N-Glycosylation Site Analysis of Proteins from Saccharomyces

Feb 14, 2014 - N-Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid Chromatography-Based En...
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N‑Glycosylation Site Analysis of Proteins from Saccharomyces cerevisiae by Using Hydrophilic Interaction Liquid ChromatographyBased Enrichment, Parallel Deglycosylation, and Mass Spectrometry Liwei Cao, Long Yu,* Zhimou Guo, Aijin Shen, Yunü Guo, and Xinmiao Liang* Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China S Supporting Information *

ABSTRACT: N-Glycosylation site analysis of baker’s yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of Nglycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC−MS/MS analysis. On the basis of the workflow, a total of 135 Nglycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG. KEYWORDS: glycoproteomics, Saccharomyces cerevisiae, hydrophilic interaction liquid chromatography, glycopeptide, mass spectrometry, yeast analysis of the deglycosylated peptides can be reduced.9−11 For the selective enrichment of glycopeptides, the hydrophilic interaction liquid chromatography (HILIC)-based method has the unique advantage of unbiased affinity for glycopeptides with various types of glycans,12−17 which compensates for the deficiency of other methods such as lectin affinity chromatography and hydrazide chemistry.18−20 Nevertheless, the glycopeptide selectivity of HILIC methods is less sufficient, particularly when compared to that of hydrazide chemistry, resulting in the coelution of nonglycosylated peptides with glycopeptides.21,22 Accordingly, improving the selectivity of the HILIC methods by refining the surface chemistry of the employed polar materials is crucial to enhance the effectiveness of the glycopeptide enrichment procedure.23−25 Following enrichment, the N-glycans attached to the glycopeptides are usually enzymatically cleaved to facilitate the analysis of the occupied N-glycosylation sites.26 During the deglycosylation by using PNGase F, the N-glycosylation site asparagine is converted to aspartic acid via deamidation. By sequencing and mapping the resulting consensus of Asp-X-Ser/Thr/Cys (X

1. INTRODUCTION The N-glycosylation is evolutionarily conserved from the baker’s yeast Saccharomyces cerevisiae to human cells.1 They share identical N-glycosylation processes occurring in endoplasmic reticulum (ER), where the N-glycans are biosynthesized and transferred to the consensus of Asn-X-Ser/Thr/Cys (X not Pro) in the target polypeptides. 2,3 The high conservation of N-glycosylation makes S. cerevisiae a valuable model organism for the mechanism elucidation of some human inherited diseases, especially the congenital disorders of glycosylation (CDG).4 CDG is mainly caused by the defects of N-glycosylation relevant genes, which alter the normal glycosylation pathways and lead to aberrant N-glycosylation site occupancy or other disorders.5,6 Therefore, N-glycosylation site analysis of S. cerevisiae proteins is of fundamental importance to reveal the pathogenesis of CDG. In principle, a typical mass spectrometry (MS)-based workflow for the N-glycosylation site analysis comprises sample preparation, glycopeptide enrichment, glycopeptide deglycosylation, as well as the LC−MS/MS analysis and data interpretation.7,8 Glycopeptide enrichment is an essential procedure to remove the nonglycosylated peptides in the proteolytic digest so that the interference to the subsequent MS © 2014 American Chemical Society

Received: October 21, 2013 Published: February 14, 2014 1485

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Figure 1. The structure of the Click TE-Cys material and the charge state of the functional groups under HILIC condition.

2.3. HILIC Enrichment of Glycopeptides by Using Click TE-Cys and ZIC-HILIC

not Pro), the former N-glycosylation sites could be indirectly deduced.27,28 However, it has been revealed recently that the deamidation of asparagine may spontaneously occur in the cell or during sample treatment, which inevitably brings falsepositive results to site assignment.29−31 At present, this issue remains unresolved even though heavy-oxygen water instead of water is applied for PNGase F digestion.18,32 In this study, we established an MS-based workflow for the analysis of N-glycosylated sites in S. cerevisiae proteins. A zwitterionic-type HILIC material named Click TE-Cys (Figure 1), which was evaluated to possess improved glycopeptide selectivity and coverage, was introduced to the workflow for the enrichment of S. cerevisiae glycopeptides. After enrichment, a parallel deglycosylation strategy, in which two endoglycosidases PNGase F and Endo Hf were employed for trimming the attached N-glycans, was implemented prior to LC−MS/MS analysis to enhance the confidence of the identified Nglycosylation sites. On the basis of the workflow, we have identified 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites from 79 S. cerevisiae proteins. Among these sites, nine novel-type sites from eight proteins, which were simultaneously identified via parallel deglycosylation, were discovered to be glycosylated with high confidence. The glycosylation site information as well as the workflow from which the information derived will contribute to the improvement of the S. cerevisiae model for the mechanism elucidation of human inherited diseases.

