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. 2024 Mar 21;96(13):5086–5094. doi: 10.1021/acs.analchem.3c04045

Mass Spectrometry Analysis of Glycopeptides Enriched by Anion Exchange-Mediated Methods Reveals PolyLacNAc-Extended N-Glycans in Integrins and Tetraspanins in Melanoma Cells

Tomislav Čaval 1, Gege Xu 1, Maryam Baniasad 1, Chih-Wei Chu 1, Rachel Rice 1, Itati Hundal 1, Gregg Czerwieniec 1, Flavio Schwarz 1,*
PMCID: PMC10993200  PMID: 38513651

Abstract

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Glycosylation is a key modulator of the functional state of proteins. Recent developments in large-scale analysis of intact glycopeptides have enabled the identification of numerous glycan structures that are relevant in pathophysiological processes. However, one motif found in N-glycans, poly-N-acetyllactosamine (polyLacNAc), still poses a substantial challenge to mass spectrometry-based glycoproteomic analysis due to its relatively low abundance and large size. In this work, we developed approaches for the systematic mapping of polyLacNAc-elongated N-glycans in melanoma cells. We first evaluated five anion exchange-based matrices for enriching intact glycopeptides and selected two materials that provided better overall enrichment efficiency. We then tested the robustness of the methodology by quantifying polyLacNAc-containing glycopeptides as well as changes in protein fucosylation and sialylation. Finally, we applied the optimal enrichment methods to discover glycopeptides containing polyLacNAc motifs in melanoma cells and found that integrins and tetraspanins are substantially modified with these structures. This study demonstrates the feasibility of glycoproteomic approaches for identification of glycoproteins with polyLacNAc motifs.

Introduction

Glycosylation is a common post-translational modification of proteins, and it impacts their polarity, solubility, structure, cellular localization, and interactions with other molecules.1,2 Thus, it is unsurprising that changes in glycosylation coincide with many diseases and aberrant glycosylation is implicated in cancer development and progression.3−6 In addition, glycoproteins have a long-standing recognition as cancer biomarkers.7−9 Poly-N-acetyllactosamine (polyLacNAc) is a linear carbohydrate polymer composed of alternating N-acetylglucosamine and galactose residues that is found mainly on tri- and tetra-antennary N-glycans and preferentially on the branch initiated by the β1–6 N-acetylglucosaminyltransferase-V encoded by the Mgat5 gene.10,11 PolyLacNAc contributes to cancer resistance against T cell killing12 and increases the stability of the PD-L1 protein at the cell surface, leading to an enhanced interaction with the PD-1 receptor and reduced cytotoxic T cell responses in cancer.13 PolyLacNAc may also exhibit context-dependent roles in cancer cells: the presence of sialyl Lewis X on polyLacNAc-elongated glycans may lead to metastasis, while the same epitope on truncated N-glycans results in the death of tumor cells in lung blood vessels.14 Despite the wealth of data connecting this glycan modification with disease, detection of polyLacNAc is complicated by its low abundance, high structural heterogeneity, and chemical–physical properties.15,16 These observations inspired us to develop a mass spectrometry (MS)-based methodology for the identification of intact glycopeptides containing polyLacNAc-elongated N-glycans in cells in a systematic manner.

Here, we first compared five anion exchange materials for enrichment of polyLacNAc-containing glycopeptides and selected the two methods that provided the overall best performance. We further tested the workflow by analyzing glycoproteins derived from cells grown in the presence of a fucosylation inhibitor or incubated with a sialidase to remove sialic acid. Next, we applied the newly developed method to malignant melanoma cells overexpressing or lacking B3GNT2, a key enzyme involved in the polyLacNAc synthesis.12 We discovered numerous proteins modified with LacNAc motifs of various lengths. Integrins, which sustain important interactions of cells with the extracellular matrix (ECM), were found to be enriched with polyLacNAc content. In addition, we found extensive polyLacNAc modification in members of the tetraspanin family that are involved in the organization of the cell membrane and modulate receptor clustering and dynamics.

Experimental Section

Cell Culture and Reagents

The A375 malignant melanoma cell line was purchased from Ubigene (Brooklyn, NY). B3GNT2 knockout and B3GNT2 overexpressing cell lines were generated by Ubigene via CRISPR-based deletion and lentivirus-based integration, respectively. Cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific) supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were maintained in a humidified incubator at 37 °C with 5% CO2. For inhibition of fucosylation, cells were cultured in medium supplemented with 0.3 mM 2-fluoro-peracetyl-fucose (2FPF; Cayman Chemicals) for 3 days.

