important to have optimized methods to unravel the glycome that combine in‐gel digestions with MALDI‐TOF‐MS. In this technical report, we investigated how the detection of in‐gel released N‐glycans could be improved by MALDI‐TOF‐MS. First, an AnchorChip target was tested and compared to ground steel target using several reference oligosaccharides. The highest signals were obtained with an AnchorChip target and D‐arabinosazone as the matrix; a LOD of 1.3 to 10 fmol was attained. Then, the effect of octyl‐β‐glucopyranoside, a nonionic detergent, was studied during in‐gel peptide‐N⁴‐(acetyl‐ß‐glucosaminyl) asparagine amidase F digestion of standard glycoproteins and during glycan extraction. Octyl‐β‐glucopyranoside increased the intensity and the amount of detected neutral as well as acidic N‐glycans. A LOD of under 7 pmol glycoprotein could be achieved.     

Analysis of Site-specific Glycosylation of Renal and Hepatic γ-Glutamyl Transpeptidase from Normal Human Tissue

The cell surface glycoprotein γ-glutamyl transpeptidase (GGT) was isolated from healthy human kidney and liver to characterize its glycosylation in normal human tissue in vivo. GGT is expressed by a single cell type in the kidney. The spectrum of N-glycans released from kidney GGT constituted a subset of the N-glycans identified from renal membrane glycoproteins. Recent advances in mass spectrometry enabled us to identify the microheterogeneity and relative abundance of glycans on specific glycopeptides and revealed a broader spectrum of glycans than was observed among glycans enzymatically released from isolated GGT. A total of 36 glycan compositions, with 40 unique structures, were identified by site-specific glycan analysis. Up to 15 different glycans were observed at a single site, with site-specific variation in glycan composition. N-Glycans released from liver membrane glycoproteins included many glycans also identified in the kidney. However, analysis of hepatic GGT glycopeptides revealed 11 glycan compositions, with 12 unique structures, none of which were observed on kidney GGT. No variation in glycosylation was observed among multiple kidney and liver donors. Two glycosylation sites on renal GGT were modified exclusively by neutral glycans. In silico modeling of GGT predicts that these two glycans are located in clefts on the surface of the protein facing the cell membrane, and their synthesis may be subject to steric constraints. This is the first analysis at the level of individual glycopeptides of a human glycoprotein produced by two different tissues in vivo and provides novel insights into tissue-specific and site-specific glycosylation in normal human tissues.     

Stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG)

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling‐based quantitative targeted glycomics (i‐QTaG) technique for the comparative and quantitative analysis of total N‐glycans using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N‐glycans using a model glycoprotein (bovine fetuin). Moreover, the i‐QTaG using MALDI‐TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of ¹³C₆/¹²C₆‐2‐aminobenzoic acid‐labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N‐glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N‐glycan peaks from i‐QTaG method showed a good linearity (R² > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2‐AA labeled N‐glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up‐regulation of the Lewis antigen (～82%) in sera from prostate cancer patients. In this proof‐of‐concept study, we demonstrated that the i‐QTaG method, which enables to achieve a reliable comparative quantitation of total N‐glycans via MALDI‐TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:840–848, 2015     

Targeted serum glycoproteomics for the discovery of lung cancer‐associated glycosylation disorders using lectin‐coupled ProteinChip arrays

To screen for glycoproteins showing aberrant sialylation patterns in sera of cancer patients and apply such information for biomarker identification, we performed SELDI‐TOF MS analysis coupled with lectin‐coupled ProteinChip arrays (Jacalin or SNA) using sera obtained from lung cancer patients and control individuals. Our approach consisted of three processes (i) removal of 14 abundant proteins in serum, (ii) enrichment of glycoproteins with lectin‐coupled ProteinChip arrays, and (iii) SELDI‐TOF MS analysis with acidic glycoprotein‐compatible matrix. We identified 41 protein peaks showing significant differences (p<0.05) in the peak levels between the cancer and control groups using the Jacalin‐ and SNA‐ProteinChips. Among them, we identified loss of Neu5Ac (α2,6) Gal/GalNAc structure in apolipoprotein C‐III (apoC‐III) in cancer patients through subsequent MALDI‐QIT‐TOF MS/MS. Furthermore, subsequent validation experiments using an additional set of 60 lung adenocarcinoma patients and 30 normal controls demonstrated that there is a higher frequency of serum apoC‐III with loss of α2,6‐linkage Neu5Ac residues in lung cancer patients compared to controls. Our results have demonstrated that lectin‐coupled ProteinChip technology allows the high‐throughput and specific recognition of cancer‐associated aberrant glycosylations, and implied a possibility of its applicability to studies on other diseases.     