An inert sieve was placed in the end of the GELoader tip. The Click TE-Cys material was slurried in acetonitrile (ACN) and packed into the GELoader tip. The resulting microcolumn was first washed with 90 μL of the washing buffer containing 50% ACN, 10 mM NH4Ac, and 2% FA. The column was then equilibrated with 90 μL of loading buffer containing 80% ACN, 10 mM NH4Ac, and 2% FA. The tryptic digests or digest mixture were dried, redissolved in 20 μL of loading buffer, and loaded onto the microcolumn. The removal of nonglycosylated peptides was implemented by rinsing the column with 60 μL of loading buffer and 100 μL of rinsing buffer containing 70% ACN, 10 mM NH4Ac, and 2% FA. The glycopeptide fraction was eluted with the eluting buffer containing 50% ACN, 10 mM NH4Ac, and 2% FA. A similar procedure was performed during glycopeptide enrichment by using ZIC-HILIC material. Briefly, after equilibrium with 90 μL of the loading buffer containing 80% ACN and 5% FA, the tryptic digest of S. cerevisiae proteins was loaded onto the microcolumn packed with ZIC-HILIC material. Then, the column was rinsed three times with 50 μL of loading buffer to remove the nonglycosylated peptides. The glycopeptides were finally obtained by washing the column with 100 μL of elution buffer containing 50% ACN and 5% FA. 2.4. Lectin Affinity Chromatography Enrichment of Glycopeptides by Using ConA Agarose

ConA agarose was packed into the sieved GELoader tip. The packed microcolumn was equilibrated with the buffer containing 20 mM Tris/HCl, 1 mM MnCl2, 1 mM CaCl2, and 0.5 M NaCl. After the tryptic digest of S. cerevisiae proteins was reconstituted and loaded, the column was rinsed with the 90 μL of the above buffer to remove the nonglycosylated peptides. The glycopeptides fraction was eluted with the buffer containing 0.2 M methyl-α-D-mannopyranoside, 20 mM Tris/ HCl, and 0.5 M NaCl.

2. EXPERIMENTAL PROCEDURES 2.1. Cell Cultures and Protein Purification

Isolated colonies were obtained from a single cell through the gradient dilution method. Wild type S. cerevisiae was grown in sterile conical flasks containing 50 mL of YPD liquid culture medium (1% yeast extract, 2% peptone, 2% dextrose) on a rotary shaker at 30 °C. Cells were grown to logarithmic phase and collected by centrifugation at 3000 rpm for 5 min. The procedure of protein purification followed a published protocol.33

2.5. PNGase F Deglycosylation

The glycopeptides fractions of S. cerevisiae were dried and redissolved in 100 mM NH4HCO3, followed by the addition of PNGase F (1 μL). The solution was incubated in 37 °C for 24 h.

2.2. Tryptic Digestion of S. cerevisiae Proteins

2.6. Endo Hf Deglycosylation

Proteins purified from S. cerevisiae (1 mg) were denatured by using 8 M urea in 50 mM NH4HCO3 for 3 h. Then the protein mixture was reduced by DTT (50 mM, 4 μL) for 2 h at 37 °C, followed by the addition of IAA (50 mM, 5 μL) for alkylation. The resulting solution was incubated in dark for 30 min and then diluted 10-fold with NH4HCO3 (50 mM) buffer. The solution was mixed with trypsin at an enzyme/substrate ratio of 1:50 (w/w) and incubated for 16 h at 37 °C.