Sample Preparation for MS Analysis

Eight million cells were washed five times with cold Dulbecco’s phosphate-buffered saline (DPBS; Gibco) and lysed with 500 μL of lysis buffer containing 6 M urea (Sigma-Aldrich), 100 mM Tris pH 8.5 (Sigma-Aldrich), 1% Triton (Sigma-Aldrich), 5 mM tris(2-carboxyethyl)phosphine (TCEP; Sigma-Aldrich), 30 mM chloroacetamide (CAA; Sigma-Aldrich), and cOmplete mini EDTA-free (Roche; 1 tablet/10 mL buffer). Cells were disrupted by sonication for 45 cycles (30 s on and 30 s off) using a Bioruptor Plus sonicator (Diagenode). Benzonase (Merck Millipore) was then added in a 1% (v/v) ratio. Cellular debris was removed by spinning the samples at 20,000g for 1 h at 4 °C, and the supernatant was subjected to methanol/chloroform precipitation by mixing 1 volume of the cell lysate supernatant with 4 volumes of methanol (Sigma-Aldrich), 1 volume of chloroform (Sigma-Aldrich), and 3 volumes of ultrapure water. The mixture was spun for 10 min at 4000g at room temperature (RT). After removal of the upper layer, 3 volumes of methanol were added, followed by a 10 min centrifugation at 4000g at RT. Following the centrifugation step, the solvent was removed and the pellet was air-dried for 10 min and then resuspended in 500 uL of digestion buffer containing 100 mM Tris-HCl pH 8.5, 5 mM TCEP, 30 mM CAA, and 0.1% RapiGest (Waters). Samples were digested with 20 ug of trypsin/Lys-C mix (Promega) overnight at 37 °C. Afterward, RapiGest was precipitated by acidification with 0.5% (v/v) trifluoroacetic acid (TFA) and the samples were passed through a Sep-Pak C18 cm3 Vac cartridge (Waters), eluted with a 50% ACN 0.1% FA solution, and subjected to glycopeptide enrichment.

For sialidase treatment, desalted samples were treated with 0.1 U (10 μL) of Arthrobacter ureafaciens sialidase (Roche) in PBS at 37 °C for 2 h. Samples were then desalted and subjected to a glycopeptide enrichment. Control samples for the sialidase experiment underwent the same process, except that they were incubated with 10 μL of PBS instead of sialidase.

To obtain plasma and crude membrane fractions, 15 million cells were processed with a Minute Plasma Membrane Protein Isolation and Cell Fractionation kit (Invent Biotechnologies, Minnesota, USA) according to the manufacturer instructions. The purified crude membrane and membrane fraction pellets were resuspended in digestion buffer (as above), and the peptides were desalted with Pierce peptide desalting spin columns (Thermo Fisher). The eluates were directly used for glycopeptide enrichment.

Glycopeptide Enrichment

Oasis MAX (Waters), SOLA SAX (Thermo Fisher), HyperSep Retain AX (Thermo Fisher), HyperSep SAX (Thermo Fisher), and AttractSPE SAX (Affinisep USA) were equilibrated by sequential addition of 1 mL of acetonitrile (ACN), 1 mL of 100 mM triethylammonium acetate, three 1 mL volumes of deionized water, and 1 mL of 95% ACN 1% TFA solution. The columns were then filled with 1 mL of 95% ACN and 1% TFA and topped up with 250 μL of desalted samples, corresponding to 300 μg of peptides and glycopeptides. Columns were then washed three times with 1 mL of a 95% ACN 1% TFA solution. Glycopeptides were eluted with 600 μL of 50% ACN 0.1% TFA solution, dried, and stored at −80 °C until MS analysis.

For HILIC enrichment, the iSPE-HILIC1 cc cartridge (Hilicon) was washed three times with 1 mL of 1% TFA solution, followed by addition of three 1 mL volumes of 80% ACN 1% TFA. One milligram of peptides and glycopeptides was added to 80% ACN and 1% TFA and the flow through was passed through the cartridge again. Samples were further washed with three 1 mL volumes of 80% ACN 1% TFA, eluted with 500 μL of 0.1% TFA, dried, and stored at −80 °C until MS analysis.

