Article pubs.acs.org/jpr
Clusterin Glycopeptide Variant Characterization Reveals Significant Site-Specific Glycan Changes in the Plasma of Clear Cell Renal Cell Carcinoma Francisca O. Gbormittah,† Jonathan Bones,§ Marina Hincapie,∥ Fateme Tousi,† William S. Hancock,*,† and Othon Iliopoulos*,⊥,# †
Barnett Institute and Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115, United States § NIBRT−The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin, Ireland ∥ Genzyme, a Sanofi Company, 45 New York Avenue, Framingham, Massachusetts 01701, United States ⊥ Center for Cancer Research at Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts 02129, United States # Division of Hematology−Oncology, Department of Medicine, Massachusetts General Hospital, Boston, Massachusetts 02114, United States S Supporting Information *
ABSTRACT: Cancer-related alterations in protein glycosylation may serve as diagnostic or prognostic biomarkers or may be used for monitoring disease progression. Clusterin is a medium abundance, yet heavily glycosylated, glycoprotein that is upregulated in clear cell renal cell carcinoma (ccRCC) tumors. We recently reported that the N-glycan profile of clusterin is altered in the plasma of ccRCC patients. Here, we characterized the occupancy and the degree of heterogeneity of individual Nglycosylation sites of clusterin in the plasma of patients diagnosed with localized ccRCC, before and after curative nephrectomy (n = 40). To this end, we used tandem mass spectrometry of immunoaffinity-enriched plasma samples to analyze the individual glycosylation sites in clusterin. We determined the levels of targeted clusterin glycoforms containing either a biantennary digalactosylated disialylated (A2G2S2) glycan or a core fucosylated biantennary digalactosylated disialylated (FA2G2S2) glycan at N-glycosite N374. We showed that the presence of these two clusterin glycoforms differed significantly in the plasma of patients prior to and after curative nephrectomy for localized ccRCC. Removal of ccRCC led to a significant increase in the levels of both FA2G2S2 and A2G2S2 glycans in plasma clusterin. These changes were further confirmed by lectin blotting of plasma clusterin. It is envisioned that these identified glycan alterations may provide an additional level of therapeutic or biomarker sensitivity than levels currently achievable by monitoring expression differences alone. KEYWORDS: Clear cell renal cell carcinoma, clusterin, site-specific analysis, glycoforms, collision-induced dissociation, N-glycan recurrence, and metastatic disease is currently incurable.1,2 Therefore, there is an urgent need to identify plasma-based markers that can be used for early detection and/or prediction of disease recurrence.
1. INTRODUCTION Clear cell renal cell carcinoma (ccRCC) accounts for approximately 75% of sporadic renal cancer. In addition, patients with Von Hippel−Lindau (VHL) disease develop multiple renal cancers exclusively of clear cell histology (ccRCC). If the disease is detected at early stages, then it can be cured by surgery. Locally advanced disease has a great risk of © 2015 American Chemical Society
Received: October 25, 2014 Published: April 8, 2015 2425
DOI: 10.1021/pr501104j J. Proteome Res. 2015, 14, 2425−2436
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Journal of Proteome Research
plasma clusterin of patients prior to RCC(+) and after RCC(−) curative nephrectomy for localized ccRCC. In this article, we validate and extend our earlier observation on glycan changes that occur in the plasma clusterin of ccRCC patients. We mapped the individual N-glycosylation sites of plasma clusterin and detected site-specific changes linked to the presence of ccRCC, using liquid chromatography followed by tandem mass spectrometry. To obtain glycan structural information, we utilized collision-induced dissociation (CID) MS/MS (MS2) fragmentation, and we verified the sequence of each glycopeptide by CID-MS3 fragmentation. We showed that an increase in the levels of both core fucosylated biantennary digalactosyl disialylated (FA2G2S2) and biantennary digalactosyl disialylated (A2G2S2) glycans best discriminates between RCC(+) and RCC(−) plasma samples.