The glycopeptides fractions of S. cerevisiae were dried and redissolved in the mixture of 10× G5 reaction buffer (4 μL) and H2O (14 μL). Then Endo Hf (2 μL) was added into the solution and incubated in 37 °C for 24 h. 2.7. Mass Spectrometric Analysis

For characterizing the human immunoglobulin G (IgG) and horseradish peroxidase (HRP) glycopeptides, MS experiments were performed on an Axima MALDI-QIT-TOF mass 1486

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tide coverage of Click TE-Cys, which are the key factors to reflect its effectiveness in glycopeptide enrichment, were evaluated prior to the application in yeast glycosylation site analysis. In the previous study, the glycopeptide selectivity of Click TE-Cys was assessed preliminarily with human IgG digest, where the nonglycosylated peptides were effectively removed from the glycopeptides.36 Herein, we further increased the complexity of the model object by mixing IgG digest with the digest of the nonglycosylated protein human serum albumin (HSA) at the different weight ratios. As shown in Figure 2A, the IgG glycopeptides could not be detected in the

spectrometer (Shimadzu Corp., Kyoto, Japan) with nitrogen pulse laser (337 nm). The mass spectrometer was operated under positive ion and reflectron mode. Argon was used as the collision gas. One microliter of samples was spotted on the MALDI plate and dried at room temperature. Then 1 μL of 2,5-dihydroxybenzoic acid (10 mg/mL in 0.1% TFA, 49.9% H2O, and 50% ACN) was applied to the target and dried. For characterizing the S. cerevisiae glycoproteome, the deglycosylated peptide samples were analyzed on an UltiMate 3000 RSLCnano system coupled online to an LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The separation was performed on a fused silica capillary column (37 cm × 200 μm i.d., Polymicro Technologies, Phoenix, AZ) packed with reversed-phase Sepax Bio-C18 particles (3 μm particle diameter, Sepax Technologies, Inc., Newark, DE) at 4100 psi. Mobile phase A (0.1% FA in water) and B (0.1% FA in 80% ACN) were used to establish the 140 min gradient as follows: 0−15 min, 2.5% B; 15−25 min, 2.5− 12.5% B; 25−110 min, 12.5−50% B; 110−130 min, 50−100% B and 130−140 min, 100% B. The flow rate was at 800 nL/ min. The MS instrument was operated in data-dependent mode with an m/z range of 100−2000 for full MS scans. The 10 most abundant ions from the MS analysis were selected for MS/MS analysis using a normalized collision energy setting of 35% and a dynamic exclusion duration of 30 s. The temperature of heated capillary and ESI voltage were 275 °C and 2.7 kV, respectively. 2.8. Data Analysis

LC−MS/MS raw files were converted to “.mgf” files by pXtract (version 1.0, one component of pFind software kit).34 The MS/MS spectra were searched using Mascot algorithm (version 2.3.0.2)35 against a composite database including both original and reversed yeast sequences in Swiss-Prot (downloaded from www.uniprot.org on August 31, 2012), including 15 572 entries. The reversed database was used to control FDR value. Mass tolerances were set as 10 ppm for parent ions and 0.5 Da for fragments. Peptides were searched using fully tryptic cleavage constraint. A maximum of two missed cleavage was allowed. Accepted peptides charge were +2, +3, and +4. Carboxyamidomethylation (C) was searched as fixed modification. (1) PNGase F: oxidation (M) and deamidation (N); (2) Endo Hf: oxidation (M) and GlcNAc (+203.0793) were searched as variable modifications. The Mascot results were filtered by pBuild (another component of pFind software kit). The filter parameters were peptide FDR ≤ 1%, number of distinct peptides ≥ 1. Since N-glycosylation often occurs at a consensus motif of Asn-X-Ser/Thr/Cys (X not Pro), the remaining peptide sequences were additionally filtered to remove those without the consensus motif. For the PNGase F deglycosylated peptides, the according product ion spectra with the Mascot score < 35 were excluded from further manual verification of the glycosylated site assignment unless the peptide sequences were identified in both replicate experiments. For the Endo Hf deglycosylated peptides, the cutoff score is set to 25 due to the relatively lower quality of the product ion spectra.