MS Analysis

Dried samples were dissolved in 0.1% formic acid (FA) and analyzed on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific) coupled with an Ultimate 3000 RSLC nanosystem LC instrument (Dionex). The binary solvent system was composed of solvent A (99.9% water and 0.1% FA) and solvent B (99.9% acetonitrile and 0.1% FA). Peptide and glycopeptide separation was achieved by a 5 min trapping/washing step on a 75 μm × 2 cm trap column (C18, 3 μm, 100 Å; Acclaim PepMap, Thermo) using solvent A at 5 μL/min, followed by a 180 min gradient on a 75 μm × 15 cm column (C18, 2 μm, 100 Å; Acclaim PepMap, Thermo) at a flow rate of 400 nL/min. The gradient was (I) held at 1% B for 5 min; (II) to 35% B over 134 min; (III) to 90% B over 11 min; (IV) held at 90% B for 5 min; (V) to 1% B over 6 min; (VI) to 90% B over 4 min; (VII) held at 90% B for 2 min; (VIII) to 1% B over 2 mi; (IX) to 90% B over 3 min and held at 90% for 2 min. The column was then re-equilibrated by a 1 min gradient back to 1% B and held for 10 min.

The MS instrument was operated in a data-dependent mode. Samples were ionized with a Nanospray Flex ion source at a spray voltage of 2800 V. The MS1 scans (m/z 400–2400) were acquired in the Orbitrap (120k resolution, 300% normalized AGC target), followed by HCD fragmentation and MS/MS acquisition of the top 20 precursors in order of intensity in the Orbitrap with an exclusion time of 20 s. The RF level was set at 50% and the HCD collision energy for the top 20 MS/MS acquisition was set at 45%, except for the sialidase-treated samples and corresponding controls, where the RF level was set at 75% and the HCD collision energy was set at 30% to obtain higher intensities of larger glycopeptides and polyLacNAc-specific fragments. The stepped HCD mode was triggered on oxonium containing MS/MS scans and was set with the stepped collision energies of 27, 35, and 43% (15k resolution, 300% normalized AGC target).

Glycopeptide Data Analysis

Glycopeptide searches were performed by Glyco-Decipher17 and Byonic (Protein Metrics). For analysis with Glyco-Decipher, trypsin was set as the enzyme, allowing for up to three missed cleavages. Cysteine carbamidomethylation was set as fixed modification, and methionine oxidation was set as variable modification. Spectrum expansion was enabled and the built-in GlyTouCan database18 was used to match glycan masses. Only glycopeptide spectrum matches with FDR < 0.01 and a minimum of three matched core fragment ions (Peptide+HexNAc, Peptide+HexNAc2···) were kept for glycopeptide identification. PolyLacNAc glycopeptides were confirmed by filtering search results for glycopeptides containing Hex(8)HexNAc(7) or larger glycan compositions and manually verifying the existence of the diLacNAc-specific fragment ion at m/z 731.27.19,20 Quantification of glycopeptide subclasses was performed with a quantification module in Glyco-Decipher based on the summed peak areas of MS1 elution profiles. For analysis with Byonic, trypsin was set as the enzyme, allowing for up to two missed cleavages. Cysteine carbamidomethylation was set as fixed modification, and asparagine deamidation and methionine oxidation were set as variable modifications. Only MS/MS spectra with a minimum of two oxonium ions were kept for glycopeptide identification. Glycan compositions identified from Glyco-Decipher searches of the same data files were combined with the glycan database. Glycopeptide spectral matches were filtered using a cutoff of Byonic score ≥300 and |Log Prob| ≥ 3 to obtain relatively confident identification results.17 Quantification of individual glycopeptides from tetraspanin-13 site N137 was performed on Skyline version 23.121 (MacCoss Lab, Department of Genome Sciences, University of Washington) by importing all glycoforms identified in any replicate of the sialidase treated or corresponding control samples and integrating the summed peak areas of the top six isotopic masses of the most abundant charge state for each glycopeptide from full MS scans.17,22 Gene ontology and pathway analyses were performed with Shiny GO version 0.77.23−25

Lectin Staining

Cells were washed with PBS, lifted with Cellstripper (Corning), and fixed by incubation in PBS containing 2% formaldehyde for 30 min at 4 °C. Cells were washed with PBS and incubated in staining buffer (PBS containing 0.2% bovine serum albumin and 1 mM EDTA) containing 5 ug/mL DyLight 649-conjugated Lycopersicon esculentum lectin (LEL, Vector Laboratories) for 30 min at 4 °C. Chitin hydrolysate (Vector Laboratories) was used at a 100 μg/mL concentration to inhibit LEL binding. Cells were washed twice with staining buffer, resuspended in staining buffer, and analyzed with a CytoFlex LX flow cytometer (Beckman Coulter). Data were analyzed by FlowJo 10 software 10 (FlowJo).

