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An efficient procedure for N-glycan analyses and detection of Endo H-like activity in human tumor specimens Erika Lattová, Joseph Bryant, Jana Skrickova, Zbynek Zdrahal, and Mikulas Popovic J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00346 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 19, 2016

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Journal of Proteome Research

1

An efficient procedure for N-glycan analyses and detection of Endo H-like activity in human tumor specimens

Erika Lattová

a*

, Joseph Bryant b, Jana Skřičková c, Zbyněk Zdráhal

a,d

, Mikuláš Popovič

b*

a

Central European Institute for Technology, Masaryk University, Kamenice 5, 625 00 Brno, CZ

b

The Institute of Human Virology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201, USA

c

Department of Respiratory Diseases and Tuberculosis, University hospital Brno, Medical Faculty, Masaryk University, 625 00 Brno, CZ

d

National centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 625 00 Brno, CZ.

*Corresponding authors: [email protected] m; Tel. +420 549-49-8425 [email protected] ; Tel. +1 401-706-5879

ACS Paragon Plus Environment


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2 ABSTRACT

Although the importance of glycosylation has been thoroughly recognized in association with a number of biological processes, efficient assessments of glycans has been hampered by both the limited size of specimens and lengthy sample preparations, particularly in clinical settings. Here we report a simple preparative method for N-glycan analyses. It involves only short one-step chloroform-methanol extraction in presence or absence of water prior to PNGase F deglycosylation. The procedure was successfully applied to the investigation of N-glycans obtained from small numbers of in vitro cultured cancer cells (≤1x105 ) and to tumor tissues, including patient biopsies of small size. MALDI-MS analysis confirmed the efficient release of all N-glycan types including complex

forms

with

poly-N-acetyllactosamine

chains.

In

addition,

non-aqueous

extraction of specimens from several established cancer cell lines, as well as patient tumor tissues, yielded high-mannose glycans with one GlcNAc moiety (Man3-9 GlcNAc), strongly suggesting preservation of enzymatic activity analogous to Endo H enzyme. In summary, the method is both a step towards the practical use of glycan profiling, and a way to detect Endo H-like activity in cancer specimens.

Key words: biopsy, cancer cells, EndoH, glycans, glycosylation, mass spectrometry, tumor tissue


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3 INTRODUCTION Glycosylation is one of the most common modes of posttranslational modifications of proteins.1 Links between changes in glycan structures and various biological functions, including malignant processes, have been reported.2-4 Better understanding of the glycosylation role in living systems can provide deeper insights into pathological processes that occur in diseases and consequently, contribute to progress in early diagnosis or targeted treatment. While different analytical approaches have been used to identify oligosaccharides, currently, mass spectrometry (MS) is routinely used.5-7 MS and other conventional approaches employed for glycoprotein investigations are frequently based on enzymatic cleavage of N-glycans by peptide-N4-(N-acetyl-beta-glucosaminyl)-asparagine amidase (N-glycosidase or PNGase F).8

Prior to this digestion, samples usually undergo multistep

treatments to enhance enzymatic deglycosylation. Commonly used protocols have applied different detergents and/or chemicals to facilitate cell lysis and denaturation of proteins and glycoproteins.9-16 These sample preparations, particularly those involving purification procedures are tedious and lengthy, and hamper the ability to include glycan screening in routine clinical evaluation. Moreover, they are difficult to successfully apply to situations with few cells, or small tissue biopsies. These limitations in cell numbers impede investigation of N-glycans by failing to generate replicate samples particularly in cases of cell subpopulations with important biological functions such as stem cell or cancer stem cells.17,18 Previously, we reported the efficient release of N-glycans from glycopeptides using cancer cells processed without using detergents or other similar reagents.19,20 These studies



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4 utilized established cancer cell lines, where specimen size was not a concern. A similar method was recently reported for generation of glycopeptides from an established human kidney cell line.21 This approach enabled detection of more complex N-glycan types in the kidney cell population freshly harvested from in vitro cultures. In the present study, we focused on further modifications of pre-digestion procedure, the primary goals being reduction of sample size and preparation time. Our method uses a mixture of chloroform-methanol (CM) for extraction of specimens and specific conditions for sample preparations prior to deglycosylation with PNGase F. This organic CM solvent has been extensively used for delipidation and protein extractions from different tissues of various species.22-27