Figure 2. MALDI MS characterization of the digest mixture of IgG and HSA treated with desalting or HILIC enrichment. (A) Mass spectrum of the desalted digest mixture of IgG/HSA (1:1, w/w); (B) mass spectrum of the glycopeptide fraction enriched from the digest mixture of IgG/HSA (1:1, w/w) by using Click TE-Cys; (C) mass spectrum of the glycopeptide fraction enriched from the digest mixture of IgG/HSA (1:10, w/w) by using Click TE-Cys. Pep1 and Pep2 stand for the peptide sequences of EEQYNSTYR and EEQFNSTFR, respectively. Blue square, N-acetylglucosamine; green circle, mannose; yellow circle, galactose; red triangle, fucose.

digest mixture of IgG/HSA (1:1, w/w) when no enrichment procedure was implemented. By contrast, 19 glycopeptides were identified in the fraction enriched from the same digest mixture by using Click TE-Cys (Figure 2B). A similar result could also be achieved with Click TE-Cys enrichment when the weight ratio of IgG/HSA digest mixture was further set as 1:10 (Figure 2C). In both cases, the nonglycosylated peptides were effectively removed, and the signals of the glycopeptides dominated the spectra. These results indicated the superior glycopeptide selectivity of the Click TE-Cys material. Note that

3. RESULTS 3.1. Assessment of the Click TE-Cys Material on Glycopeptide Selectivity and Coverage

Selective enrichment of glycopeptides aims to remove the nonglycosylated peptides and minimize the glycopeptide loss during enrichment.23 Therefore, the selectivity and glycopep1487

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Table 1. Peptide Sequence and Glycan Composition of the HRP Glycopeptides Enriched by Using Click TE-Cysa

a

Asterisk indicates that sites N216 and N228 were simultaneously glycosylated with the glycan marked with the asterisk.

Figure 3. (A) Comparison of PNGase F and Endo Hf deglycosylation on the quantities of different types of identified glycosylated sites; (B) comparison of the Click TE-Cys and ZIC-HILIC enrichment on the glycosylation site coverage; (C) comparison of the Click TE-Cys and ConA enrichment on the glycosylation site coverage.

compatibility of the material for glycopeptides with different hydrophilicities. As shown in Table 1, 11 glycopeptides that cover six glycosylation sites were identified after enrichment. The peptide backbones of the identified glycopeptides varied in the lengths from 6 to 28 amino acid residues, indicating that Click TE-Cys is capable of capturing glycopeptides with broad hydrophilicity difference.

sialylated glycopeptides were not found in Figure 2B,C. This is possibly due to the postsource loss of sialic acid in MALDI-MS, since the sialylated glycopeptides were detectable in ESI−MS (Supplementary Figure 1, Supporting Information), which were confirmed by the oxonium ions of sialic acid (m/z 292) generated from the dissociation of these glycopeptides in MS/ MS analysis (Supplementary Figure 2, Supporting Information). To evaluate the glycopeptide coverage of Click TE-Cys, we made use of HRP as the model protein, which contains eight glycosylation sites attached with xylose and/or fucose modified glycans.37 The proteolytic digest of HRP contains glycopeptides with various peptide lengths, which exhibited diverse hydrophilicity under HILIC conditions. Therefore, the effectiveness of enriching HRP glycopeptides reflects the

3.2. General Information on the Identified N-Glycosylation Sites of the Proteins in S. cerevisiae

Because of the advantages of Click TE-Cys in glycopeptide selectivity and coverage, the material was employed to establish the workflow for glycosylation site analysis of the proteins in baker’s yeast S. cerevisiae. In addition, parallel deglycosylation strategy by using two different endoglycosidases, that is, PNGase F and Endo Hf, was incorporated in the workflow as 1488

