HCD - American Chemical Society

Article pubs.acs.org/jpr

Higher Energy Collision Dissociation (HCD) Product Ion-Triggered Electron Transfer Dissociation (ETD) Mass Spectrometry for the Analysis of N‑Linked Glycoproteins Charandeep Singh, Cleidiane G. Zampronio, Andrew J. Creese, and Helen J. Cooper* School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. S Supporting Information *

ABSTRACT: Large scale mass spectrometry analysis of N-linked glycopeptides is complicated by the inherent complexity of the glycan structures. Here, we evaluate a mass spectrometry approach for the targeted analysis of N-linked glycopeptides in complex mixtures that does not require prior knowledge of the glycan structures or pre-enrichment of the glycopeptides. Despite the complexity of N-glycans, the core of the glycan remains constant, comprising two N-acetylglucosamine and three mannose units. Collision-induced dissociation (CID) mass spectrometry of Nglycopeptides results in the formation of the N-acetylglucosamine (GlcNAc) oxonium ion and a [mannose+GlcNAc] fragment (in addition to other fragments resulting from cleavage within the glycan). In ion-trap CID, those ions are not detected due to the low m/z cutoff; however, they are detected following the beam-type CID known as higher energy collision dissociation (HCD) on the orbitrap mass spectrometer. The presence of these product ions following HCD can be used as triggers for subsequent electron transfer dissociation (ETD) mass spectrometry analysis of the precursor ion. The ETD mass spectrum provides peptide sequence information, which is unobtainable from HCD. A Lys-C digest of ribonuclease B and trypsin digest of immunoglobulin G were separated by ZIC-HILIC liquid chromatography and analyzed by HCD product ion-triggered ETD. The data were analyzed both manually and by search against protein databases by commonly used algorithms. The results show that the product ion-triggered approach shows promise for the field of glycoproteomics and highlight the requirement for more sophisticated data mining tools. KEYWORDS: glycosylation, tandem mass spectrometry, HCD, ETD, ZIC-HILIC, post-translational modification, N-linked glycosylation

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INTRODUCTION

glycosylation, and therefore glycosylation characterization is a quality control parameter in the biotechnology industry. Glycosylation may be N-linked (through asparagine) or Olinked (through serine or threonine). A third, and rare, type is C-linked (through tryptophan). N-linked glycosylation involves co-translational transfer of a 14 saccharide carbohydrate followed by post-translational processing of the glycan.12 Nlinked glycans have high-mannose, complex or hybrid-type structures and occur within the consensus sequence N-X-S/T, where X is any amino acid except proline. Typically glycoprotein characterization involves partial or total deglycosylation of the glycoprotein followed by separate analyses of the protein and glycan entities.13,14 Peptide Nglycosidase F (PNGase F) is the most frequently used deglycosylation enzyme.14,15 Recent developments in mass spectrometry have allowed confident assignment of glycosyla-

Glycosylation is one of the most important post-translational modifications of proteins. Apweiler et al. estimated that ∼50% of human proteins are glycosylated.1 A more recent statistical study of the SWISS-PROT database suggests that ∼20% are glycoproteins. That study also revealed that N-linked glycosylation was the third most abundant and O-linked glycosylation the seventh most abundant protein modification.2 Glycoproteins and glycopeptides play myriad roles in nature. They are involved in cell−cell recognition3,4 and are implicated in the development of many diseases and disorders, including cancer and HIV. For example, the HIV coat N-linked glycoproteins gp120/41 play an important role in disguising the viral identity from the human immune system.5,6 Similarly, glycoproteins have been found to be associated with inflammation and inflammatory disorders.7−9 Many therapeutic biological drugs comprise glycoproteins.10,11 Such biotechnology products are faced with the problem of improper © 2012 American Chemical Society

