Evaluation of Different N-Glycopeptide Enrichment Methods for N

2 Aug 2016 - In this work, we identified a total of 3446 unique glycosylation sites conforming to the N-glycosylation consensus motif (N-X-T/S/C; X â‰...
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Evaluation of Different N‑Glycopeptide Enrichment Methods for N‑Glycosylation Sites Mapping in Mouse Brain Chengqian Zhang,†,‡,§ Zilu Ye,†,‡,§ Peng Xue,† Qingbo Shu,†,‡ Yue Zhou,†,‡ Yanlong Ji,†,‡ Ying Fu,†,‡ Jifeng Wang,† and Fuquan Yang*,†,‡ †

Laboratory of Protein and Peptide Pharmaceuticals & Laboratory of Proteomics, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China ‡ University of Chinese Academy of Sciences, Beijing100049, China S Supporting Information *

ABSTRACT: N-Glycosylation of proteins plays a critical role in many biological pathways. Because highly heterogeneous N-glycopeptides are present in biological sources, the enrichment procedure is a crucial step for mass spectrometry analysis. Five enrichment methods, including IP-ZIC-HILIC, hydrazide chemistry, lectin affinity, ZIC-HILIC-FA, and TiO2 affinity were evaluated and compared in the study of mapping N-glycosylation sites in mouse brain. On the basis of our results, the identified N-glycosylation sites were 1891, 1241, 891, 869, and 710 and the FDR values were 3.29, 5.62, 9.54, 9.54, and 20.02%, respectively. Therefore, IPZIC-HILIC enrichment method displayed the highest sensitivity and specificity. In this work, we identified a total of 3446 unique glycosylation sites conforming to the N-glycosylation consensus motif (N-X-T/S/C; X ≠ P) with 18O labeling in 1597 N-glycoproteins. N-glycosylation site information was used to confirm or correct the transmembrane topology of the 57 novel transmembrane Nglycoproteins. KEYWORDS: N-glycosylation sites, ZIC-HILIC, lectins, TiO2, hydrazide, mouse brain

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INTRODUCTION N-Glycosylation is one of the most common and significant post-translational modifications (PTMs) of proteins. Nglycosylation affects protein folding, trafficking, stability, and interactions with other molecules and plays an important role in many cellular processes.1,2 Therefore, the accurate large-scale identification of N-glycopeptides and N-glycosylation sites is of vital importance. Because there is a minor portion of Nglycopeptides in comparison with peptides after digestion, specific and efficient enrichment strategies are developed for analyzing complex protein mixtures, such as lectin affinity,3,4 TiO2 affinity,5,6 hydrophilic interaction chromatography (HILIC),7,8 and hydrazide chemistry.9,10 On account of different characteristics of these methods, in recent years, a combination of two or three of these methods is commonly applied, primarily to increase the coverage of N-glycosylation sites. Parker et al. parallelly applied hydrazide chemistry, TiO2, and ZIC-HILIC with and without ion-pairing agent from ex vivo rat left ventricular myocardium.11 Combined IP-ZICHILIC with non-IP ZIC-HILIC and ZIC-HILIC contributed more N-glycosylation site information than either hydrazide capture or TiO2. In addition, IP-ZIC-HILIC obtained more identified N-glycopeptides than the other methods. Zhu et al. combined click maltose-HILIC and improved hydrazide chemistry to comprehensively identify 4783 N-glycosylation sites corresponding to 2210 N-glycoproteins in human liver.12 © XXXX American Chemical Society

Their results showed enrichment complementarities of these two methods, and click maltose-HILIC provided a little higher enrichment capacity than hydrazide chemistry. Li et al. combined hydrazide chemistry and ZIC-HILIC methods in the N-glycosylation sites mapping of the secretome of human metastatic hepatocellular carcinoma cell lines.13 In this work, the two enrichment methods were somewhat complementary, and ZIC-HILIC provided a much higher number of identified N-glycosylation sites than hydrazide chemistry. Xue et al. compared N-glycosylation sites from lectins and hydrazide chemistry in HepG2 cells, and their results showed that lectin enrichment method was better than hydrazide chemistry method.14 Moreover, the two methods did not show significant complementarity. A comprehensive comparison of these different N-glycopeptides enrichment methods is still missing. The brain serves as the center of the mammal nervous system and is composed of neurons, glial cells, and blood vessels and has specialized functions in neural signal transmission. N-Glycosylation has been associated with a spectrum of biological processes in brain including the nervous system development, synaptic transmission, learning, memory, and processing of sensory information.15,16 It has been reported that direct loss of GlcNAc transferase alters the structure and Received: February 5, 2016