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well. On the basis of the workflow, a total of 135 glycosylated sites mapping to 79 proteins were identified in S. cerevisiae (Supplementary Table 1, Supporting Information). Upon comparison to the Uniprot Knowledgebase, the types of the 6, 93, and 36 sites out of the 135 identified sites were classified as known, potential and novel, respectively. The amount of the sites identified via PNGase F deglycosylation is higher than that of the sites identified via Endo Hf deglycosylation (125 to 77), as shown in Figure 3A. This is possibly due to the interference to the ionization efficiency and tandem MS fragmentation of the Endo Hf deglycosylated peptides caused by the remaining reducing-end GlcNAc.38 Meanwhile, 67 glycosylated sites including 4 known, 54 potential, and 9 novel were simultaneously identified via both deglycosylation procedures, which accounted for approximately 50% of the overall identified sites (Figure 3A). The identification of these sites is of higher confidence in comparison to that of the other sites identified individually via PNGase F or Endo Hf deglycosylation. The glycosylation site coverage of Click TE-Cys was compared to that of ZIC-HILIC as well as the ConA material, whose specificity is matched with the high-mannose-type glycans linked to yeast proteins. As shown in Figure 3B, Click TE-Cys has exhibited slightly higher glycosylation site coverage than ZIC-HILIC (125 to 117 sites). Among these sites, only 71 of them are overlapped, which indicates the diverse glycopeptide specificities even between two HILIC materials. Meanwhile, the glycopeptide selectivity of Click TECys (43%) was also relatively higher than that of ZIC-HILIC (33%). In comparison to the Con A material, however, Click TE-Cys has exhibited remarkably higher glycosylation site coverage (125 to 45 sites), and 67% of the sites identified by using ConA can also been identified by using Click TE-Cys, as shown in Figure 3C. The 135 sites identified via the established workflow were derived from four separate LC−MS/MS analysis of the samples treated with four independent enrichment and deglycosylation procedures including two PNGase F and two Endo Hf digestions. For PNGase F, the overlapping sites of the two replicate digestions comprised 84% of the overall 125 sites, and for Endo Hf the proportion is 69%, suggesting the relatively good reproducibility of the workflow (Supplementary Figure 3, Supporting Information). Of the 79 proteins identified herein, 29 proteins were found with more than one glycosylated sites (Supplementary Figure 4, Supporting Information). The cellular component, molecular function, as well as the involved biological process of the 79 proteins are summarized in Figure 4, according to the Gene Ontology information. For cellular component, more than half of the proteins were distributed in the ER and plasma membrane, followed by those from the cell wall, vacuole, and other organelles (Figure 4A). The molecular functions of these proteins are rather diverse, with glycosyltransferase and glycosidase activity at the top-two position, followed by those with protease/peptidase and oxidoreductase activity (Figure 4B). The situation of molecular function had certain correlation with that of the biological process, where cell wall structuring (15%) and glycosylation (11%) comprised the first and second position (Figure 4C), since a majority of the identified proteins with glycosidase activities are involved in cell wall structuring and glycosyltransferases are mainly involved in glycosylation. We compared the identified sites and the mapped proteins with those identified previously by Bertozzi et al.39 Though the scales of the two data sets are rather similar (sites 135 to 133

Figure 4. Functional analysis of the identified 79 proteins according to Gene Ontology. (A) Cellular component; (B) molecular function; and (C) biological process.

and proteins 79 to 58), only 39 sites from 24 proteins overlapped, accounting for 29% and 30% of the sites and proteins identified here, respectively. Meanwhile, we also compared the proteins identified here to those identified via fluorescent lectin binding of protein microarrays by Snyder and Zhu et al., where 30 proteins out of the 79 proteins identified here were matched.40 Apart from the influence of colony types and culture conditions on the sources of the yeast proteome, the differences of the identified results may suggest that different approaches have certain complementation in global analysis of the S. cerevisiae glycoproteome. 3.3. Assignment of the Novel-Type Glycosylation Sites by Parallel Deglycosylation

Identification of novel glycosylation sites via the glycoproteomic methods is of referential significance to the elucidation of glycoprotein structure and biological functions. Herein, a total of 36 novel-type sites were identified from 25 S. cerevisiae proteins by using the established workflow, which accounted for 27% and 32% of the overall identified sites and proteins, respectively. Among these sites, the occupation of nine sites that were simultaneously identified from PNGase F and Endo Hf deglycosylation was confirmed with high confidence. The proteins to which these sites belong include Prb1p (a protease 1489

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peptide at m/z 1191.08 (2+) in Figure 5B. In some cases, neutral loss of GlcNAc may also occur in the fragments (Supplementary Figures 7B and 8B, Supporting Information). For all the other novel-type sites identified individually by PNGase F or Endo Hf deglycosylation, further validation is required due to the possible false-positive results caused by the deglycosylation procedures.

in vacuole), Alg12p (a mannosyltransferase in ER membrane), Msc1p (a protein involved in reciprocal meiotic recombination and distributed in ER, mitochondrion and plasma membrane), Pom152p (a component of nuclear pore complex), Heh2p (a protein integral to nuclear inner membrane), Sga1p (a glucoamylase in vacuole), and two VEL1 family members. Note that site Asn387 in Prb1p, Asn483 in Alg12p, Asn569 in Pom152p, and Asn91 in the two VEL1 family members were also identified via ZIC-HILIC enrichment. Figure 5 shows the