Protein glycosylation is the most common post-translational protein modification (PTM), and aberrant protein glycosylation has been shown to be associated with several malignancies.3−6 Glycoprotein-based biomarkers have become widely used in the clinical setting;7−9 for example, monosialylated alpha-fetoprotein (AFP) and carbohydrate antigen 19-9 (CA 19-9) are currently used as biomarkers for hepatocellular carcinoma and stomach cancer, respectively. Although these markers are widely used, they have low sensitivity and/or specificity; their clinical application, therefore, requires the combination of these biomarkers with additional ones in order to create a panel with an optimal receiver operating curve (ROC).10,11 A major challenge in glycoproteomic studies is the low concentration of the potentially relevant candidate glycoprotein marker(s) in the blood. In addition, the attachment of glycans in multiple N-glycan sites of a protein (macroheterogeneity) and the variable number and levels of N-glycans at one or more occupancy sites (microheterogeneity) further complicate glycoproteomic investigation. The most common approaches for undertaking Nglycosylation studies include either oligosaccharide profiling or glycopeptides analysis. In oligosaccharide profiling, total Nglycans are released from a single purified/enriched glycoprotein, typically using PNGaseF treatment followed by either labeled or unlabeled chromatographic and/or MS analysis. In glycopeptide analysis, a single glycoprotein or multiple proteins is digested, and the resulting glycopeptides are subsequently enriched followed by LC−MS analysis.12−15 While glycan structural information is obtained using the oligosaccharide profiling approach, no knowledge about glycan site attachment can be derived. In contrast, a glycopeptide-focused method may provide information regarding both the glycan structure and the site of glycan attachment.16−18 A limitation to this latter approach, however, is that, due to inherent analytical complexity, data analysis is often time-consuming and tedious, especially when several glycopeptides with multiple glycan attachment sites are involved. Tandem mass spectrometry analysis of glycopeptides, combined with PNGaseF-assisted glycan release, provides information on direct, site-specific oligosaccharide heterogeneity of glycoproteins19 and glycopeptide sequence information. Thus, this approach allows for site-specific glycoform comparison between clinically relevant specimens and corresponding controls. Observations of site-specific glycosylation changes may enhance our understanding of glycoprotein function and improve glycan detection specificity for therapeutic targets, as previously noted.20−26 The significance of clusterin differential expression across many cancers, especially breast, prostate, ovarian, and renal cell carcinoma, has been reported previously.27−32 In ccRCC, loss of the VHL tumor suppressor gene has been associated with a hypoxia-inducible factor (HIF)-independent upregulation of clusterin in tumor samples.33 Preliminary evidence suggests that strong expression of clusterin in surgically removed ccRCC tissue may correlate with shorter recurrence-free survival.34 In contrast to studies examining the expression of clusterin in surgically removed ccRCC tumors, few studies have addressed the importance of circulation clusterin in ccRCC detection or in monitoring disease progression. Our previous work examined plasma clusterin glycosylation in ccRCC.35 We profiled total N-glycans released from immunoaffinity-enriched plasma clusterin and observed glycosylation changes in the
2. MATERIALS AND METHODS 2.1. Materials
Capture select clusterin resin and Capture select Protein G were provided by BAC B. V. (Netherlands) and Life Technologies, Inc. (Carlsbad, CA), respectively. Human clusterin ELISA kit was purchased from R&D systems, Inc. (Minneapolis, MN). POROUS beads for conjugation were purchased from Applied Biosystems (Framingham, MA). Sequencing-grade trypsin and Glu-C endopeptidase were purchased from Promega (Madison, WI). All lectins used throughout this study were obtained from Vector Laboratories (Burlingame, CA). HPLC-grade water, acetonitrile, and all other buffer reagents were purchased from Sigma-Aldrich (St. Louis, MO). 2.2. Clear Cell Renal Cell Carcinoma (ccRCC) Plasma Sample Collection and Preparation
Plasma samples from 20 ccRCC patients both before (RCC(+)) and after (RCC(−)) nephrectomy (±RCC, n = 40) were collected at Massachusetts General Hospital (MGH) (Boston, MA). Patients provided informed consent to the corresponding Institutional Review Board (IRB) approved protocol. Pathology reports after nephrectomy were used to verify the diagnosis of ccRCC for each patient. A summary of the ccRCC plasma samples used for this study is presented in Table 1. Immediately after plasma collection, samples were aliquoted and frozen at −80 °C until further analysis. To ensure consistency, each sample was not thawed more than twice. 2.3. Clusterin Immunoaffinity HPLC Purification
Clusterin glycoprotein was isolated from plasma samples through immunoaffinity antibody capture. Immunoaffinity HPLC purification of clusterin consisted of three PEEK columns packed in-house using high liquid pressure as reported earlier.35 Prior to clusterin purification, the concentration of clusterin in each sample was determined using a specific clusterin ELISA assay for each of the RCC(+) and RCC(−) plasma samples. Fifty microliters of each (before nephrectomy, n = 20; after nephrectomy, n = 20) ccRCC plasma sample was centrifuged at 8000g to precipitate mucins and other particulates. Purification of clusterin from plasma samples was performed using a semiautomated three-column multidimensional platform, as previously described. Briefly, we used a 44 min online 2D HPLC platform, which combines albumin and IgG depletion using CaptureSelect HSA and Protein G ligands immobilized on POROS chromatographic media, followed by immunocapture of clusterin on an agarose bead functionalized 2426
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Journal of Proteome Research Table 1. Patient Information of RCC Plasma Samplesa patient ID
protocol no.