Gene Expression Analysis

RNA were isolated using a Monarch total RNA miniprep kit (New England Biolabs) according to the manufacturer’s recommendation. RNA were reverse-transcribed into cDNA by incubation with 10 mM DTT, oligo d(T), SuperScript IV First-Strand Synthesis System (Thermo Fisher), and 0.5 mM dNTPs. The qPCR reactions were performed using a TaqMan gene expression assay (Thermo Fisher Scientific) with primers selective for B3GNT2 or bACT (Thermo Fisher) using a QuantStudio 3 system (Thermo Scientific).

Data Visualization

Data visualization and statistical analysis were performed with GraphPad Prism 10. Venn diagrams were drawn with https://bioinformatics.psb.ugent.be/webtools/Venn/. Protein structure prediction was obtained from the AlphaFold database.26,27 Glycan modeling was performed by GLYCAM.28 PyMOL (Schrödinger) was used for structure visualization.

Results and Discussion

Comparison of Anion Exchange Columns for the Enrichment of Glycopeptides Carrying PolyLacNAc Motifs

First, we sought to identify an effective method for the identification of glycopeptides containing polyLacNAc. The most common approaches for glycopeptide analysis are based on enrichment by hydrophilic interaction chromatography (HILIC) and anion exchange chromatography. Whereas the latter generally provides higher glycopeptide yield compared to HILIC,29 the term “anion exchange” encompasses multiple commercially available solutions, which are often used interchangeably.29−32 To investigate the enrichment preference to polyLacNAc-containing glycopeptides of anion exchange-based methods, we selected five column materials, including (1) mix-mode anion exchange (MAX), (2) strong anion exchange SOLA (SOLA), (3) HyperSep retain anion exchange (RAX), (4) HyperSep strong anion exchange (hySAX), and (5) affinisep strong anion exchange (afSAX).

As a first pass, each cartridge was tested by using 300 μg of glycopeptides derived from whole cell lysates of A375 melanoma cells. The glycopeptide fraction was estimated for each preparation as the number of MS2 oxonium ions containing scans, which are indicative of glycopeptides, divided by the total number of MS2 scans: the five columns exhibited comparable values in the 82–89% range (Table S1). Next, we analyzed glycopeptide composition in each sample and found that SOLA and RAX cartridges provided the highest number of unique glycopeptides, followed by afSAX, MAX, and hySAX (Figure 1A). Each of the approaches resulted in comparable profiles of high mannose, complex, truncated, and modified glycan types (Figure S1), excluding potential enrichment biases based on the glycosylation structure. Melanoma cells were found to predominantly express high-mannose-type N-glycans, in agreement with previous reports.33,34 To investigate whether the higher number of glycopeptides represents a similar increase in unique glycoprotein identifications, or rather a more detailed characterization of same glycoproteins, we compared the numbers of unique glycoprotein identifications among the enrichment approaches. SOLA and RAX resulted in a higher number of unique glycoproteins, albeit the differences were less pronounced compared to unique glycopeptide identification, indicating that SOLA and RAX provide a more comprehensive coverage of peptide glycoforms (Figure 1B).

Figure 1.

Figure 1

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Performance of anion exchange columns in the enrichment of glycopeptides containing polyLacNAc. (A) Overlap of the glycopeptides enriched by MAX, RAX, SOLA, afSAX, or hySAX. (B) Overlap of glycoproteins identified with the different approaches. (C) Number of glycopeptides containing ions indicative of the polyLacNAc motifs enriched by the columns.

To evaluate the enrichment specificity for polyLacNAc glycopeptides, we compared the number of identified polyLacNAc glycopeptides by each method. SOLA showed the best performance, followed by comparable results from afSAX, MAX, and RAX (Figure 1C). Additionally, we calculated two levels of enrichment specificity: the percentage of polyLacNAc-containing peptides among identified glycopeptides and the percentage of polyLacNAc-containing peptides among total peptides. Among the five enrichment methods, SOLA exhibited the highest specificity for polyLacNAc-containing glycopeptides, both among glycopeptides and among total peptides, followed by afSAX, MAX, and RAX (Table 1). HySAX had the lowest specificity. Interestingly, we observed that the percentage of polyLacNAc-containing peptides among identified glycopeptides for each enrichment method was comparable with the corresponding ratio of MS2 spectra containing m/z 731.27, which corresponds to a (HexNAcHex)2 repeat and is a signature fragment ion of polyLacNAc, among all MS2 spectra. Higher numbers of peptide and glycopeptide identifications were obtained by Byonic compared with Glyco-Decipher for all of the enrichment methods, likely because Glyco-Decipher uses more stringent criteria for glycopeptide searches. Additionally, we found that the time required for the samples to pass through the columns by gravity flow varied based on the material, ranging from 20 min for RAX and hySAX to 60 min for SOLA and MAX and to 90 min for afSAX. This aspect represents an important practical consideration when designing large-scale experiments.