N-linked glycosylation profiling of pancreatic cancer serum using capillary liquid phase separation coupled with mass spectrometric analysis

Glycoproteins play important roles in various biological processes including intracellular transport, cell recognition, and cell-cell interactions. The change of the cellular glycosylation profile may have profound effects on cellular homeostasis and malignancy. Therefore, we have developed a sensitive screening approach for the comprehensive analysis of N-glycans and glycosylation sites on human serum proteins. Using this approach, N-linked glycopeptides were extracted by double lectin affinity chromatography. The glycans were enzymatically cleaved from the peptides and then profiled using capillary hydrophilic interaction liquid chromatography coupled online with ESI-TOF MS. The structures of the separated glycans were determined by MALDI quadrupole ion-trap TOF mass spectrometry in both positive and negative modes. The glycosylation sites were elucidated by sequencing of PNGase F modified glycopeptides using nanoRP-LC-ESI-MS/MS. Alterations of glycosylation were analyzed by comparing oligosaccharide expression of serum glycoproteins at different disease stages. The efficiency of this method was demonstrated by the analysis of pancreatic cancer serum compared to normal serum. Ninety-two individual glycosylation sites and 202 glycan peaks with 105 unique carbohydrate structures were identified from approximately 25 mug glycopeptides. Forty-four oligosaccharides were found to be distinct in the pancreatic cancer serum. Increased branching of N-linked oligosaccharides and increased fucosylation and sialylation were observed in samples from patients with pancreatic cancer. The methodology described in this study may elucidate novel, cancer-specific oligosaccharides and glycosylation sites, some of which may have utility as useful biomarkers of cancer.     

Intact Glycopeptide Analysis of Influenza A/H1N1/09 Neuraminidase Revealing the Effects of Host and Glycosite Location on Site‐Specific Glycan Structures

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site‐specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi‐5 cells. The data indicate that three of four potential N‐linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site‐dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.     

A novel endo‐β‐N‐acetylglucosaminidase releases specific N‐glycans depending on different reaction conditions

Milk glycoproteins are involved in different functions and contribute to different cellular processes, including adhesion and signaling, and shape the development of the infant microbiome. Methods have been developed to study the complexities of milk protein glycosylation and understand the role of N‐glycans in protein functionality. Endo‐β‐N‐acetylglucosaminidase (EndoBI‐1) isolated from Bifidobacterium longum subsp. infantis ATCC 15697 is a recently isolated heat‐stable enzyme that cleaves the N‐N′‐diacetyl chitobiose moiety found in the N‐glycan core. The effects of different processing conditions (pH, temperature, reaction time, and enzyme/protein ratio) were evaluated for their ability to change EndoBI‐1 activity on bovine colostrum whey glycoproteins using advanced mass spectrometry. This study shows that EndoBI‐1 is able to cleave a high diversity of N‐glycan structures. Nano‐LC‐Chip–Q‐TOF MS data also revealed that different reaction conditions resulted in different N‐glycan compositions released, thus modifying the relative abundance of N‐glycan types. In general, more sialylated N‐glycans were released at lower temperatures and pH values. These results demonstrated that EndoBI‐1 is able to release a wide variety of N‐glycans, whose compositions can be selectively manipulated using different processing conditions. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1323–1330, 2015     

Impact of cultivation conditions on N‐glycosylation of influenza virus a hemagglutinin produced in MDCK cell culture

Manufacturers worldwide produce influenza vaccines in different host systems. So far, either fertilized chicken eggs or mammalian cell lines are used. In all these vaccines, hemagglutinin (HA) and neuraminidase are the major components. Both are highly abundant glycoproteins in the viral envelope, and particularly HA is able to induce a strong and protective immune response. The quality characteristics of glycoproteins, such as specific activity, antigenicity, immunogenicity, binding avidity, and receptor‐binding specificity can strongly depend on changes or differences in their glycosylation pattern (potential N‐glycosylation occupancy as well as glycan composition). In this study, capillary gel electrophoresis with laser‐induced fluorescence detection (CGE‐LIF) based glycoanalysis (N‐glycan fingerprinting) was used to determine the impact of cultivation conditions on the HA N‐glycosylation pattern of Madin–Darby canine kidney (MDCK) cell‐derived influenza virus A PR/8/34 (H1N1). We found that adaptation of adherent cells to serum‐free growth has only a minor impact on the HA N‐glycosylation pattern. Only relative abundances of N‐glycan structures are affected. In contrast, host cell adaptation to serum‐free suspension growth resulted in significant changes in the HA N‐glycosylation pattern regarding the presence of specific N‐glycans as well as their abundance. Further controls such as different suppliers for influenza virus A PR/8/34 (H1N1) seed strains, different cultivation scales and vessels in standard or high cell density mode, different virus production media varying in either composition or trypsin activity, different temperatures during virus replication and finally, the impact of β‐propiolactone inactivation resulted—at best—only in minor changes in the relative N‐glycan structure abundances of the HA N‐glycosylation pattern. Surprisingly, these results demonstrate a rather stable HA N‐glycosylation pattern despite various (significant) changes in upstream processing. Only the adaptation of the production host cell line to serum‐free suspension growth significantly influenced HA N‐glycosylation regarding both, the type of attached glycan structures as well as their abundances. Biotechnol. Bioeng. 2013; 110: 1691–1703. © 2013 Wiley Periodicals, Inc.     