4. DISCUSSION A glycoproteomic workflow for N-glycosylation site analysis in S. cerevisiae proteins was established based on HILIC enrichment and parallel deglycosylation of N-linked glycopeptides. As far as HILIC enrichment of glycopeptides is concerned, the effectiveness is mainly dependent on the discrepant hydrophilic interactions provided by the employed polar materials.41,42 Under HILIC conditions, the stagnant water-layer that is formed on the surface of the polar material facilitates the retention of glycopeptides attached with hydrophilic glycans, while the nonglycosylated peptides with less hydrophilicity tend to be retained in the bulk solvent and are eluted earlier.43 Therefore, highly hydrophilic material with the stable formation of a water-layer on its surface is more likely to provide discrepant partitioning interaction for the isolation of the glycosylated peptides from their nonglycosylated counterparts.44 The Click TE-Cys material was synthesized by linking the polar amino acid cysteine to the surface of silica via “Thiolene” click reaction.36 Under HILIC conditions, the amino and carboxylic acid group of the linked cysteine are readily ionized to carry positive and negative charges, respectively, resulting in the zwitterionic property of Click TE-Cys, as shown in Figure 1. Different from other zwitterionic materials such as ZICHILIC or ZIC-cHILIC, the distribution of the zwitterions carried by Click TE-Cys is parallel to the surface of the silica.36 In our previous study on polar compounds (e.g., saccharides) separation, Click TE-Cys exhibited higher hydrophilicity than ZIC-HILIC or the Click Maltose material customized in our lab23 (data not shown). Therefore, the high hydrophilicity of Click TE-Cys, which derives from the unique surface structure and charge distribution, is possibly responsible for the superior selectivity and glycopeptide coverage of the material. For the deglycosylation of N-glycopeptides, PNGase F is the most frequently used endoglycosidase in glycoproteomic workflows. PNGase F digestion is a deamidation process, through which the occupied N-glycosylation site asparagine is converted to aspartic acid in addition to the removal of the attached N-glycans.18 The resulting mass shift of 0.9840 Da in the deglycosylated peptides can be detected by MS as the marker for the former occupied sites.28 However, PNGase F digestion is not the only approach to generate the deamidated asparagine. The deamidation of asparagine may also occur spontaneously in natural cell activities, which leads to the interference to the assignment of glycosylated sites.45 The use of heavy oxygen water as the solvent for PNGase F digestion, which adds a mass difference of 2.9890 Da to the peptide, can be helpful to distinguish the spontaneous deamidation from PNGase F deamidation of the asparagine residues.46,47 However, a recent report by Larsen et al. revealed that the introduction of H218O cannot exclude the false-positive results derived from the chemical deamidation during sample preparation.32 Apart from PNGase F, Endo H/Hf is another important type of endoglycosidase for the cleavage of highmannose or hybrid N-glycans from proteins. Although the remaining reducing-end GlcNAc residue on the Endo H/Hf

Figure 5. (A) MS/MS spectrum and fragment assignment of the PNGase F deglycosylated peptide (M + 2H)2+ at m/z 1091.52; (B) MS/MS spectrum and fragment assignment of the Endo H f deglycosylated peptide (M + 3H)3+ at m/z 862.08.

assignment of the glycosylated sites mapped to protein Sga1p by interpreting the MS/MS spectra of the according deglycosylated peptides. There were two asparagine residues (Asn166 and Asn177) containing in the consensus of Asn-X-Ser/ Thr/Cys (X not Pro) in the peptide backbone. The occupation of site Asn177 was assigned according to the mass difference of the y9 and y10 ions generated from the fragmentation of PNGase F deglycosylated peptide in Figure 5A. Meanwhile, the fragment ions of y9 and y11 from Endo Hf deglycosylated peptide (Figure 5B) confirmed the occupation of this site since the mass difference of the two ions indicated the existence of a threonine and a GlcNAc-linked asparagine residue. The assignment of site Asn166, however, was deduced by the mass difference between the molecular ions and the most proximal fragment ions (y17 in Figure 5A and y18 in Figure 5B), due to the lack of adjacent fragment ions around the site. The mass differences in both spectra proved that site Asn166 was occupied simultaneously with site Asn177. Other than the fragment ions derived from the dissociation between amino acid residues, the ions of Endo Hf deglycosylated peptide with neutral loss of the attached N-GlcNAc can be observed as well, such as the 1490