Table 1. Specificity of Enrichment of PolyLacNAc-Containing Glycopeptide of Anion Exchange Columns from A375 Cell Lysatesa.

        polyLacNAc-containing GPs among total GPs
 polyLacNAc-containing GPs among total peptides
        Glyco-Decipher
 Byonic
      
method MS2 scans MS2 scans withm/z731.27 ratio (%) polyLAcNAc-containing GP GPs ratio (%) polyLAcNAc-containing GP GPs ratio (%) polyLAcNAc-containing GP peptides ratio (%)
SOLA 153979 3514 2.28 37 1929 1.92 41 2474 1.66 41 3136 1.31
afSAX 153190 1662 1.08 17 1506 1.13 14 2036 0.69 14 3046 0.46
hySAX 149855 571 0.38 6 1023 0.59 6 1515 0.40 6 3227 0.19
MAX 151015 1548 1.03 15 1208 1.24 12 1602 0.75 12 2656 0.45
RAX 153359 1237 0.81 16 1862 0.86 14 2470 0.57 14 4344 0.32
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a

The percentage of polyLacNAc-containing peptides among identified glycopeptides was calculated using results obtained by Glyco-Decipher and Byonic. The two search engines yielded comparable percentages. The percentage of polyLacNAc-containing peptides among total peptides was calculated using search results by Byonic as Glyco-Decipher does not search for non-glycosylated peptides. The ratio of MS2 spectra containing m/z 731.27, which corresponds to a (HexNAcHex)2 repeat and is a signature fragment ion of polyLacNAc, among all MS2 spectra, is also indicated.

Altogether, these data indicate that SOLA offers a superior performance in the identification of both total and polyLacNAc-containing glycopeptides. RAX outperformed the other columns in terms of speed and overall glycopeptide identification and achieved comparable performance for polyLacNAc-containing glycopeptides among the remaining four enrichment methods (RAX, MAX, afSAX, and hySAX). Therefore, SOLA and RAX were selected for subsequent in-depth evaluation.

In-Depth Characterization of the Performance of SOLA and RAX for the Enrichment of PolyLacNAc-Containing Glycopeptides

Next, we sought to thoroughly characterize the ability of SOLA and RAX to support glycopeptide analysis of the A375 melanoma cell line. Both SOLA and RAX yielded approximately 80% of glycopeptides with high mannose (Figure 2A). SOLA enriched a relatively higher portion of complex-/hybrid-type glycans, and RAX enriched relatively more glycopeptides with truncated N-glycans, indicating that RAX retains more hydrophobic glycopeptides with smaller glycan motifs, probably due to its mixed mode of action. The two cartridges enriched comparable percentages of glycopeptides that are both sialylated and fucosylated (Figure 2B). RAX showed a minor preference toward glycopeptides that lack sialic acid and fucose.

Figure 2.

Figure 2

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Evaluation of glycopeptide patterns enriched by SOLA and RAX. (A) Glycan types enriched by SOLA and RAX. Minor differences in complex-/hybrid-type and truncated N-glycans were observed (unpaired t test, p = 0.007 and p = 0.006, respectively). (B) Relative abundance of fucosylated and sialylated glycopeptides in complex-/hybrid-type N-glycopeptides. SOLA and RAX overall enriched similar glycosylation patterns, with minor differences in sialylated/fucosylated glycans (p = 0.066) and nonfucosylated/nonsialylated glycans (p = 0.047). (C) SOLA and RAX enable the detection of changes in fucosylated glycopeptides. SOLA and RAX exhibited minor differences in enrichment of 2FPF-treated samples (p = 0.009). (D) SOLA and RAX enable the detection of changes in sialylated glycopeptides. Search results from samples treated with sialidase and enriched by SOLA or RAX, plotted as a proportion of all complex/hybrid N-glycopeptides. Differences at baseline were detected (p = 0.0012). (E) SOLA enables the identification of a higher number of glycopeptides (GPs) containing polyLacNAc compared to RAX. Collision energy was set to 30% to allow for favorable fragmentation patterns. PolyLacNAc detection was based on the presence of the signature 731 m/z ion and a structure of Hex8HexNAc7 or higher. SOLA outperformed RAX with and without pretreatment of glycopeptides with sialidase (p = 0.0105 and p = 0.0004, respectively). The mean value and standard deviation of four biological replicates are represented in each panel. The Welch unpaired t test was calculated.