Carbohydrate‐binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol‐anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N‐glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin–VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high‐affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose‐specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.     

An improved protocol for N-glycosylation analysis of gel-separated sialylated glycoproteins by MALDI-TOF/TOF

Different glycoforms of some proteins have been identified as differential spots for certain diseases in 2-DE, indicating disease-related glycosylation changes. It is routine to determine the site-specific glycosylation of nonsialylated N-glycoproteins from a single gel spot, but some obstacles still exist in analyzing sialylated glycoproteins due to the lability and higher detection limit of acid glycans in MALDI-TOF/TOF analysis. Thus, we present an improved protocol here. Tryptic glycopeptides were separated and subjected to MALDI-TOF/TOF analysis, resulting in the identification of site-specific glycosylation of high-intensity glycopeptides. Sequential deglycosylation and desialylation were used to improve the identification of glycosylation sites and desialylated glycans. The site-specific glycosylation of large glycopeptides and low-intensity glycopeptides was deduced based on the masses of glycopeptides, deglycosylated peptides and desialylated glycans. By applying it to 2-DE separated human serum, the difference of N-glycosylation was successfully determined for α1-antitrypsin between different gel spots.     

Identification of Plasma Membrane Glycoproteins Specific to Human Glioblastoma Multiforme Cells Using Lectin Arrays and LC‐MS/MS

Glioblastoma, also known as glioblastoma multiforme (GBM), is the most malignant type of brain cancer and has poor prognosis with a median survival of less than one year. While the structural changes of tumor cell surface carbohydrates are known to be associated with invasive behavior of tumor cells, the cell surface glycoproteins to differentiate the low‐ and high‐grade glioma cells can be potential diagnostic markers and therapeutic targets for GBMs. In the present study, lectin arrays consisting of eight lectins were employed to explore cell surface carbohydrate expression patterns on low‐grade oligodendroglioma cells (Hs683) and GBM cells (T98G). Griffonia simplicifolia I (GS I) was found to selectively bind to T98G cells and not to Hs683 cells. For identification of the glioblastoma‐specific cell surface markers, the glycoproteins from each cell type were captured by a GS I lectin column and analyzed by LC‐MS/MS. The identified proteins from the two cell types were quantified using label‐free quantitative analysis based on spectral counting. Of cell surface glycoproteins showing significant increases in T98G cells, five proteins were selected for verification of both protein and glycosylation level changes using Western blot and GS I lectin‐based immunosorbent assay.     

Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths

Post-translational modifications such as glycosylation are important for changing the properties and functions of proteins. To analyze the importance of glycosylation during cold stress in rice, a proteomics approach was used. Proteins extracted from the basal part of rice leaf sheaths were separated by two-dimensional polyacrylamide gel electrophoresis, and subjected to lectin blot analysis using concanavalin A. From a total of 250 detected proteins, 22 reacted with the lectin, suggesting that they were N-glycosylated proteins. To determine how N-glycosylation of these proteins is affected by cold stress, rice seedlings were incubated at 5°C for 48 h, and proteins extracted from the basal parts of leaf sheaths were analyzed by the lectin blot assay. Cold stress changed the reactivity toward the lectin for 12 of the 22 glycoproteins. The identity of the 12 proteins was determined by protein sequencing and mass spectrometry with the majority of these glycoproteins being categorized as involved in energy production. Furthermore, calreticulin, one of the 12 glycoproteins, was also phosphorylated as a result of cold stress. These results indicate that cold stress of the basal parts of rice leaf sheaths changes the glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates the quality control of other proteins.     