Moreover, the use of non-aqueous CM extraction can effectively

remove lipids and preserve activities of a few membrane bound enzymes while aqueous CM extraction renders these enzymes inactive. 28 Thus, the principal difference between our approach and conventionally-used methods targeting N-glycan analysis, is the use of a one-step CM and/or CM with water (CMW) extraction, which is performed in lieu of detergent/chemical treatment. This extraction is followed by deglycosylation mediated by enzymatic digestion with analysis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The modifications of the extraction procedure using CM allowed us: (i) to shorten sample preparation time; (ii) to monitor PNGase F-released glycans prior to purification; (iii) to perform analyses of samples containing few cells (≤1x105 ) including biopsies of small sizes; and finally, (iv) to reveal the generation of high-mannose glycans with one GlcNAc residue instead of having an intact chitobiose core in CM extracts of several cancer specimens. This uncommon cleavage generated under condition of PNGase F deglycosylation is most likely attributable to Endo H-like


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5 enzyme(s) reaction, which can be effectively neutralized by the CMW specimen extractions. The glycan profiles achieved with the application of CMW extractions are fully comparable to glycan profiles obtained by conventional methods. . EXPERIMENTAL SECTION

Cell Culture The

following

human

cell lines maintained

in vitro

were used

for this study:

choriocarcinoma cells JEG-3 (ATCC HTB-36),29 RL(B) non-Hodgkin B-lymphoma (ATCC CRL2261),30 TIB223 fibrous histiocytoma derived from metastatic lesion of lung (ATCC TIB223 GCT),31 HTB-182 squamous cell carcinoma (NCI-H520, ATCC HTB182); small cell lung cancer (SCLC) HTB-171 (NCI-H446, ATCC HTB-171) and nonSCLC (NSCLC) HTB-177 (NCI-H460, ATCC HTB-177) large cell carcinoma,32 H358 bronchoalveolar carcinoma (NCI-H358, ATCC CRL-5807)33 and LuCa-6 adenocarcinoma. This LuCa-6 cell line was derived from the pleural effusion from a non-smoker patient and was established at the Institute of Human Virology (IHV), University of Maryland School of Medicine (UMSM).34 The authentication of the LuCa-6 cell line was performed by the HLA typing kit Micro SSPT M HLA Class I and II ABDR DNA typing (One Lambda Inc., California) in the Department of Pathology, UMSM. The authenticated cell lines, with exception of LuCa-6, were obtained from American Type Culture Collection (ATCC) and maintained in RPMI1640 with supplements (10% inactivated fetal bovine serum, 1% penicillin/streptomycin, 2mM Glutamax) at the IHV. Two cell lines, HTB-177 and H358, also from ATCC were kindly provided by S. Ostrand-Rosenberg.35 The cell line TIB223



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6 was cultured in McCoy’s medium.31 Cell lines obtained from ATCC were routinely tested for mycoplasma and documented to be negative. The cultured cells in our laboratory were monitored for presence of mycoplasma using the mycoplasma detection kit MycoAlert T M PLUS (Lonza Walkersville, Inc., Maryland). Cultures of the cell lines growing adherently were expanded in vitro using T-75 flasks (Corning Life Sciences) in the culture media as indicated above. Viable cell counts were determined on a hemacytometer by the trypan blue exclusion method. The adherent cell lines, with exception of the RL (B) lymphoma line growing in suspension, were once washed with cold Dulbecco phosphate buffer solution (D-PBS) in T-75 flasks and cells subsequently were detached mechanically from surfaces of flasks into cold D-PBS. Cells released into suspension as well as the RL (B) cell line were pelleted and washed with cold D-PBS by centrifugation 3-times. After third washing and pipetting off the supernatant, the pelleted cells were snap-frozen at -180o C in liquid nitrogen, lyophilized and stored at -25o C.

Tumor Tissue Mice used for this study were housed under standard conditions in the animal facility at the Institute of Human Virology. All experiments were performed according to the University of Maryland IACUC guidelines, under an approved research protocol. Human tumor tissues (xenotransplants) of the HTB-182 cell line were obtained by s. c. inoculation of 106 cells per mouse into three 10 week-old female NSG (NOD-SCID- γc-/-) mice. Tumor growth in mice was evaluated weekly by measuring tumor size. Tumor tissues were obtained from grown tumors of 1.5 to 2.5 g weight that were removed and minced into small pieces with scissors, washed extensively (5-times) with cold D-PBS by


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7 centrifugation. The pelleted tumor tissues were frozen in liquid nitrogen at -180o C and lyophilized prior to processing for N-glycan analyses.