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DOI: 10.1021/acs.jproteome.6b00098 J. Proteome Res. XXXX, XXX, XXX−XXX

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Journal of Proteome Research function of Drosophila neuromuscular junctions as well as locomotory behavior.17 N-glycoproteins are widely located in the cell surface, intracellular, and are also abundantly secreted. N-glycoproteins secreted or expressed on the cell surface could be potential drug targets for a variety of neoplastic diseases.18 A comprehensive mapping of N-glycosylation sites of mammal brain will be of particular importance in the study of the function of N-glycoproteins in brain. During the past few years, post-translational modifications such as glycosylation19 and phosphorylation20 have been incorporated to predict and correct the 3D membrane structure of protein.21,22 Knowledge of protein structure provides crucial information for understanding protein function as well as location information. Membrane proteins typically contain a number of transmembrane alpha helices and connect to other molecules by intra- and extracellular loops. Although many bioinformatics tools have been developed to predict protein topology,23−25 only a few proteins have experimentally determined structures regarding extracellular versus cytoplasmic in UniProt. The N-glycosylation site of proteins is restricted to locate in the extracellular region of plasma membrane; therefore, the site information used as constraint condition is significant to provide experimental evidence.26,27 In the study of N-glycosylation sites mapping of mouse brain, five enrichment methods, including lectin affinity, TiO2 affinity, ZIC-HILIC-FA (with 0.1% formic acid in mobile phases), IPZIC-HILIC (with 0.1% trifluoroacetic acid as ion-pairing reagent in mobile phases), and hydrazide chemistry, were evaluated and compared. The incorporation of H218O improves the confidence in the N-glycosylation site assignment.28 The sensitivity of each method was evaluated based on the number of identified N-glycosylation sites; the specificity of each enrichment method was evaluated based on the calculated FDR. The systematic methodological evaluation facilitates a more complete understanding of these enrichment methods.

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remaining debris and unbroken cells were removed by centrifugation at 14 000g at 4 °C for 10 min. Protein yield was quantified by BCA assay. Clarified supernatants were transferred to 30 kDa ultrafiltration units and rinsed three times with UA (8 M urea in 0.1 M Tris/HCl pH 8.5). Total protein was reduced with 10 mM 1,4-dithiothreitol (DTT) at 37 °C for 1 h and subsequently alkylated in 40 mM iodoacetamide (IAA) for 30 min at room temperature in the dark. The filters were washed twice with UA, followed by three washes with 50 mM NH4HCO3. Finally, trypsin was added to 300 μL of 50 mM NH4HCO3 to each filter at a 1:50 trypsin-to-protein mass ratio at 37 °C overnight. The samples were collected at 14 000g for 10 min, and the digestion process was stopped by acidifying the peptide solution. All tryptic peptide samples were desalted on an Oasis HLB SPE column and then lyophilized under vacuum. Enrichment Methods for N-Glycopeptides

ZIC-HILIC Enrichment (IP-ZIC-HILIC and ZIC-HILIC-FA) and Deglycosylation. Two different mobile phases were chosen to investigate the glycopeptide enrichment efficiency of ZIC-HILIC SPE. For ZIC-HILIC-FA, binding buffer (0.1% FA, 19.9% H2O, 80% ACN, v/v) and elution buffer (0.1% FA, 99.9% H2O, v/v) were used. For ZIC-HILIC-TFA, binding buffer (0.1% TFA, 19.9% H2O, 80% ACN) and elution buffer (0.1% TFA, 99.9% H2O) were used. Here TFA was performed as ion-pairing reagent, so this method was also named as IPZIC-HILIC. For the ZIC-HILIC enrichment, the ZIC-HILIC beads were first activated with 200 μL of elution buffer for 30 min and then washed with binding buffer twice. About 200 μg tryptic peptides mixture was dissolved in 500 μL of binding buffer and mixed with 10 mg activated ZIC-HILIC resin at a 1:50 peptide-to-material mass ratio in a microcentrifuge tube.30 The tube was shaken over a vortex mixer for 1 h. The slurry was transferred to a 200 μL tip plugged with C8 membrane. The flow-through (FT) fraction was collected by centrifuging at 1000g for sequential enrichment. Subsequently, the resin was washed six times with 100 μL of binding buffer and eluted five times with 100 μL of elution buffer. The eluted peptides were dried by vacuum centrifugation and then dissolved in 50 mM NH4HCO3 buffer (H218O) and deglycosylated with PNGase F at 37 °C for 2.5 h. Lectin Affinity Enrichment and Deglycosylation. NGlycopeptides were enriched by lectin affinity following the protocol of Mann3 with some modifications. Three agarosebound lectins (33 μL of ConA, 28.55 μL of WGA, and 50 μL of RCA I) were packed in the Pierce spin columns and activated with 50 mM NH4HCO3 in 10% (v/v) ACN. 200 μg peptides was mixed with the lectins at a 1:2 peptide-to-lectin mass ratio and incubated at 4 °C overnight and then washed with 100 μL of 50 mM NH4HCO3 in 10% (v/v) ACN and 50 mM NH4HCO3 six times, respectively, by centrifugation. The FT fractions were assembled for further sequential enrichment experiment. Finally, deglycosylated peptides were released via PNGase F digestion in 300 μL of 50 mM NH4HCO3 buffer (H218O) at 37 °C for 2.5 h. Hydrazide Chemistry Enrichment and Deglycosylation. Hydrazide chemistry was performed according to the procedure described in by Zhang,31 with some modifications. In brief, 200 μg peptides were dissolved in binding buffer (0.5% formic acid, 60% ACN) and oxidized with 10 mM NaIO4 at room temperature in the dark for 1 h. The solution was diluted 12-fold, and excess NaIO4 was removed by desalting using an Oasis HLB column. The elution solution was collected directly