sex
38 46 48 50 53 62 64 75 88 108 116 124 135 170 192 223 235 251 253 261
01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-130 01-131 01-132 01-130 01-130 01-130 01-130 01-130 01-130
female female male female male male female female female male male male female male female male female male male female
a
tumor size 8.5 5.5 7.2 6.3 8.0 7.5 8.2 8.5 3.5 6.0 7.5 8.5 5.5 6.2 8.0 7.0 4.5 5.2 4.7 8.2
× × × × × × × × × × × × × × × × × × × ×
6.3 5.0 4.0 5.0 7.0 7.5 8.0 7.5 3.5 4.0 4.5 6.0 4.0 5.0 4.5 6.5 4.5 5.0 4.2 7.5
× × × × × × × × × × × × × × × × × × × ×
6.0 5.0 3.0 4.5 6.0 6.0 7.2 7.0 3.5 3.5 3.0 5.5 3.0 3.5 4.0 5.0 3.0 4.5 4.0 6.0
an alternating fashion. After destaining, 10 mM (dithiothreitol) DTT for disulfide bond reduction was added to gel pieces and incubated for 45 min at room temperature followed by 1 h alkylation using 50 mM iodoacetamide (IAA) at room temperature in the dark. Gel pieces were washed three times with 0.1 M NH4HCO3, pH 7.6, briefly washed with acetonitrile, and reduced to dryness by vacuum centrifugation. Trypsin and Glu-C (12.5 ng/μL) or PNGase F (1unit per 25 μL) enzymes were prepared separately in 50 mM NH4HCO3, pH 7.6, and added to gel pieces for a 12 h incubation at 37 °C. Glycosylated peptides (Trypsin and/or Glu-C digest) were extracted following incubation with 5% v/v formic acid/50% v/v acetonitrile twice, whereas deglycosylated peptides (PNGase F + Trypsin and/or Glu-C peptides) were extracted with water and 5% v/v formic acid/50% v/v acetonitrile. Extracted peptides were dried completely by vacuum centrifugation and reconstituted in 20 μL of 0.1% formic acid in water prior to LC−MS/MS analysis.
clinical diagnosisb ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC ccRCC
2.6. C18 Reversed-Phase Nano-LC−MS/MS Analysis
Reconstituted glycosylated and deglycosylated peptides were loaded onto a C18 reverse-phase capillary column (150 mm × 75 mm i.d.) (New Objective, Woburn, MA) self-packed with 5 μm, 300 Å C18 silica bonded stationary phase (MICHROM Bioresources, Inc., Auburn, CA). Liquid chromatography separation was performed using a Dionex Ultimate 3000 HPLC system (LC Packings-Dionex, Marlton, NJ) at a constant flow rate of 200 nL/min. Mobile phase A was 0.1% v/v formic acid in HPLC-grade water, and mobile phase B was 0.1% v/v formic acid in acetonitrile using the following gradient: 15 min at 2% mobile phase B for sample loading, a linear increase from 5% mobile phase B to 40% mobile phase B in 60 min, 40% mobile phase B to 80% mobile phase B in 15 min, and lastly 80% mobile phase B to 2% mobile phase B in 10 min. All mass spectrometry experiments were performed on an LTQ-Orbitrap Elite instrument (Thermo Fisher Scientific, San Jose, CA). The mass spectrometer was operated in datadependent fashion with an automatic switch between the full MS survey (scan 1) acquired over the range of 400−2000 m/z, followed by CID-MS/MS fragmentation on the six most abundant precursor ions. The mass spectrometer was operated using the following parameters: precursor ion isolation width of 3 Th, mass resolution at 60 000 for 400 m/z, 35% normalized collision energy, 2.1 kV spray voltage, and capillary temperature of 210 °C. Dynamic exclusion was activated with the following parameters: 1 repeat count, repeat duration of 30 s, exclusion list size of 100, exclusion duration of 45 s, and exclusion mass width of 1.0 m/z low and 1.50 m/z high. The instrument was calibrated and tuned with Thermo Scientific calibration mix.