Biopsy Human lung cancer tissues have been obtained by means of bronchoscopy following the protocol approved by the Ethics Committee of University Hospital Brno (CZ). The examination was conducted by a flexible bronchoscope and tissue samples were collected for histological and molecular-genetic examination. Tissues determined for glycan analysis were extensively washed with cold PBS and then stored at -80o C.

Release of N-glycans from Cell and Tissue Specimens Three following protocols were applied and samples with the same quantity of cells were processed for comparative profiling (usually up to 1 mg of dry material or up to 1x107 cancer cells): 1. Lyophilized cancer cells or tumor tissue were resuspended in 1x Reaction or Tris buffer (80-150 µL) and heated at 100o C for 5 min (water bath) in the presence of a denaturation solution accompanying enzyme (Prozyme). After cooling detergent solution (2.5 µL) was added, mixed with PNGase F (1-3 mU, Prozyme or Roche) and incubated at 37o C for 1-12 h. During the incubation time, the reaction sample was frequently mixed.

After that, the

digest was cooled on ice (2 hours) and insoluble precipitate was pelleted by centrifugation. The supernatant was next purified (SPE). 2. Ethanol extraction was performed according to a protocol described previously11 with the important omission of steps regarding the use of detergents and reductive alkylation.



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8 Instead, lyophilized cancer cells or tumor tissue were resuspended in the 75 % ethanol, sonicated (5 min) and left in the freezer for 2 hours. After centrifugation (5 min), the supernatants were discarded or evaporated and examined by an electrophoretic method. Crude pellets after removing solvent residues (vacuum evaporation) were resuspended in 5 mM ammonium bicarbonate (AB) and followed by deglycosylatiom (PNGase F, 1- 3 mU) at 37o C for 1-12 h. After centrifugation the supernatant was analyzed or purified. 3. New protocol based on chloroform-methanol extraction a) Samples were first mixed with chloroform-methanol-water (CMW), preferably in the ratio 8:4:1, 300 µL, and sonicated for 5 min at room temperature. After centrifugation, 5 min, 3000 g, the supernatant layers were pipetted out and the crude sediments were dried under vacuum to remove traces of the organic solvent (~5 min). To dried pellets, 5 mM AB (80-150 µL) was added and incubated with PNGase F (1-3 mU; 37o C for 1-12 h). During/after incubation, the digest was briefly centrifuged and the supernatant was analyzed or purified by solid phase extraction (SPE) prior to MS analysis. b) Protocol variant permitting N-glycan profiling with detection of Endo H-like enzymatic activity - Samples were first mixed with chloroform-methanol (CM) (2:1; 300 µL) under non-aqueous conditions, then sonicated and processed as described in a).

Desialylation Sialic acid residues were removed with neuraminidase (Clostridium perfringens, Roche) at 37o C for 1-3 h, usually followed immediately deglycosylation with/without deactivation of PNGase F.


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9 Solid Phase Extraction (SPE) Glycans released from samples were purified and enriched on nonporous graphitized carbon cartridges (Supelco). The cartridges were first washed with 80% acetonitrile with 0.1% TFA followed by deionized water, 10 mL of each. Digested samples were applied to the columns, and after a short adsorption time, the columns were washed with deionized water, 6 x 1000 μL. Oligosaccharides were eluted with a solution of 40% ACN with 0.1 % TFA and all fractions were evaporated prior to analysis.

PHN Derivatization The labeling of oligosaccharides was performed by applying on-target derivatization method,36 modified to implement the advantages of Anchorchip technology – the hydrophobic surface of the target prevents spot spreading during room temperature drying that makes this derivatization faster and more convenient for routine analysis. Glycan fractions were reconstituted in deionized water (10-100 µL) and 1-1.5 µL of diluted sample was loaded onto the wet spot with matrix ATT/PHN.HCl (0.8 µL; 4mg of ATT and 2 mg of PHN.HCl dissolved in 300 µL of 40% ACN in deionized water) predeposited on the AnchorChip target and immediately followed by addition of 0.5-0.8 µL derivatization reagent (10 µL of PHN dissolved in 50 µL deionized water and 5 µL ACN). The target was kept at room temperature until the spot had dried (5-10 min).