MATERIALS AND METHODS

Chemicals and Materials

H218O (97% 18O) was purchased from Cambridge Isotope Laboratories (Andover, MA). Sequencing-grade modified trypsin was purchased from Promega (Madision, WI). PNGase F (500 000 units/mL) was from New England Biolabs (Ipswich, MA). Hydrazide resin was purchased from Bio-Rad (Hercules, CA), Titanium dioxide (10 μm) was purchased from GL Science (GL Science, Japan), lectins were from Vectorlabs (Burlingame, CA), and ZIC-HILIC (10 μm, 100 Å) was from Merck (Darmstadt, Germany). Pierce Spin Columns were from Thermo Fisher Scientific (San Jose, CA). Oasis HLB solid reverse-phase columns were from Waters (Bedford, MA). All other chemicals and solvents used were obtained from Sigma (St. Louis, MO), and all solutions were made with ultrapure Milli-Q water (Millipore, Bedford, MA). Protein Extraction and Digestion

Eight-week-old C57BL/6J mice were euthanized by decapitation, and total brains were removed and stored at −80 °C until further use. An N-glyco-FASP protocol described by Zielinska and Wisniewski was modified and used to enrich Nglycopeptides for further analysis.29 In brief, a total of three mouse brains were pooled and homogenized using a homogenizer with extraction buffer containing 4% SDS (W/ V) in 0.1 M Tris-HCl (pH 7.6), followed by incubation at 95 °C for 3 min. The lysate was sonicated three times, and the B

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Figure 1. Schematic diagram of different N-glycopeptides enrichment methods.

into the hydrazide resin spin columns (50 μL of 50% slurry for each sample) and incubated at room temperature overnight with rotation. After the reaction, the resin was washed three times with 50% ACN, 1.5 M NaCl, water, and 50 mM NH4HCO3 buffer sequentially. Finally, deglycosylated peptides were released via PNGase F digestion in 300 μL of 50 mM NH4HCO3 buffer (H218O) at 37 °C for 2.5 h. TiO2 Affinity Enrichment and Deglycosylation. NGlycopeptides were enrichment by TiO2 affinity following the protocol of Larsen.5 200 μg tryptic peptides was treated twice with 20 U of alkaline phosphatase in 100 μL of 50 mM NH4HCO3, at 37 °C for 1 h, and the sample volume was then adjusted to 1 mL by adding 900 μL of TiO2 loading buffer (1 M glycolic acid in 80% ACN, 5% TFA, 15% H2O) to avoid coenrichment of phosphorylated peptides. The TiO2 bead slurry was prepared by weighing 10 mg of TiO2 and adding 100 μL of ACN. Twelve μL of the slurry was added above peptides solution and incubated for 30 min at room temperature with gentle shaking. The beads were washed with 400 μL of loading buffer, 400 μL of washing buffer 1 (80% ACN, 1% TFA, 19% H2O), and 400 μL of washing buffer 2 (20% ACN, 0.1% TFA, 79.9% H2O), respectively, to get rid of nonglycopeptides. The N-glycopeptides were eluted by incubating the beads with 400 μL of elution buffer (25% NH4OH, pH 11.3) with shaking at room temperature for 20 min. The supernatant containing Nglycopeptides was collected by centrifugation at 1000g for 5 min and then dried in a vacuum centrifuge. The dried peptides were dissolved in 50 mM NH4HCO3 buffer (H218O) and deglycosylated with PNGase F at 37 °C for 2.5 h. Sequential Enrichment (IP-ZIC-HILIC/Lectin Affinity and Lectin Affinity/IP-ZIC-HILIC). The flow-through (FT) fraction from IP-ZIC-HILIC was lyophilized and dissolved in 50 mM NH4HCO3, followed by lectin affinity enrichment. The FT fraction from lectin affinity enrichment was lyophilized and dissolved in 500 μL of binding buffer (0.1% TFA, 19.9% H2O, 80% ACN), followed by IP-ZIC-HILIC enrichment.