Received: March 15, 2012 Published: July 16, 2012 4517

dx.doi.org/10.1021/pr300257c | J. Proteome Res. 2012, 11, 4517−4525

Journal of Proteome Research

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tion sites in intact glycopeptides. Whereas collision induced dissociation (CID) results in loss of the glycan from the peptide backbone often at the expense of sequence fragments, electron transfer dissociation (ETD) and electron capture dissociation (ECD) produce sequence fragments that retain the modification, thereby enabling site localization.16−18 Several methods have been developed which combine CID and ETD to better characterize glycopeptides.19−21 Following the implementation of beam-type CID, known as higher energy collision dissociation (HCD),22 in hybrid ion trap-orbitrap mass spectrometry, Segu and Mechref considered its application to glycosylation analysis.23 They found that HCD generated distinct Y1 ions (peptide+GlcNAc) that were useful in glycopeptide assignment and that, because HCD overcomes the 1/3 m/z cutoff associated with ion trap CID, glycan oxonium ions were detected. Hart-Smith and Raftery exploited the presence of these oxonium ions in HCD spectra to confirm the assignments of low abundance glycopeptides.24 Scott et al. combined HCD and CID for the analysis of the Campylobacter jejuni N-linked glycoproteome,21 and Zhao et al. have combined HCD and ETD for the analysis of O-glycosylation.25 The analysis of specific post-translational modifications in proteomics is complicated by the fact that the analysis is performed against the background of unmodified peptides and peptides with modification other than those of interest. One approach is to exploit characteristic fragmentation associated with different tandem mass spectrometry techniques to target the analysis. For example, we have shown that neutral loss triggered (NL) ECD may be used for the targeted analysis of phosphopeptides.26,27 Eluting peptides are subjected to CID, and only if a loss of phosphoric acid is observed is ECD triggered. Similarly, we have shown that neutral loss triggered ETD may be applied to the targeted analysis of citrullinated peptides.28 The NL ET/CD approach is not suitable for the analysis of glycosylation because of the heterogeneity of the modification. As described above, N-linked glycosylation encompasses numerous structures; however, the core of the glycan is constant. The glycan is linked to the peptide via an Nacetylglucosamine unit. Therefore, if the CID fragmentation conditions are sufficiently harsh, the GlcNAc oxonium ion will be observed and this can be used as the trigger for ETD. This product ion-triggered ETD approach overcomes the challenge of glycan heterogeneity and allows targeted analysis of glycopeptides. The ETD spectrum provides information on site of glycosylation and peptide sequence and the corresponding CID spectrum (in this case HCD) provides information on glycan structure. The method does not necessarily require any pre-enrichment of the glycopeptide sample. The HCD product ion-triggered ETD (HCD PI ETD) approach was applied by Steentoft et al.29 in their analysis of O-glycosylation in “SimpleCell” lines. The cell lines were engineered such that all O-glycosylation presents as N-acetylgalactosamine (GalNAc). As a result of the homogeneity of the glycosylation, HCD acted simply as a trigger for ETD. Here, we evaluate online ZIC HILIC liquid chromatography HCD PI ETD for the N-linked glycoproteins RNase B and human IgG. The glycans have complex structures, and therefore, as well as providing a means for targeting the analysis, the approach yields structural information about both the glycan and peptide. The data were analyzed both manually and by search against protein databases by commonly used algorithms (SEQUEST, Zcore, Mascot).

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EXPERIMENTAL SECTION

Samples

Ribonuclease B from bovine pancreas (RNase B) and human immunoglobulin G isolated from human serum (IgG) (SigmaAldrich, Dorset, U.K.) were used without further purification. For each protein, 1 mg/mL solutions were prepared in 0.1% formic acid (J. T. Baker, Holland), and 100 μL of these protein solutions was incubated with 40 μL of 100 mM ammonium bicarbonate (Sigma-Aldrich, Dorset, U.K.) and 50 μL of 10 mM dithiothrietol (Sigma-Aldrich) (in 100 mM ammonium bicarbonate) for 1 h at 56 °C. Then, 25 μL of 50 mM iodoacetamide (Sigma-Aldrich) (in 100 mM ammonium bicarbonate) was added, and the suspensions were incubated in the absence of light at room temperature for 45 min. RNase B protein solution was incubated with 10 μL of 12 ng/μL endoproteinase Lys-C (sequencing grade from Lysobacter enzymogenes; Roche Diagnostics GmbH, Germany), and IgG protein solution was incubated with 10 μL of 12 ng/μL trypsin (Promega, Madison, WI) overnight at 37 °C. The reaction was quenched by addition of 10 μL of 10% formic acid. Digested samples were desalted using ZipTip micropipet C18 tips (Millipore, U.K.). Tips were washed with 10 μL of acetonitrile and re-equilibrated with 0.1% trifluoroacetic acid (TFA) (Sigma-Aldrich, Dorset, U.K.). The samples were loaded onto the C18 packing and desalted by washing with 0.1% TFA buffer. The bound peptides were eluted with 10 μL of 50:50 0.1% TFA/acetonitrile. The peptides were dried and resuspended in 0.1% formic acid prior to LC−MS/MS analysis. HCD PI ETD MS/MS Analysis of Lys-C Digest of Bovine Pancreatic Ribonuclease B