Average age of 52 years. bccRCC, clear cell renal cell carcinoma.
with an anti-clusterin camelid single-chain antibody. Enriched clusterin fractions were desalted using an R1 reversed-phase column, and the concentration of enriched clusterin was determined using a BCA assay (Thermo Fischer Scientific, San Jose, CA) following the manufacturer’s instructions. 2.4. Lectin Blot Assay of Purified Clusterin
In order to verify the identified clusterin fucosylation changes between plasma samples obtained before and after nephrectomy, we performed lectin blot assays using biotinylated Aleuria aurantia lectin (AAL) (Vector Laboratories, Burlingame, CA). Purified clusterin (1 μg) was loaded on a 10% Mini-PROTEAN TGX Tris/Glycine SDS precast gel (Bio-Rad Laboratories, Hercules, CA). Following completion of the electrophoretic run, proteins were transferred to 0.2 μm nitrocellulose miniformat using a Bio-Rad transfer-blot turbo transfer system at 2.5 A constant voltage for 3 min. The nitrocellulose membrane was blocked in carbo-free protein-based blocking solution (Vector Laboratories, Inc. Burlingame, CA) for 1 h at room temperature. After three washes in Tris-buffered saline containing 0.5% Tween-20 (TBST), the membrane was incubated with the biotinylated lectin (AAL) at a concentration of 1 μg/mL for 1 h, followed by three washes with TBST. The membrane was then incubated for 1 h in 1 μg/mL streptavidin−HRP (Vector Laboratories, Burlingame, CA). Lectin blots were visualized using ECL western blotting reagents (GE Healthcare), and images were captured with a Fluorchem SP system (Alpha Innotech, Santa Clara, CA).
2.7. Data and Statistical Analysis
Gel loading amounts of clusterin were normalized based upon the ELISA quantification data. Lectin blot densities of RCC(+) and RCC(−) purified clusterin samples were evaluated using ImageJ, version 1.47 (http://rsbweb.nih.gov/ij/download. html). Experimental deglycosylated peptides and glycosylated peptides were identified by searching LC−MS/MS raw data with a combined clusterin sequence (SwissProt P10909) and annotated human database (release 2012_1) using the SEQUEST algorithm (Thermo Electron Corp., San Jose, CA) incorporated within the Thermo Fisher Proteome Discoverer 1.3 suite. Acceptance criteria for peptide identifications were as follows: singly, doubly, and triply charged peptides were accepted for identification if Xcorr values were ≥1.9, 2.5, and
2.5. 1DE SDS-PAGE and Enzymatic Digestion
Ten micrograms of purified clusterin from each sample was loaded on a 10% Bis-Tris SDS-PAGE gel (Novex NuPAGE, Life Technologies) and separated for 45 min at 200 V. Resulting protein bands were visualized with SimplyBlue safe stain Coomassie G-250 stain (Life Technologies, Grand Island, NY). Gel bands corresponding to clusterin (based on western blot verification) were excised and cut into ∼1 mm2 pieces followed by destaining with 500 μL of 0.1 M ammonium bicarbonate (NH4HCO3), pH 7.6, and acetonitrile (ACN) in 2427
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Table 2. Enzymatic Glycopeptides and Nonglycopeptides Identified after PNGase-F Treatment by Nano-LC−MS/MS Analysis
* N-Linked glycopeptides not identified in replicate sample analysis; N/D designates the conversion of glycosylated peptides to deglycosylated peptides (i.e., the conversion of asparagine (N) to aspartic acid (D) during the process of deglycosylation). ‡Carbarmidomethylatedcysteine.