Mass Spectrometry Analysis. Each deglycosylated peptides sample was dissolved in 5 μL of 0.1% FA and loaded onto an in-house packed C18 trap column (100 μm ID × 2 cm, 5 μm, Reprosil-Pur C18 AQ, Dr. Maisch, Germany) and then separated on an in-house packed C18 analytical column (75 μm ID × 20 cm, 3 μm, Reprosil-Pur C18 AQ, Dr. Maisch). Mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in ACN) were used to establish the 78 min gradient comprised of 5−8% B, 8 min; 8−22% B, 50 min; 22−32% B, 12 min; 32− 95% B, 1 min; 95% B, 7 min at a constant flow rate of 280 nL/ min. EASY-nLC 1000 HPLC system (Thermo Scientific) was connected to a Q Exactive mass spectrometer (Thermo Scientific). The mass spectrometer was operated in positive ion mode with data-dependent mode. Full-scan MS spectra (from m/z 300 to 1600) were acquired in the Orbitrap at a high resolution of 70 000 (m/z 200) with an automatic gain control (AGC) of 3 × 106 and a maximum fill time of 60 ms. The 20 most intense ions were sequentially isolated and fragmented in the HCD collision cell with normalized collision energy of 27%. Fragmentation spectra were acquired in the Orbitrap analyzer with a resolution of 17 500 at m/z 200. Ions selected for MS/ MS were dynamically excluded for a duration of 40 s. Peptides carrying from two up to five positive charges were chosen for fragmentation. All raw data were viewed in Xcalibur v2.2. Data Analysis. The raw data were analyzed with Proteome Discovery v1.4 (Thermo Scientific) using Sequest HT search engine for protein identification and Percolator for FDR (false discovery rate) against a UniProt Mouse protein database (50 837 entries, updated on 06-2013) with a common contaminant database (215 entries). Enzyme was set to trypsin allowing N-terminal cleavage to proline, and two missed cleavages were allowed. Database searches were performed with the following parameters: precursor mass tolerance was up to 10 ppm and the product ion mass was up to 0.02 Da. Cysteine carbamidomethylation was set as a fixed modification and Nterminal acetylation, methionine oxidation, deamidation of C

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Journal of Proteome Research asparagine (+0.9840 Da), and deglycosylated asparagine 18O labeling (+2.9890 Da) were set as variable modifications. FDR analysis was performed with Percolator, and q < 1% was set for protein identification based on the decoy database search results. The peptide confidence level was set at high for peptides filter. An in-house Perl program was used to count the number of N-glycosylation sites by mapping corresponding deglycosylated peptides to the protein based on Proteome Discovery v1.4. Function Annotation. Subcellular and function categories were based on the annotations of PANTHER Web site.32 The topology of identified transmembrane glycoproteins was predicted using TMHMM 2.0.24 Pathway analysis was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway collection.33

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RESULTS AND DISCUSSION

Figure 2. Workflow for the mapping of N-glycosylation sites with different enrichment methods.