Mass Spectrometry (MS) MS analysis was performed on the MALDI-TOF/TOF instrument (UltrafleXtreme, Bruker Daltonic) equipped with a Smartbeam-II laser, and used in positive reflectron mode. The



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10 instrument was calibrated externally using calibration standards over a mass range of 8005000 Da. For each spectrum, 10000-20000 laser shots were accumulated. The spectra were acquired using the FlexControl software. Raw data were processed by using an instrument software tool (FlexAnalysis 3.4, Bruker). Structural assignments of the glycan structures shown in the figures were derived from their molecular masses, MS/MS fragmentation patterns and knowledge of biosynthetic glycan pathways. In MS/MS spectra, the assignment of fragment ions, is based on the nomenclature reported by Domon and Costello.37 In MS spectra, glycan peaks are annotated with structures composed from key symbols used for monosaccharides - the symbolic nomenclature used by the Consortium for Functional Glycomics - http://www.functionalglycomics.org. The reproducibility of results was verified from experiments repeated minimally in triplicates. For comparative profiling, the resulting glycan signals, categorized according to their m/z values, were normalized to 100%. The relative abundances of individual glycans were calculated by using the Microsoft Excel software system. The recorded peaks with assigned structures of polyhexoses - (Hex)n , were excluded from the evaluation.

RESULTS and DISCUSSION

Method Optimization To assess capacity of N-glycan extractions from specimens using CMW, we compared this method (Protocol 3a) to the extraction of N-glycans using detergent in combination with denaturation reagents (Protocol 1) and to the extraction method using 75% ethanol (Protocol 2). The schematic outline of the procedure based on CM extraction for analyses


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11 of N-glycans in cancer cells from established cell lines and tumor specimens is illustrated in Scheme 1A. For each procedure we used 1x106 cells of the human choriocarcinoma line JEG-3. MS analysis confirmed the efficient release of high-mannose, hybrid and complex glycans (Figure S1). Those with poly-N-acetyllactosamine chains were also detected, mostly when enzymatic desialylation followed the PNGase F deglycosylation. As shown in Figure 1, the average relative intensity peaks of N-glycans obtained by using three different pre-digestion methods were highly comparable. To verify that the procedure in question does not result in losses of glycoproteins/proteins, two cell lines of NSCLC (HTB-177 and H358) were subjected to the CMW and CM method and compared to the alcohol-based extraction. Following centrifugation of total cell lysates, supernatants pipetted off from the crude sediments were tested for the presence of proteins and glycoproteins using SDSPAGE electrophoresis. No significant bands were observed in supernatants from the CM and CMW extractions, just low molecular weight compounds were recorded (Figure S2). Some bands of weak intensities with higher MW were noticed in supernatant samples using the ethanol extraction. It should be pointed out that none of the protocols using classical detergents permitted detection of oligosaccharides before purification, unless samples were treated with organic solvents. Even short one-step treatment (3-5 min) with CM or CMW (optimal ratios 2:1 or 8:4:1) showed distinct advantages. In connection with PHN labeling38 , it was possible to detect released glycans even in unpurified digests. The effectiveness of this step is depicted e.g. in the analysis of PNGase F treatment of the JEG-3 choriocarcinoma cells. Glycans detected from a 1 μl test sample taken during deglycosylation step (Figure S3-A) showed a profile comparable to that obtained after SPE purification (Figure S3-B). Another



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12 example of unpurified digest testing is shown on the spectra recorded from a tumor tissue (xenotransplant) of the lung squamous cell carcinoma line HTB-182. The main glycans correspond to high-mannose structures (Figure S4-A). After desialylation, the additional detected peaks with high intensities corresponded to complex bi, tri and tetra antennary glycans (m/z 1753, 1899, 2118, 2264, 2630; Figure S4-B). The results indicate that these originally sialylated oligosaccharides and high-mannose structures are the most abundant glycans in cells of the squamous cell carcinoma HTB-182. The advantage of glycan monitoring prior to purification was also observed in samples extracted with ethanol, although N-glycan detections were less reliable. On the other hand, the CMW/CM method provided consistency and high reproducibility in recording glycans with m/z up to 3000 Da.