BGAL1 depletion boosts the level of β‐galactosylation of N‐ and O‐glycans in N. benthamiana

Glyco‐design of proteins is a powerful tool in fundamental studies of structure–function relationship and in obtaining profiles optimized for efficacy of therapeutic glycoproteins. Plants, particularly Nicotiana benthamiana, are attractive hosts to produce recombinant glycoproteins, and recent advances in glyco‐engineering facilitate customized N‐glycosylation of plant‐derived glycoproteins. However, with exception of monoclonal antibodies, homogenous human‐like β1,4‐galactosylation is very hard to achieve in recombinant glycoproteins. Despite significant efforts to optimize the expression of β1,4‐galactosyltransferase, many plant‐derived glycoproteins still exhibit incomplete processed N‐glycans with heterogeneous terminal galactosylation. The most obvious suspects to be involved in trimming terminal galactose residues are β‐galactosidases (BGALs) from the glycosyl hydrolase family GH35. To elucidate the so far uncharacterized mechanisms leading to the trimming of terminal galactose residues from glycans of secreted proteins, we studied a N. benthamiana BGAL known to be active in the apoplast (NbBGAL1). Here, we determined the NbBGAL1 subcellular localization, substrate specificity and in planta biological activity. We show that NbBGAL1 can remove β1,4‐ and β1,3‐galactose residues on both N‐ and O‐glycans. Transient BGAL1 down‐regulation by RNA interference (RNAi) and BGAL1 depletion by genome editing drastically reduce β‐galactosidase activity in N. benthamiana and increase the amounts of fully galactosylated complex N‐glycans on several plant‐produced glycoproteins. Altogether, our data demonstrate that NbBGAL1 acts on galactosylated complex N‐glycans of plant‐produced glycoproteins.     

Comparison of sialylated N‐glycopeptide levels in serum of pancreatic cancer patients,acute pancreatitis patients,and healthy controls

Serum protein glycosylation is known to be affected by pathological conditions, including cancer and inflammatory diseases. Pancreatic cancer patients would benefit from early diagnosis, as the disease is often detected in an advanced stage and has poor prognosis. Searching for changes in serum protein site‐specific glycosylation could reveal novel glycoprotein biomarkers. We used Sambucus nigra lectin affinity chromatography to enrich α‐2,6 sialylated tryptic N‐glycopeptides from albumin‐depleted sera of pancreatic cancer patients, acute pancreatitis patients, and healthy individuals, and compared their relative abundance using ultra performance LC‐MS. Relative quantitation was done using the spectrum processing software MZmine. Identification was performed on the web‐based tool GlycopeptideID, developed for in silico analysis of intact N‐glycopeptides. Seventeen high‐abundance serum proteins, mainly acute‐phase proteins, and immunoglobulins, with total 27 N‐glycosylation sites, and 62 glycoforms, were identified. Pancreatitis patient sera contained 38, and pancreatic cancer patients sera contained 13 glycoform changes with statistical significance (p < 0.05). In pancreatitis, up to tenfold changes were found in some glycoforms, and in pancreatic cancer, threefold. Analysis showed that the changes often concerned one or two, but not all, N‐glycosylation sites in a specific glycoprotein. In conclusion, the analysis shows that pancreatic cancer, and acute pancreatitis are associated with changes in concentrations of intact sialylated N‐glycopeptides derived from acute‐phase proteins, and immunoglobulins, and that changes are site specific.     

Automated measurement of site‐specific N‐glycosylation occupancy with SWATH‐MS

Asparagine‐linked glycosylation is a common post‐translational modification of proteins catalyzed by oligosaccharyltransferase that is important in regulating many aspects of protein function. Analysis of protein glycosylation, including glycoproteomic measurement of the site‐specific extent of glycosylation, remains challenging. Here, we developed methods combining enzymatic deglycosylation and protease digestion with SWATH‐MS to enable automated measurement of site‐specific occupancy at many glycosylation sites. Deglycosylation with peptide‐endoglycosidase H, leaving a remnant N‐acetylglucosamine on asparagines previously carrying high‐mannose glycans, followed by trypsin digestion allowed robust automated measurement of occupancy at many sites. Combining deglycosylation with the more general peptide‐N‐glycosidase F enzyme with AspN protease digest allowed robust automated differentiation of nonglycosylated and deglycosylated forms of a given glycosylation site. Ratiometric analysis of deglycosylated peptides and the total intensities of all peptides from the corresponding proteins allowed relative quantification of site‐specific glycosylation occupancy between yeast strains with various isoforms of oligosaccharyltransferase. This approach also allowed robust measurement of glycosylation sites in human salivary glycoproteins. This method for automated relative quantification of site‐specific glycosylation occupancy will be a useful tool for research with model systems and clinical samples.