Experimental Design and Data Analysis

The aim of this study was to evaluate the performance of the five most commonly used enrichment strategies for the mapping of N-glycosylation sites of mouse brain. All enrichment experiments and replicates were performed from the same pooled and extracted protein samples to reduce experimental variability from the sample source. All enriched glycopeptides were deglycosylated with PNGase F in H218O to enable the identification of N-glycosylation sites with higher sensitivity. In addition, the deglycosylation reaction time was reduced from overnight to 2.5 h, which greatly decreases the potential influence of spontaneous deamidation during PNGase F treatment. Each enrichment method was independently prepared in triplicate and analyzed by three LC−MS/MS runs. For the lectin affinity enrichment method, three kinds of lectins, concanavalin A (ConA) recognizing high-mannose type N-glycans, wheat germ agglutinin (WGA) recognizing GlcNAc or sialic acid, and Ricinus communis agglutinin I (RCA I) recognizing galactosylated complex-type N-glycans, were selected.34−36 The three lectins have a broad spectrum of recognition for glycans epitopes. HILIC is a good one-step Nglycopeptide-enrichment method without chemical modification and has no need to use any glycosidase to specifically release N-glycopeptides from the solid support. Among the various HILIC materials, ZIC-HILIC has been shown to possess high selectivity for the enrichment of glycopeptides.37 TFA, as an ion-pairing reagent, increases the difference in hydrophilicity between glycopeptides and nonglycosylated peptides in the IP-ZIC-HILIC method.7 However, ZICHILIC-FA method was included in this study as well to obtain comprehensive understanding of ZIC-HILIC enrichment method. TiO2 affinity enrichment and hydrazide chemistry enrichment methods were processed following the procedures previously reported with modifications. Figure 1 is the schematic diagram of different enrichment approaches.7,38,39 Figure 2 shows the workflow for N-glycosylation sites analysis with different enrichment methods. On the basis of the FASP method, the protein mixture was reduced and alkylated in 30 kDa ultrafiltration units. 200 μg tryptic peptides was used for each enrichment procedure. To enable the identification of N-glycosylation sites with higher accuracy, all enriched Nglycopeptides were deglycosylated with PNGase F in H218O. Deglycosylated peptides were analyzed by LC−MS/MS. Each enrichment method was repeated three times.

In total, 3446 unique N-glycosylation sites containing the motif (N-X-T/S/C; X ≠ P) as well as the 18O labeling assigned to 1597 N-glycoproteins were identified in this study. Information for the unique N-glycosylation sites was listed in Supplementary Table 1. Compared with previous work,3 we got 1629 N-glycosylation sites (containing the motif and 18O labeling) in common. The combination of our results could improve the data set of N-glycosylation sites in mouse brain tissue. Comparison of Five Enrichment Methods and Sequential Enrichment Methods

Table 1 and Supplementary Table 1 show the summary of identification of unique N-glycosylation sites using different enrichment methods. The statistical results include the number of the identified N-glycosylation sites with 18O labeling, number of the identified N-glycosylation sites conforming to the characteristic motif (N-X-T/S/C; X ≠ P), number of the Nglycosylation sites conforming to the motif and 18O labeling, and the FDR values of each method. The results indicate that the IP-ZIC-HILIC is the most efficient enrichment method for N-glycopeptides. ZIC-HILIC, unbiased toward the N-glycopeptides and without initial chemical derivation, has been widespread used and highly regarded. It has been previously reported that the ion-pairing reagent used in the IP-ZIC-HILIC method increases the difference in hydrophilicity between glycosylated peptides and nonglycosylated peptides. Therefore, the reduced suppression of the glycopeptides ionization caused by the coenriched hydrophilic nonglycopeptides renders IPZIC-HILIC method superior to the ZIC-HILIC-FA method.7 The hydrazide chemistry method is the second most efficient method for enrichment. However, these top two methods also have some limitation. IP-ZIC-HILIC method might result in the loss of N-glycopeptides with high hydrophobic peptide moieties or N-glycopeptides not having minimum degree of local hydrophilicity to enable ZIC-HILIC retention.40 For the hydrazide chemistry method, periodate oxidation converts the cis-diol groups of multiple monosaccharide residues to aldehydes as well as oxidizes methionine (Met), carbamidomethylated cysteine (CamCys), and tryptophan (Trp) residues.37,41,42 However, compared with standard database search, adding oxidized Trp and CamCys as variable modifications could not increase the number of identified ND

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Journal of Proteome Research Table 1. Summary of Identified N-Glycosylation Sites Using Different Enrichment Methods enrichment methods IP-ZIC-HILIC 1 IP-ZIC-HILIC 2 IP-ZIC-HILIC 3 IP-ZIC-HILIC hydrazide 1 hydrazide 2 hydrazide 3 hydrazide ZIC-HILIC-FA 1 ZIC-HILIC-FA 2 ZIC-HILIC-FA 3 ZIC-HILIC-FA lectin 1 lectin 2 lectin 3 lectin TiO2 1 TiO2 2 TiO2 3 TiO2