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Figures 1−11 as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

deglycosylated peptide facilitates the unambiguous assignment of the occupied glycosylation site, it has a negative effect on the ionization efficiency and MS detection of the resulting peptides.48 Furthermore, the remaining GlcNAc is labile in tandem MS fragmentation, and its neutral loss may interfere with the dissociation of the peptide backbone, from which the crucial information for site assignment is derived.38 Herein, parallel deglycosylation was implemented to amend the deficiencies of the two glycosidases. The combination use of PNGase F and Endo Hf not only increased the quantity of identified sites but also significantly improved the reliability of the sites, especially for those identified simultaneously through both deglycosylation approaches. Among these sites, in particular, the identification of nine novel-type sites from eight proteins may shed new light on the investigation of protein structure and biological functions. N-Glycosylation site analysis of S. cerevisiae is of special significance to elucidate the molecular mechanism of human inherited disease CDG. A majority of the discovered CDG cases are caused by the gene defects in N-glycosylation pathways.6 Because of the conservation of N-glycosylation in ER, the defective genes in human CDG can be located by using S. cerevisiae as the model organism, whose genes relevant to Nglycosylation have almost been clearly identified, and the mutants for certain N-glycosylation genes are available.1,49,50 In particular, the S. cerevisiae mutants with mutations of the genes involved in the biosynthesis and transfer of the lipid linked oligosaccharides to the nascent polypeptides are favorable for the discovery of gene defects in CDG type I (CDG-I), which usually leads to the disorders of N-glycosylation site occupancy.51,52 The analytical workflow presented here, which is capable of profiling the occupied N-glycosylation sites in S. cerevisiae proteins, has the potential to describe and distinguish the molecular phenotypes of the mutants with different mutated glycosylation genes. These molecular phenotypes on glycosylation site occupancy are valuable references for the screening of the S. cerevisiae mutant relevant to a certain subtype of CDG-I. Meanwhile, restoration of the disordered glycosylation site occupancy by the expression of the normal human gene analogous to the mutated gene in the according S. cerevisiae mutant is convincing evidence for the confirmation of the deficient gene in CDG-I, as demonstrated by Korner et al.53 Compared to the traditional method for monitoring the glycosylation alteration, where only the status of several glycoproteins could be observed, the presented workflow has the advantage of providing more comprehensive and more distinct information on the alteration of glycosylation site occupancy at the cellular scale, which is more convincing for the validation of CDG-related genes. To this end, it is believed that the established workflow together with the profile of N-glycosylated sites will contribute to the improvement of the S. cerevisiae model for revealing the pathogenesis of CDG.

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AUTHOR INFORMATION

Corresponding Authors

*(X.L.) Tel, +86-0411-84379519; fax, +86-0411-84379539; email, [email protected]. *(L.Y.) Tel, +86-0411-84379541; fax, +86-0411-84379539; email, [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors want to thank Dr. Bo Wang for assistance with the Orbitrap Elite MS experiments. This work was supported by National High Technology Research and Development Program of China (863 Program, 2012AA020203) and National Natural Science of China (Grant Nos. 21135005 and 21205116).

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ABBREVIATIONS ER, endoplasmic reticulum; CDG, congenital disorders of glycosylation; MS, mass spectrometry; HILIC, hydrophilic interaction liquid chromatography; LC−MS/MS, liquid chromatography coupled to tandem mass spectrometry; PNGase F, peptide N-glycosidase; Endo Hf, endoglycosidase Hf; IgG, immunoglobulin G; HRP, horseradish peroxidase; HSA, human serum albumin; ConA, concanavalin A; ACN, acetonitrile; FA, formic acid; TFA, trifluoroacetic acid; GlcNAc, N-acetylglucosamine; HexNAc, N-acetylhexosamine; Hex, hexose; Fuc, fucose; Xyl, xylose

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary experimental procedures; Supplementary Table 1: Identification of the occupied glycosylation sites and the mapped proteins in S. cerevisiae by using the established workflow. Supplementary Table 2: Identification of the occupied glycosylation sites and the mapped proteins in S. cerevisiae by using ZIC-HILIC enrichment, followed by PNGase F deglycosylation and LC−MS/MS analysis. Supplementary 1491

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