3.8, respectively; delta CN value (ΔCN) > 0.1; and peptide probability score > 0.95. Other search parameters were as follows: carbamidomethylation (C) as fixed modification, deamidation (N) as variable modification, full trypsin as the enzyme specificity, maximum 2 missed cleavages allowed, precursor ion mass tolerance of 5 ppm, and fragment ion mass tolerance of 0.8 Da. Glycan site occupancy was determined by the combination of automatic database search followed by peak area analysis of the extracted ion chromatogram (EIC) of deglycosylated and glycosylated peptides. CID-MS2 and CIDMS3 fragmentation patterns allowed for manual annotation, characterization, and relative quantitation of glycoforms. GlycoWorkbench,36 version 1.1.3480, was used for glycan structure generation and evaluation of the MS/MS fragmentation of glycan structures. Data analysis and graphic generation were performed using Microsoft Excel 2010. A p value < 0.05 was considered to be statistically significant in all statistical analyses.
biantennary digalactosyl disialylated glycan (A2G2S2) were observed in RCC(−) samples. 3.1. Development of the Analytical Approach
Reversed-phase nanoliquid chromatography mass spectrometry analysis of glycopeptides is challenging due to the hydrophilic nature of glycopeptides and the associated structural complexity imparted by variability in the glycan structures present.38 Glycopeptides, unlike unmodified peptides, generally do not ionize well with electrospray, and their signals are easily suppressed by coeluting nonglycosylated peptides.39 It is therefore important to use an analytical approach that completely isolates the glycoprotein under study in order to facilitate and improve glycopeptide characterization. The differences in ionization of glycosylated and nonglycosylated peptides have been described previously by Stavenhagen et al.,40 highlighting the do’s and do nots of this application. To this end, we first determined the total clusterin concentration in ccRCC plasma (before and after nephrectomy) by an ELISA assay (Supporting Information Figure S1). An average concentration of 290 ± 5 μg/mL was observed. Importantly, there was no significant difference in total plasma clusterin concentration in this cohort of patients prior to and after nephrectomy (Supporting Information Figure S1). This finding highlights the fact that measurement of total clusterin cannot inform on the presence of ccRCC. In contrast, glycoproteomic assays designed to capture specific glycoforms of clusterin may contribute to ccRCC disease monitoring. Below, we describe changes in clusterin glycosylation that may inform such assays. We utilized a semiautomated immunoaffinity purification platform to generate clusterin-enriched extracts from patient plasma. To evaluate the performance and efficiency of this platform, we analyzed pooled reference female plasma purchased from Bioreclamation (Jericho, NY) at different loading amounts (50−100 μL). Testing the loading capacity helped to ensure minimal sample loss and minimal sample-tosample carryover during each clusterin purification process. After SDS-PAGE analysis, 50 μL of ccRCC plasma, corresponding to 14 ± 2 μg of clusterin as estimated based on ELISA, was established to be the optimal loading amount,
3. RESULTS AND DISCUSSION Clusterin is a heavily glycosylated protein (approximately 30% w/w carbohydrate), harboring seven potential N-linked glycosylation sites.37 Since glycosylation alterations are directly linked to several disease states, understanding the glycan status may serve as a biomarker for the specific disease. Our previous work characterized the overall glycan modifications of clusterin isolated from patient samples prior to and after curative nephrectomy. In the current study, we employed nano-LC−MS/MS, integrated with lectin blotting, to investigate and quantify site-specific changes of previously observed clusterin glycans. 1DE SDS-PAGE gel loading of purified clusterin was estimated based upon the clusterin concentration determined by ELISA in RCC(+) and RCC(−) plasma samples. Sialylated bi- and triantennary glycans (monosialo, disialo, and trisialo), with or without fucose, were the major glycan types identified. A significant increase in the levels of a core fucosylated biantennary digalactosyl disialylated glycan (FA2G2S2) and a 2428