Detection Limit and Reproducibility of the Method Rahman at el. have recently reported a simple method for isolation of glycoprotein Nglycans from mammalian cells based on using filter-aided removal of detergent, termed also FANG.15 The authors demonstrated that samples containing 2-5x105 cells were sufficient to accomplish a medium throughput analysis of N-glycans. Our present study indicates that glycan profiles can be efficiently obtained even from smaller cell number with a simpler preparation method and in a shorter time. Sample preparation time with the CM or CMW extraction can be accomplished in about 30 minutes. The spectrum of N-glycans depicted in Figure 2 was recorded from 1x10 5 cells of the JEG-3 choriocarcinoma cell line. As evidenced by the spectrum, this cell number was sufficient to detect even minor glycans with high extensions of N-acetyllactosamine epitopes (Figure 2C). A decline in detection of these high MW glycans was observed when


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13 the same amount of cells underwent ethanol extraction, and an even more prominent drop was noted when preparative conditions utilized detergents (Figure S5). To determine accurately the cell number requirement (the detection limit) for the CMW extraction method, we performed a quantitative assessment of N-glycan profiling with cell samples from the lung cancer cell line HTB-171 and from the non-Hodgkin B-lymphoma line RL(B). Following washing with D-PBS and determining cell counts for each cell line, we prepared a series of samples by dilution corresponding to cell concentrations from 2x106 to 2x103 cells of the HTB-171 cell line and from 1x107 to 1x103 cells per sample of the RL(B) cell line, respectively. Results of N-glycan detections in these two cell lines are summarized in Table 1. A schematic illustration of oligosaccharide types, their structures and abbreviations are depicted in Scheme S1. As shown, high mannose (HM), hybrid (HB), complex (CP) and complex poly-N-acetyllactosamine (CPL) glycans were readily detected in HTB-171 cell samples containing 1x105 cells. The range of detected N-glycans in the HTB-171 cell samples with 2x106 to 2x103 cells are listed in Table S1. A drop in detection of CPL glycans was recorded at a concentration of 2x104 cells. In contrast, the RL(B) cell population

in

samples

with

concentration

from 1x107

predominantly the high-mannose glycans (e.g. Figure 3C).

to

1x103

cells exhibited

In general, the detection limit

for the non-Hodgkin lymphoma line RL(B) was 1x103 cells. On the other hand, 1x105 cells were needed of the HTB-171 cell line to obtain highly reproducible results for all four types of N-glycans including minor CPL components. It should be pointed out that the FANGS preparation method utilized for N-glycan analyses 3.8x105 Chinese hamster ovary (CHO) cells.15 However, no finding of polylactosamine glycans was reported in this particular study using the CHO cells, though



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14 detection of these glycans in our study of human cancer cells showed significant dependence on cell numbers. The FANGS method was developed and refined using the well-defined wild-type and mutant CHO cell system for the investigation of N-glycan profiles and could detect qualitative differences between variants of the CHO cell population. Yet, in addition to detailed analyses of the CHO cells, the study was very limited with regard to human cells.15 Considering the N-glycome study of the cultured human embryonic stem cells (hESC) by Satomaa et al, their analysis was performed using 1x105 cells harvested from the hESC cultures.39 Besides species differences and disparities in methods used in these two studies, the detection limit discrepancy for hESC 39 and CHO cells15 could also be explained by considerable divergence in the N-glycan phenotypes between these two cell types. Indeed, in our study of human cancer cells, notable differences were observed in N-glycan profiles between several cell lines. The most striking difference was found in the case of non-Hodgkin lymphoma cell population RL(B) expressing predominantly the HM glycans (~ 99%), as compared to other cancer cell lines. This narrow N-glycan phenotype of RL(B) cells most likely accounts for low cell number requirements for the detection limit. In the case of RL(B) lymphoma the lowest cell number necessary for determining the N-glycan profile was 103 cells. Evidently, the differentiation and tissue of cancer cell origin plays an essential role in formation of N-glycan phenotypes having a direct impact on cell number requirements for the detection limit. To further validate the efficiency of the CM and CMW extraction methods, a quantitative comparison of N-glycans was performed using samples of other cancer cell lines. In addition to the RL(B) and choriocarcinoma JEG-3 cells, two NSCLC cell lines (LuCa-6 and HTB-177) were processed under the same conditions using all three


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15 preparative procedures (Protocol 1, 2 and 3). The results are summarized in Table S2. In the case of the three human cancer cell lines (JEG, HTB-177 and LuCa-6), the concentration of 1x106 cells was sufficient to detect all N-glycan types even with the extraction procedure using detergents. However, only 1x10 5 cells were required to obtain comparable glycan profiles when samples were subjected to the CM or CMW extractions. The difference between methods was noticeable at 1x10 5 cells and particularly at lower cell concentrations. Conditions with limited cell numbers favor the extraction procedure based on chloroform-methanol. Importantly, although no significant discrepancy in N-glycan profiles was recorded using either the CM or CMW preparation, the CM extraction procedure consistently generated high-mannose glycans with one GlcNAc moiety in a number of specimens (Table S2 and next sections).