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O-labeled sites 1977 1992 2106 2025 1493 1369 1139 1334 982 956 976 971 906 971 997 958 760 745 766 757

Motif

Motif & 18O

FDR (%)

1915 1942 2009 1955 1452 1347 1143 1314 956 957 970 961 946 985 1023 985 888 874 901 888

1850 1879 1944 1891 1377 1284 1063 1241 872 862 874 869 840 908 925 891 703 698 729 710

3.39 3.24 3.23 3.29 5.17 4.68 7.00 5.62 8.79 9.92 9.90 9.54 11.2 7.82 9.58 9.54 20.83 20.14 19.09 20.02

a18 O-labeled sites refer to the number of the identified N-glycosylation sites displaying an 18O mass shift; Motif refers to number of deamidated sites with the characteristic motif (N-X-T/S/C; X ≠ P); Motif & 18O refers to the number of the N-glycosylation sites containing the motif as well as the 18O labeling. FDR refers to the calculated FDR values of each method. Numbers in bold are the average values of three technical replicates in each method.

Figure 3. (A) Venn diagram of the identified N-glycosylation sites between IP-ZIC-HILIC and other methods. The result of each method is the summation of three technical replicates. (B) Average numbers of identified N-glycosylation sites in IP-ZIC-HILIC and the sequential enrichment methods.

glycosylation sites in our study (Supplementary Table 3) because much wider search space will lead to more complicated searching algorithm and the reduction of search sensitivity.42 As shown in Table 1, lectin affinity method yielded similar results to that of ZIC-HILIC-FA. Even though three kinds of lectins were employed, it is impossible to completely cover all types of N-glycans. The TiO2 affinity method yielded the lowest number of N-glycosylation sites because TiO2 does not bind all forms of N-glycans but is highly selective for sialic acid containing glycopeptides.6 It is worth mentioning that the phosphatase-treated procedure could not remove all phosphate groups from the peptide samples, which may influence the enrichment of N-glycopeptides. Because the IP-ZIC-HILIC method displayed the highest enrichment efficiency, we next investigated the overlap of unique N-glycosylation sites between IP-ZIC-HILIC and the other four methods. Figure 3A shows that the four methods (hydrazide chemistry, ZIC-HILIC-FA, lectin affinity, and TiO2) appeared a certain degree complementarity with IP-ZICHILIC. To investigate the complementarity, we performed a sequential enrichment using IP-ZIC-HILIC/lectin affinity and lectin affinity/IP-ZIC-HILIC. The flow-through (FT) fractions from IP-ZIC-HILIC or lectin affinity were enriched by lectin affinity or IP-ZIC-HILIC. The results (Figure 3B, Supplementary Table 4) indicate that IP-ZIC-HILIC alone is almost as effective as the sequential enrichment methods because the sequential methods do not substantially increase the number of identified N-glycosylation sites.

deamidation spontaneously converts asparagine into aspartic acid, resulting in an identical mass increment.43 To discriminate between spontaneous and enzymatic deamidations for largescale analysis, we used two commonly applied strategies: First, N-glycosylation sites were confirmed on the basis of the (N-XT/S/C; X ≠ P) motif,44 as the presence of asparagine in the specific consensus sequence of this motif is essential for the glycan to be transferred to a nascent polypeptide.45 However, the outcome of this strategy is negatively influenced by spontaneous deamidation at the glycosylation motif. Second, the enzymatic deglycosylation was performed in the presence of H218O to get rid of the interference by spontaneous deamidation occurring during sample preparation.4,46 As a consequence, an increase in the mass of ΔM = 2.9890 Da was used as a criterion to confirm N-glycosylation sites.3 However, spontaneous deamidation could still occur with 18O incorporation during PNGase F treatment overnight, especially within N-G, N-S, and N-D sequences.47,48 In this study, we applied both of the two strategies to confirm the N-glycosylation sites. The 18O-labeled N-glycosylation sites conforming to the consensus motif (N-X-T/S/C; X ≠ P) were confirmed as final results. To investigate the accuracy of each method, we compared their false discovery rates (FDRs). The 18O-labeled Nglycosylation sites conforming to the consensus motif (N-XT/S/C; X ≠ P) were defined as “true” results, and the glycosylation sites conforming to N-X-S/T/C motif without

Impact of Spontaneous Deamidation on Different Methods

During MS data analysis, N-glycosylation sites are identified on the basis of a mass increment of ΔM = 0.9840 Da. However, E

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Figure 4. (A) Distribution of identified N-glycosylation site motifs (N-X-T/S/C; X ≠ P). (B) Number distribution of the number of N-glycosylation sites for per identified N-glycoprotein.