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Journal of Proteome Research and the purity of isolated clusterin was estimated to be 70% (data not shown). The final step in the purification process was achieved by 1DE SDS-PAGE of the affinity chromatographypurified sample in which some higher and lower MW impurities were removed (data not shown). Proteomic analysis of the major band that contained clusterin, as identified by the expected MW and western blotting, showed that the isolated clusterin displayed a 95% purity. We did identify small amounts of glycoproteins, such as apolipoprotein E and complement C3, but at levels that would not significantly affect our glycopeptide analysis. The effect of these low-level contaminants was further mitigated by the analysis of the glycan attached to the given clusterin peptide and the ability to assess purity of a selected precursor ion by MS/MS analysis. A successful proteolytic digestion strategy depends on the glycoprotein under study. In some instances, trypsin is sufficient for complete digestion; however, some glycoproteins require a combination of proteolytic enzymes for efficient digestion.41 In a preliminary study, we investigated the efficiency of two proteolytic enzymes, trypsin and Glu-C, for the digestion of purified clusterin treated with PNGase-F (before or after protease digestion) to select the appropriate enzyme for this study. Trypsin digestion on its own generated three glycopeptides (30% overall sequence coverage). Similarly, Glu-C digestion on its own generated two glycopeptides (17% overall sequence coverage). Combined digestion with Glu-C and trypsin generated five potential glycopeptides (and >80% overall sequence coverage). A detailed summary of identified glycopeptides using an optimized protein digestion procedure involving combination of Glu-C and trypsin enzymes is presented in Table 2. Unfortunately, neither the aspartic acid form (after PNGase F digestion) nor the asparagine (N) form of N-linked potential glycopeptides sites 103 and 145 was identified in replicate samples (Table 2). The reason for the lack of detection is unclear but could be related to the length and number of hydrophobic residues in these glycopeptides (ELPGVCNETMMALWEECKPCLK, 2696.1786; QLEEFLNQSSPFYFWMNGDRIDSLLE, 3178.4825) as well as Glu-C enzyme specificity, which may be responsible for low recovery during LC−MS analysis. CID tandem mass spectrometry has been applied successfully to the analysis of glycopeptides, although measurement of low levels of glycopeptides still remains a challenge. In this study, we used both data-dependent CID LC−MS analysis and targeted CID LC−MS for the characterization of clusterin glycopeptides (Supporting Information Figure S2). Datadependent analysis of PNGase-F treated and untreated samples produced a candidate list of glycopeptides, glycan site occupancy, and glycoforms, all of which are available for further analysis. In order to characterize and quantify specific glycan structures and their corresponding glycopeptide backbones, we used CID-MS2 and -MS3 fragmentation. This targeted approach improved our ability to quantify specific glycopeptides and glycoforms (i.e., same peptide with different glycans) present in low levels, such as fucosylated glycans.
with slight modifications, as presented in Supporting Information Figure S2. Briefly, we perform a database search using SEQUEST algorithm against clusterin sequence (UniProt P10909) on PNGase-F treated and untreated mass spectrometry data, with Asn (N) deamidation set as variable modification. Deamidation at N residues that may occur as a result of sample manipulation was first evaluated in PNGase-F untreated samples from database search results. No observations of induced deamidation (N → D) were found on peptides identified with an NXS/T sequon after this initial database search; therefore, mass defect resulting from PNGase-F treatment (N → D) (deglycosylation) was used for initial screening to identify glycopeptides in samples treated with PNGase-F. In addition, in order to locate glycopeptides’ elution periods, we extracted glycan oxonium ions with m/z 366, 528, and 657 from data-dependent CID MS/MS spectra of samples that had not been subjected to PNGase-F treatment. Glycopeptides with partial glycan occupancy were observed as both the nonglycosylated peptides (Asn) and the corresponding Asp-containing peptides after PNGase-F treatment. Estimation of the degree of occupancy at each previously identified specific peptide site was based on peak areas of the extracted ion chromatogram for deglycosylated and nonglycosylated peptides (Supporting Information Figure S3), based on the assumption of similar MS response factors for the glycosylated versus deglycosylated peptides, as previously described.26 Of the seven potential glycosylation sites, five sites were observed and validated in replicate samples: three were fully glycosylated, one was partially glycosylated, and one was not glycosylated (Table 2). N-Linked sites 86, 291, and 374 were found to be fully occupied, showing 92, 90, and 96% occupancy, respectively, whereas N-linked site 354 exhibited partial glycosylation with 75% glycan occupancy. An average variation of