Difference between CM and CMW Extraction The investigation of N-glycans in several samples which underwent the CMW or CM extractions showed high comparability in N-glycan profiles. As noted above (Table S2; Protocol 3a,b), an important difference was observed at the level of HM glycan detections (Scheme 1B). Cell samples from choriocarcinoma JEG, histiocytoma TIB223, lung cancer cell lines SCLC HTB-171 and two NSCLC (HTB-177 and LuCa-6) when subjected to the CM extraction (in the absence of water) produced intensive peaks matching to HM-glycans with one GlcNAc residue - Man5-10 GlcNAc.

An example of detecting these structures

corresponding to m/z values 1144, 1306, 1468, 1630 and 1792 is shown in the cell sample of TIB 223 in Figure 3A. We use the term “truncated” forms to designate these uncommonly cleaved high-mannose structures generated in the presence of PNGase F.



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16 None, or only minor peaks corresponding to truncated forms, were detected in the MS spectra of samples that were denatured before digestion with detergent or 75 % ethanol (Figure S6-A). To substantiate the fact that the uncommon cleavage of N-glycans in the presence of PNGase F is associated with the samples in question, a ribonuclease was used as a substrate and subjected to the CM extraction. No truncated forms were detected in the ribonuclease samples, just typical oligo-mannose structures with the intact chitobiose core (Man5-10 GlcNAc2 ; Figure S6-C). The structural differences between high-mannose glycans typically observed after the PNGase F cleavage versus their truncated forms were deduced from their molecular masses and confirmed by MALDI-TOF-MS/MS analysis (Figure S7). The presence of high-mannose glycans with one GlcNAc moiety would be expected if Endo H recombinant enzyme was used for deglycosylation.40 This enzyme is known to cleave the bond in the diacetylchitobiose core of the oligosaccharide between two GlcNAc subunits. However, in our study only PNGase F was utilized and thus, the cleaved asparagine linked oligosaccharides were expected to have the intact chitobiose core (Scheme 2). Kurihara et al. reported that using non-aqueous CM extractions on the freezedried brain preserved enzymatic activities of four enzymes.28 Our detection of Endo H-like activity support and extend the original observation of Kunihara et al., since the four enzymes are not involved directly in processing of N-glycans. Currently available results strongly suggest that the CM extraction procedure has the capacity to preserve a broader scale of active enzymes not only in brain tissues but also in cancer cells originating from different tissues. Performing extractions at room temperature did not hamper Endo H-like enzymatic activities but increased temperature did. No truncated forms were detected when samples were subjected to heat-denaturation after the CM extraction. An example of this is


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17 apparent in the analysis of N-glycans in the TIB223 (Figure 3AB). Interestingly, in contrast to the cancer cell lines noted above, glycan profiles for the non-Hodgkin B-lymphoma line RL(B) were identical, regardless of the processing method used. As shown in Figure 3C, only the typical high-mannose glycans (m/z 1347, 1509, 1671, 1833, and 1995) were observed without detecting truncated forms.

Applicability in Clinical Settings The successful N-glycan profiling of specimens from the HTB-182 tumor tissue and several samples containing cancer cells in low quantities strongly suggested that the CM extraction procedure can be applicable for the investigation of N-glycans in biopsy materials of small sizes. The detailed analyses of 1-2 mm biopsies from human malignant lung tissues obtained via bronchoscopy from two patients, one diagnosed with SCLC (Figure 4A) and the other one with spinocellular lung carcinoma (Figure 4B) confirmed the presence of high-mannose, hybrid and two-tetra antennary complex structures with zero, one or more fucose residues. The significant presence of high-mannose (m/z 1347, 1509, 1671, 1833 and 1995), and core fucosylated glycans with none or one galactose (m/z 1007, 1575 and 1737) is frequently associated with malignancy. As recently reported,41,42 these oligosaccharides were downregulated in the healthy lungs, but increased presence of them was found in tumor tissues of patients with lung cancer. Moreover, our biopsy study of two patients revealed also the presence of truncated forms (Figure 4, m/z values in red) that were detected in several cancer cell lines including lung cancer cells originating from both, SCLC and NSCLC histological types.