O labeling were defined as “false” results. The FDR was calculated by dividing the number of false results by the number of all identified glycosylation sites in accord with the motif. The identification of glycosylation sites as “false” mainly occurred as a result of nonspecific adsorption. H218O used during the deglycosylation procedure increases the confidence in the identification of the correct sites. Furthermore, it allows for evaluation of the influence of spontaneous deamidation on different enrichment methods. The five methods resulted in the FDR at 3.29% (IP-ZIC-HILIC), 5.62% (hydrazide), 9.54% (ZIC-HILIC-FA), 9.54% (lectin affinity), and 20.02% (TiO2). The fact that the IP-ZIC-HILIC method resulted in the lowest FDR indicates its high accuracy for N-glycopeptide enrichment. In this study, the TiO2 method shows the highest FDR, which is different from the literature.47 Our results suggest that TiO2 affinity enrichment is not an ideal method for N-glycopeptide enrichment due to unacceptable levels of nonspecific binding of nonglycopeptides, resulting in high FDR values. Chemical deamidation could occur at both glutamine and asparagine residues under strongly acidic, neutral, and basic conditions. The generated glutamic acid and aspartic acid are acidic amino acids. We speculate that the ability of TiO2 to adsorb acidic amino acid is responsible for this phenomenon.38 However, TiO2 affinity enrichment is still an efficient and simple method for the selective purification of SA-containing glycopeptides. It is worth mentioning that the purity of H218O (97% 18O) and back-exchange would influence the FDR values. These negative influences were inevitable in all methods, resulting in an overestimation of FDR values. However, the relative accuracy of the comparison among five methods will not be affected. 18

New Information for Transmembrane N-glycoprotein Topology Prediction by N-Glycosylation Analysis

In our work, among the identified glycoproteins, 980 were annotated in UniProt KB as known glycoproteins and 617 as novel. 164 of the 617 novel N-glycoproteins are annotated as transmembrane (TM) proteins, few of which have detailed topology information in UniProt. We applied the information on identified N-glycosylation sites information to provide evidence of orientation prediction of the 164 novel TM Nglycoproteins. On the basis of the subsection of the “Topology” information from UniProt, 57 of the 164 novel TM N-glycoproteins only possess one helix domain. For these single spanning TM proteins, N-glycosylation site information has been used to determine the spatial orientation (Supplementary Table 5). There are almost no inconsistent results of confirmed extracellular domains with the results from TMHMM program except three proteins. For multispanning TM proteins, the reliable site information could be used to confirm some potential extracellular domains as the site information could not cover the whole sequence. In addition, the demonstrated topology structure information could be used as a filter for glycosylation site confirmed. N-Glycosylation modification, a crucial part of the primary protein structure, cannot be read from the gene yet provides significant information for understanding protein function as well as secondary structure topological information. Applying this information facilitates the locating of the extracellular segment based on prior topology knowledge and results in improvement of prediction accuracy. Functional Annotation of Identified N-Glycoproteins

Cellular components and molecular functions for the 1597 Nglycoproteins identified in the mouse brain were clustered by PANTHER (Figure 5A,B). About 65% of the N-glycoproteins are membrane or extracellular proteins. The majority of the Nglycoproteins were involved in binding activity, catalytic activity, receptor activity, and transporter activity. Further pathway analysis by KEGG mapped seven significant pathways (p < 0.05) (Figure 5C). Several pathways are closely related to the network of neurons such as neuroactive ligand−receptor interaction, axon guidance, and ECM−receptor interaction.

Identification of N-Glycosylation Sites of Mouse Brain Tissue

In this study, we totally identified 3446 unique N-glycosylation sites that matched with the N-glycosylation consensus motif (N-X-T/S/C; X ≠ P) as well as 18O labeling. The Web sitebased programs motif-X49 and Weblogo50 were used to characterize the motif composition of identified N-glycosylation sites. Figure 4A shows the distribution of the three motifs at 58.6% (N-X-T), 40% (N-X-S), and 1.4% (N-X-C). These results are similar to those from previous reports.4,12 We identified one single N-glycosylation site in 53% of all glycoproteins and two N-glycosylation sites in 22% of those (Figure 4B).