To evaluate the quality of the glycopeptide identification workflow, we analyzed samples from cells grown in the presence of the fucose inhibitor 2-fluoro-peracetyl-fucose (2FPF). Glycopeptide enrichment by SOLA from untreated samples resulted in complex-type N-glycans being 63% fucosylated, while the RAX-based method produced complex-type N-glycans that were 55% fucosylated (Figure 2C). 2FPF treatment drastically decreased the proportion of complex N-glycans carrying fucose (from 63 to 14% in the SOLA-based experiment and from 55 to 9% in the RAX-based experiment), but did not completely abolish fucosylation in the conditions tested, in agreement with previous reports.35 The proportions of sialylated glycopeptides from samples treated or untreated with 2FPF were comparable (Figure S2). Additionally, we treated samples with sialidase to remove sialic acids. SOLA and RAX captured glycopeptides with 90 and 75% sialylation, respectively (Figure 2D). Sialidase treatment completely removed sialic acid (Figure 2D), while the proportion of fucosylated glycopeptides from the same samples remained unchanged (Figure S2).

In addition, since polyLacNAc is found in complex-type N-glycans of secreted or membrane-resident glycoproteins, we tested if membrane fractionation would increase the proportion of these N-glycans and exclude the contributions of endoplasmic reticulum and Golgi compartments, which are a main source of high-mannose N-glycans.1,36 Based on oxonium ion analysis, we observed a higher proportion of glycopeptides in the crude membrane and plasma membrane fractions compared to whole cell lysates (Table S2), likely due to removal of cytosolic proteins that may interfere with the enrichment. Intriguingly, plasma membrane fractionation, despite yielding about 40 μg total peptides in the conditions tested, enabled the identification of approximately 2500 glycopeptides after enrichment by SOLA, and about 1900 by RAX, with 1400 overlapping between the two enrichment methods. We observed a 1.6-fold increase in the proportion of complex-type glycans compared with whole cell lysates by SOLA (Figure S3), indicating that membrane fractionation increases the enrichment efficiency of complex-type N-glycans, albeit at the cost of a significant increase in the amount of the material required. Of note, we did not observe an increased number of polyLacNAc-containing MS2 spectra and glycopeptides in crude and plasma membranes compared to the whole cell lysate.

Finally, we compared SOLA and RAX for their ability to enrich glycopeptides containing the polyLacNAc motif. SOLA provided 3-fold higher numbers of unique glycopeptides compared to RAX (Figure 2E). Intriguingly, incubation of glycopeptides with sialidase enabled the identification of 2–3 times higher number of glycopeptides, possibly thanks to increased signals due to the improved ionization efficiency after sialic acid removal and the reduction in the size and heterogeneity of the glycoforms.37 For this set of experiments, we found that a lower HCD collision energy and a higher RF voltage improved both the overall glycopeptide identification and the confidence of detecting polyLacNAc-containing glycopeptides, in agreement with previous reports.37 To further improve the analysis of polyLacNAc-extended glycopeptides, an alternative washing solvent with different organic compositions (stronger solvents) may be used during glycopeptide enrichment to remove glycopeptides with smaller glycan motifs, such as high mannose, and retain larger and more anionic glycopeptides. However, a stronger washing solvent may introduce higher variability into the enrichment process.

Taken together, these data indicate that SOLA- and RAX-based enrichment methods are effective and robust strategies for capturing changes in glycosylation and polyLacNAc-containing glycopeptides in large-scale glycoproteomics, despite minor differences in enrichment preferences toward different classes of glycan motifs.