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18 CONCLUSIONS We demonstrate two variations of an efficient preparative procedure for N-glycan analyses: (a) the CMW aqueous extraction for studying glycan profiles, and (b) the CM non-aqueous procedure making it possible to uncover an Endo H-like enzymatic activity via the detection of truncated high-mannose forms. The reported conditions are suitable for applying PHN derivatization enabling screening of the released N-glycans even without a purification step; informative MS analysis can be accomplished within 4 hours. Even when applying a purification step, detailed N-glycan profiles can be obtained in less than a day. The performance of both variants of the procedure was validated by analyses of different specimens: glycoproteins, tumor tissues, and cultured cancer cells requiring low cell numbers. Most importantly, we showed applicability of the method for the investigation of N-glycans released from small biopsies of lung cancer patient tissues. Moreover, the detection of truncated forms in patients’ lung cancer tissues and in several cell lines clearly demonstrated the presence of enzymatic activity strongly resembling the Endo H enzyme. To our knowledge, this is the first description of such uncommon cleavage of highmannose glycans from cell and tissue specimens under conditions of PNGase F deglycosylation. Currently, detailed study and potential applicability of the CM/CMW method in clinical settings are in progress.

ASSOCIATED CONTENT

Supporting information Additional supporting figures, scheme1 and tables are available free of charge on the ACS publication website at http://pubs.acs.org.


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19 Figure S1: Selected MS profiles obtained with different preparative procedures Figure S2: SDS-PAGE electrophoresis Figure S3: MS spectra of N-glycans recorded from unpurified versus purified digest Figure S4: MS spectra recorded from unpurified digests of human tumor HTB-182 Figure S5: MS spectra of complex poly-N-acetyl-lactosamine glycans Figure S6: Influence of preparative conditions on the preservation of Endo-H like activity Figure S7: Tandem spectra of high-mannose glycan with composition Man5 GlcNAc1-2 Scheme S1: Illustration of N-glycan structures and their abbreviations Table S1: The list of N-glycans detected in human cancer HTB-171 cell line Table S2: Comparison of N-glycans detected in different cell lines

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was carried out with the support of Proteomics Core Facility of CEITEC – Central European Institute of Technology under CIISB project, ID number LM2015043, funded by the Ministry of Education, Youth and Sports of the Czech Republic; the project CEITEC 2020 (LQ1601) with financial support from the Ministry of Education, Youth and Sports of the Czech Republic under the National Sustainability Programme II, and the University of Maryland School of Medicine through departmental funding of Animal Model Division of the Institute of Human Virology. Z.Z. thanks for the support of Czech Science Foundation project (no. P206/12/G151). We wish to thank D. Fridrichova for technical assistance with SDS experiments and R.C. Gallo is appreciated for his support of the project.



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23 FIGURE LEGENDS

Figure

1.

Comparison of N-glycan profiles obtained

from the

JEG-3 human

choriocarcinoma cells (~1x106 cells used for each experiment) with three different preparative conditions: bars in blue color represents the procedure using detergent and denaturation reagents (Protocol 1), bars in green denote the method based on extraction with 75% ethanol (Protocol 2) and bars in red represent chloroform-methanol-water (8:4:1). N-glycan compositions are depicted on the horizontal axis (H-hexose, NGlcNAc, F-fucose). The graphs were obtained from triplicate analysis for each protocol and represent the averaged relative intensities with shown error bars (±SD). Figure 2. MALDI-TOF-MS spectrum of SPE purified N-glycans released from human choriocarcinoma cells JEG-3 corresponding to 1x105 cells: A) at m/z 1000 - 2700; B) m/z between 2500-5000; and C) at m/z 3600 - 5000.

The sample was extracted with

chloroform-methanol (2:1) and denatured by heating in a waterbath (~95o C) prior to deglycosylation. All ions are as MNa+. Because of space not all structures are depicted. Suggested glycan structures not currently represented within the human and mouse glycan databases are indicated by a red asterisk. Key symbols:

Fuc;

GlcNAc;

Man;

Gal;

Figure 3. MALDI-TOF-MS spectra of N-glycans obtained from samples (106 cells) subjected to extraction with chloroform-methanol (2:1): A) TIB223 incubated with PNGase F after extraction; B) TIB223 after extraction was denaturated with heating (95o C, waterbath) prior to incubation with PNGase F; and C) Non-Hodgkin B-lymphoma RL(B) after CM extraction underwent to incubation with PNGase F. Glycans are labeled with PHN (+90.06 increases) as described in the experimental section. Peaks with m/z values in red color correspond to compositions Man4-9 GlcNAcPHN. Figure 4. MALDI-TOF/TOF-MS spectra of N-glycans from lung tissue obtained by bronchoscopy from non-smoker patients: A) 40-year-old woman diagnosed with SCLC, and B) 68-year-old man diagnosed with spinocellular carcinoma. N-glycans were released from biopsies (1x2mm) extracted with CM; after incubation for 2 hours with



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24 PNGase F, the digest was SPE purified and labeled with PHN (+90.06 Da).