Major Groups of Identified N-glycoproteins in Mouse Brain Tissue

On the basis of our results, receptor and transporter proteins are two major groups of identified N-glycoproteins in mouse F

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Journal of Proteome Research

proteins.15 Thus, comprehensive characterization about Nglycosylation status is important for the understanding of the mechanisms of synapses. In a previous study, N-glycoproteome of murine synaptomes was investigated with an identification of 375 N-glycoproteins.51 Our work covered 81% N-glycoproteins of their results, which showed high coverage of synaptic membrane proteins. IP-ZIC-HILIC alone covered 73% N-glycoproteins, providing a potential capacity to investigate the synaptic membrane Nglycoproteins. It is worth mentioning that our results covered all N-glycosylation sites of the neurotransmitter receptors in their data except two sites. For 90% of the receptor proteins, we identified more N-glycosylation sites than their results.

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CONCLUSIONS In our study, five N-glycopeptide enrichment methods were used and compared for the enrichment and profiling of Nglycosylation sites in mouse brain. It is very important to have a direct comparison of different N-glycopeptide enrichment methods to evaluate their strengths and faults. IP-ZIC-HILIC was proved to have a higher coverage and lower FDR value for assigning N-glycosylation sites than other four methods. In total, 3446 unique N-glycosylation sites containing the motif (N-X-T/S/C; X ≠ P) as well as the 18O labeling in 1597 Nglycoproteins were identified in mouse brain tissue. We applied the N-glycosylation sites information to increase the accuracy in prediction of transmembrane topology. Because N-glycosylation always plays a key role in brain development and homeostasis, our work should facilitate the functional research of brain.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.6b00098. Figure S-1. N-glycosylated receptors or transporters in pre- and postsynaptic neurons. Table S-3. Statistical results of N-glycosylation sites numbers with or without oxidized Trp, and CamCys as variable modifications in hydrazide methods. Table S-6. N-glycosylation sites and the role of N-glycosylation on major groups of receptor and transporter N-glycoproteins. (PDF) Table S-1. Detailed information about the identified 3446 unique N-glycosylation sites that conforming to the N-glycosylation consensus motif (N-X-T/S/C; X ≠ P) and 18O labeling. (XLSX) Table S-2. Detailed information about the identified Nglycosylated proteins and N-glycosylation sites from mouse brain in each method. (XLSX) Table S-4. Detailed information about the identified Nglycosylated proteins and N-glycosylation sites in sequential methods. (XLSX) Table S-5. Predicted topology information on the 57 identified novel single spanning transmembrane Nglycoproteins. (XLSX)

Figure 5. (A) Cellular component annotations for the identified Nglycoproteins. (B) Molecular function annotations for the identified N-glycoproteins. (C) Bioinformatics analysis of identified Nglycoproteins using KEGG pathways database. (p < 0.05, only the ratios greater than 3% are shown).

brain tissue (Supplementary Table 6). Various kinds of receptors and transporters proteins participate in key steps including neurotransmitter release, reception, and uptake. Among 1597 identified N-glycoproteins, 272 are receptor proteins. Simultaneously, the ligand−receptor interaction pathway is the most enriched pathway according to KEGG pathway analysis. Only 196 identified receptor proteins were annotated as N-glycoproteins in Uniprot. Among 79 identified transporters, 67 are the membrane transport proteins of solute carrier (SLC) group, which is the largest multispanning membrane group in mouse brain, half of which are not annotated as N-glycoprotein in UniProt. These heavily N-glycosylated receptors or transporters are always found in pre- and postsynaptic neurons in synapses to perform their functions (Supplementary Figure 1). Neurotransmitter receptors modulate the actions of excitatory and inhibitory neurotransmitters, while transporters mediate neurotransmitter concentrations. N-Glycosylation can affect synaptic processes via distinct effects on the function of synaptic

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: 86-10-64888581. G

DOI: 10.1021/acs.jproteome.6b00098 J. Proteome Res. XXXX, XXX, XXX−XXX

Article

Journal of Proteome Research Author Contributions

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C.Z. and Z.Y. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Z. S. Xie, X. Ding, L. L. Niu, T. X. Cai and other members in the Laboratory of Proteomics of Core Facilities for Protein Science, Institute of Biophysics, Chinese Academy of Sciences, for their excellent technical support. We thank Dr. Torsten Juelich for language editing. This work was supported by grants from the National Basic Research Program of China (Grant Nos. 2012CB966803 and 2014CBA02003) and Novo Nordisk-CAS Research Fund (NNCAS-2015-11), and the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDA12030202).

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DOI: 10.1021/acs.jproteome.6b00098 J. Proteome Res. XXXX, XXX, XXX−XXX