Identification of Glycoproteins Containing PolyLacNAc in Melanoma Cells

Next, we applied our platform to gain insight into the proteins modified with the polyLacNAc motif. PolyLacNAc can be challenging to distinguish from an increase in N-glycan branching: for example, triantennary N-glycans with a single polyLacNAc repeat have identical composition and mass to those of a tetra-antennary N-glycan. Therefore, we searched for the signature oxonium ion at m/z 731.27 and restricted our search to structures containing 7 or more N-acetylhexosamine and 8 or more hexose residues, also to account for a lack of data on the specificity of the m/z 731.27 ion. We identified a number of glycoproteins with the polyLacNAc motif including the DR alpha chain of HLA class II histocompatibility antigen (HLA-DRA) (Figure 3A). A detailed look into the HLA-DRA glycopeptide spectrum matches revealed a species containing 9 hexoses, 8 N-acetylhexosamines, 3 N-acetylneuraminic acids, and 1 fucose. The presence of an m/z 731 signal was manually confirmed (Figure S4). Similar patterns were observed for glycopeptides derived from the mannose-6 receptor (M6PR) site N83 (Figure 3B), integrin β-1 (ITGB1) site N520 (Figure 3C), and CD63 antigen (CD63) site N130 (Figure 3D). Sialidase treatment enabled the detection of glycopeptides with a remarkably high number of LacNAc repeats and a high intensity of the diagnostic ion (Figure S5).

Figure 3.

Figure 3

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Analysis of polyLacNAc-containing glycoproteins in melanoma cells. (A–D) Exemplary spectra of polyLacNAc-elongated glycopeptide from (A) HLA-DRA N103, (B) M6PR N83, (C) ITGB1 N520, and (D) CD63 N130. The glycan represents a compositional assignment and highlights the 731.27 m/z fragment (HexNAcHex)2 indicative of polyLacNAc elongation. (E) KEGG pathway analysis of all of the glycoproteins detected in this study. (F) KEGG pathway analysis of the subset of detected glycoproteins containing polyLacNAc.

We then compared the number of MS/MS spectra containing m/z 731.27 (±0.01) in melanoma cells lacking a functional B3GNT2 (B3GNT2 KO) or overexpressing the enzyme (B3GNT2 OE), and found a 3-fold difference (294 vs 890 spectra by RAX, 1173 vs 3611 spectra by SOLA). Cell staining with the LEL lectin specific for LacNAc repeats also indicated that removal of B3GNT2 did not completely abolish polyLacNAc biosynthesis in these cells and that overexpression of the glycosyltransferase did not result in a substantial increase in polyLacNAc (Figure S6). These data suggest that other members of the B3GNTX family contribute to polyLacNAc synthesis, that factors other than B3GNT2 control the synthesis of the glycan motif, or that the glycopeptide substrates of B3GNT2 were not comprehensively monitored in the current study.

To explore the potential roles of polyLacNAc-bearing glycoproteins in A375 cells, we analyzed a subset of proteins that contained polyLacNAc motifs (Table S3) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. Interestingly, in contrast with pathway analysis of the entire glycoproteome, we observed a strong enrichment of polyLacNAc-modified glycoproteins in the “ECM–receptor interaction” pathway, followed by the “other glycan degradation” and the “lysosome” pathways (Figure 3E,F). To gain deeper insights into the ECM–receptor interaction pathway, we examined the glycoproteins involved in this pathway and found polyLacNAc modification on integrin α1, α3, α5, and αV, as well as integrins β1 and β6, along with their respective ligands vitronectin, tenascin, CD44, and thrombospondin 1 (Figure S7). As integrins serve as crucial adhesion receptors capable of bidirectional signaling across the plasma membrane and there is evidence of the impact of integrin glycosylation on their function,38−40 these findings represent an intriguing avenue for further studies.41

Altogether, these data indicate that several surface proteins are modified with polyLacNAc in melanoma cells and suggest that this glycan motif might exert direct or indirect effects on cellular activities such as adhesion, migration, differentiation, proliferation, and apoptosis.

In-Depth Characterization of PolyLacNAc Modifications in Tetraspanin-13

After developing methods for detecting polyLacNAc-elongated N-glycans and demonstrating their feasibility in identifying target glycoproteins, our next objective was to investigate the extent of polyLacNAc modification in selected glycoproteins and to approach its potential physical or functional implications. We focused on tetraspanin-13 (TSPAN13) as it carried extensive polyLacNAc modification (Figure 4A). The tetraspanin family consists of 33 transmembrane proteins with a variable number of glycosylation sites in the extracellular domains (Figure S8). Their function as membrane-organizing proteins42 relies on lateral protein–protein interactions in cis on the cell membrane, with integrins being one of their key interactors.43 We combined protein structure prediction with GLYCAM-based glycan modeling to gain insights into the polyLacNAc-elongated N-glycan on TSPAN13, and we found that it protruded from the extracellular loop of TSPAN13 (Figure 4A). Considering the highly dynamic nature of glycans, it is conceivable that changes in glycosylation in this region may exert functional consequences.

Figure 4.