SCHEME LEGENDS

Scheme 1. Schematic outline of: (A) the procedure based on chloroform-methanol extraction for the investigation of N-glycans in cancer cell lines and tumor tissue specimens; and (B) the influence of extraction conditions on the cleavage of highmannose glycans and its role in Endo H-like activity detection in the cancerous specimens (red line).

Scheme 2. Schematic illustration of differences between the cleavage by PNGase F and Endo H, two specific glycosidases commonly used for the investigation of glycoproteins.


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25

Table 1. Quantitative comparison of N-glycans detected by MALDI-TOF/TOF-MS in HTB-171 and RL(B) cell lines at different concentration of cells.

Cell line

Number of cells

HM

HB

CP

CPL

6

+

+

+

+

6

+

+

+

+

5

+

+

+

+

5

+

+

+

+

5

+

+

+

+

5

+

+

+

+

4

+

+

+

±

3

+

+

+

-

3

+

+

+

-

7

+

-

-

-

6

+

-

-

-

5

+

-

-

-

4

+

-

-

-

3

+

-

-

-

2x10 1x10 HTB-171 small cell lung cancer

5x10 2.5x10

2x10 1x10 2x10 5x10 2x10

1x10 1x10 RL(B) non-Hodgkin B-lympoma

Detected N-glycans*

1x10 1x10 1x10

* The schematic illustration of abbreviations used for glycan types can be seen in the Scheme S1. High-mannose (HM), hybrid (HB), complex (CP), complex polylactosamine with masses > 3200 (CPL) For more details about N-glycan structures analyzed in HTB-171 cancer cells see the Table S1 (+) N-glycans repeatedly detected; (±) detected < 3900 Da; (-) detected sporadically or signal intensities S/N < 3.



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Supernatant

A. Cancer specimens + CHCl3+CH 3OH+H 2O or CHCl3+CH 3OH

Cancer pellets +

PNGase F

PHN labelling MALDI-MS, MS/MS

medium-throughput screening

Purification

PHN labelling MALDI-MS, MS/MS (deep gly can profiling)

B. 1. CHCl3+ CH3OH+ H2O 2. PNGase F

1.CHCl3+ CH3OH

Scheme 1. Schematic outline of: (A) the procedure based on chloroform- methanol extraction for the investigation of N-glycans in cancer cell lines and tumor tissue specimens; and (B) the influence of extraction conditions on the cleavage of high- mannose glycans and its role in Endo H-like activity detection in the cancerous specimens (red line).


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[%]

Protocol 1 Protocol 2 Protocol 3a

20

15

10

5

0

Figure 1


100

3600

3800 3250

5x

50

4000

* 3500

6x 3750

6x 7x

6x 8x

4200 7x

4000

4400 4250

6x

7x

* *

4600

Figure 2

ACS Paragon Plus Environment 4500

7x

4800

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4x

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4x

2200 2400 2600

7x

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5x 1800

4658.7

3000 3x

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1995.8

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 Intens. [a.u.]

Journal of Proteome Research Page 28 of 32

A

m/z

5x 8x

B

2x

*

7x m/z

8x 8x

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m/z

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Endo H

Asn

0-9

PNGase F

Scheme 2. Schematic illustration of differences between the cleavage by PNGase F and Endo H, two specific glycosidases commonly used for the investigation of glycoproteins.


1000

* 1347.5

1.0 1509.6

1833.7

1250

1500

2.0

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2250

Figure 3


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Journal of Proteome Research Page 30 of 32

x10 4

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x10 4

A

2800

2800

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*

0.0 m/z


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1. CHCl3 + CH3OH+ H2O 2. PNGase F

1. CHCl3+ CH3OH 2. PNGase F

For TOC only


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Efficient Procedure for N-Glycan ... - ACS Publications

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