Figure 4

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Tetraspanin-13 is a carrier of polyLacNAc-elongated N-glycans. (A) Structural model of tetraspanin-13 with a polyLacNAc-elongated N-glycan detected at site N137. The site N113 was not detected, probably because it is included in a very long tryptic peptide. (B) Performance of SOLA and RAX in enriching glycopeptides with the N137 site of tetraspanin-13. Pie charts represent the relative abundance of glycans with ≥ Hex8HexNAc7 (orange) or ≤ Hex7HexNAc6 (black). The bars represent the average value of four biological replicates, and the error bars represent the standard deviation. (C) Performance of SOLA and RAX in enriching desialylated glycopeptides with the N137 site of tetraspanin-13. The pie charts represent the relative abundance of glycans with ≥ Hex8HexNAc7 (orange) or ≤ Hex7HexNAc6 (black). The bars represent the average value of four replicates, and the error bars represent the standard deviation.

Strikingly, in-depth glycosylation analysis resulted in the detection of extensive polyLacNAc elaboration of TSPN13, with masses indicative of up to 7 LacNAc repeats (Figure 4B,C and Figure S9). Whereas robust evidence of extensive polyLAcNAc elaboration was obtained with both SOLA and RAX columns, SOLA was more efficient than RAX at enriching glycopeptides with high polyLacNAc, and RAX appeared to favor bi-, tri-, and tetra-antennary glycans with limited polyLacNAc extension. Remarkably, a recent interactome analysis of galectin-3, a lectin that binds to lactose and LacNAc repeats, revealed interactions with multiple tetraspanins, integrins, and other proteins,44 some of which were determined to carry polyLacNAc in the current study (e.g., BSG, neuroplastin, integrin α1, integrin β1, CD44 antigen, leukocyte surface antigen CD47, and CD9 antigen). This precedent, combined with the data presented here, provides further support for the existence of an integrin/tetraspanin/galectin interaction axis and highlights the potential role of polyLacNAc in modulating the integrin–tetraspanin interplay.

Altogether, these findings showcase the applicability of the methods described above for in-depth characterization of the polyLacNAc extension at specific sites, supporting the characterization of the biological roles of this sugar motif.

Conclusions

In this work, we demonstrated that different anion exchange materials enrich overlapping but not identical glycopeptides and that SOLA and RAX cartridges exhibit superior performance for the identification of glycopeptides containing polyLacNAc. In-depth comparisons of these two methods revealed that SOLA is more efficient in enriching larger glycopeptides with more LacNAc repeats. Removal of sialic acid allows the identification of a higher number of glycopeptides with the polyLAcNAc motif by SOLA and RAX. We applied these methods to melanoma cells expressing the B3GNT2 glycosyltransferase and identified a variety of glycoproteins with polyLacNAc repeats, including integrins and tetraspanins, that are involved in the organization of receptors and signaling adaptors at the cell membrane. This study provides a guideline for the selection of appropriate methods for enrichment of glycopeptides containing polyLacNAc for mass spectrometry analysis and demonstrates the feasibility of glycoproteomic approaches for the identification of proteins with these glycan motifs.

Acknowledgments

We thank all the colleagues at InterVenn Biosciences for fruitful scientific discussions and support during the planning and execution of this work and the preparation of this manuscript. We are grateful to Carolyn Bertozzi and Carlito Lebrilla for input and advice during the execution of this study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.3c04045.

  • Additional glycopeptide spectra, glycopeptide searches, and cell staining data (PDF)

  • List of glycoproteins containing polyLacNAc identified in this study (XLSX)

Author Contributions

# T.Č. and G.X. contributed equally to this paper.

Author Contributions

Conceptualization: T.Č. and F.S. Methodology: T.Č., G.X., M.B., and G.C. Data generation and analysis: T.Č., G.X., M.B., R.R., C.-W.C., I.H., and F.S. Manuscript writing and reviewing: T.Č., G.X., C.-W.C., M.B., R.R., and F.S. All authors contributed to the article and approved the submitted version.

The authors declare the following competing financial interest(s): All authors are or were employees of InterVenn Biosciences during the study period. InterVenn Biosciences identifies biomarkers and develops diagnostic tests.

Supplementary Material

ac3c04045_si_001.pdf (1.2MB, pdf)
ac3c04045_si_002.xlsx (29.5KB, xlsx)

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Supplementary Materials

ac3c04045_si_001.pdf (1.2MB, pdf)
ac3c04045_si_002.xlsx (29.5KB, xlsx)

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