Samples were analyzed using a Dionex 3000 Ultimate nano-LC (Dionex, Sunnyvale, CA) liquid chromatograph coupled with a Triversa Nanomate nanospray source (Advion Biosciences, NY) and further interfaced to an LTQ Orbitrap Velos ETD mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Endoproteinase Lys-C digested peptides from bovine pancreatic Ribonuclease B were separated using a zwitterionic hydrophilic interaction liquid chromatography nano column, 150 mm × 75 μm, 5 μm, 200 Å (ZIC-HILIC, SeQuant, U.K.). Five microliters of the desalted peptide mixture was injected onto the column. The gradient was 90% A to 60% A from 0 to 20 min, from 60% to 55% A in 20−25 min, and finally from 55% A to 90% A in 25−46 min at a flow rate of 350 nL/min. Mobile phase A was 0.1% formic acid in acetonitrile (J.T. Baker, Holland), and mobile phase B was 0.1% formic acid in water (J.T. Baker, Holland). The peptides eluted directly into a LTQ Orbitrap Velos ETDTM mass spectrometer. The mass spectrometer performed a full FT-MS scan (m/z 380−1600) and subsequent HCD MS/MS scans of the 40 most abundant ions above an absolute signal intensity threshold of 500 counts. If peaks at m/z 204.09 (HexNAc oxonium ions) or 366.14 (HexHexNAc oxonium ions) (±m/z 0.05) were within the top 20 most abundant peaks, a supplemental activation (sa) ETD MS/MS scan of the precursor ion in the linear ion trap was triggered. Precursor isolation width was 3 m/z. Full scan mass spectra were recorded at a resolution of 30,000 at m/z 400. HCD MS/MS spectra were recorded at a resolution of 7500 at m/z 400. Automatic gain control (AGC) was used to accumulate sufficient ions. For survey scans, AGC target was 1 × 106 (maximum injection time 1 s). For HCD and ETD, AGC target was 1 × 105 (maximum inject time 100 and 25 ms, respectively). HCD was performed at a normalized 4518



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collision energy of 35%.22,30 ETD was performed with fluoranthene ions. AGC target for fluoranthene ions was 1 × 105 (maximum fill time 50 ms). Precursor ions were activated for 130 ms (charge dependent activation time was enabled). Supplemental activation was performed with a normalized collision energy of 25%. Dynamic exclusion repeat count was set to 1 with duration of 180 s. Data acquisition was controlled by Xcalibur 2.1 (Thermo Fisher Scientific).

Scheme 1. Known Glycoforms of IgG

HCD PI ETD MS/MS Analysis of Tryptic Digest of Human IgG

Desalted (ZipTip micropipet C18 tips; Millipore, U.K.) tryptic peptides from human IgG were analyzed as above with the exception that mobile phase A was 80:20 acetonitrile/5 mM ammonium acetate, and mobile phase B was 5 mM ammonium acetate. Data Analysis

Data were analyzed using the workflow option of Proteome Discoverer 1.3 (Thermo Fisher Scientific). The ribonuclease B ETD data were searched using SEQUEST, Zcore and Mascot, and the immunoglobulin G ETD data by SEQUEST, as described below. Data (ETD and HCD) were also analyzed manually in Xcalibur 2.1 (Thermo Fisher). SEQUEST Search Parameters. Ribonuclease B data were searched against the bovine database (36,638 entries) created from the NCBI non-redundant database (updated June 2, 2011). Immunoglobin G data were searched against the human database (207,759 entries) created from the NCBI nonredundant database (updated November 19, 2009). Monoisotopic precursor mass tolerance was 10 ppm, and monoisotopic fragment mass tolerance was 0.8 Da. Number of missed cleavages was set to a maximum of 2. Fixed modification was cysteine carbamidomethylation (+57.021 Da). Variable modifications for the ribonuclease B data were methionine oxidation (+15.995 Da), and asparagine glycosylation: Hex5 HexNAc 2 (+1216.423 Da), Hex 6 HexNAc 2 (+1378.476 Da), Hex 7 HexNAc 2 (+1540.528 Da), Hex 8 HexNAc 2 (+1702.581 Da) and Hex 9 HexNAc 2 (+1864.634 Da). Variable modifications for the IgG data were glycosylation of asparagine: G0 (+1298.476 Da), G1 (+1460.529 Da), G2 (+1622.582 Da), G0F (+1444.534 Da), G1F (+1606.587 Da), G2F (+1768.640 Da), G1FSA (+1897.682 Da), G2FSA (+2059.735 Da), G0FB (+1647.613 Da), G1FB (+1809.666 Da), G2FB (+1971.719 Da); and HexNAc (+203.079 Da) (Ser/Thr), and methionine oxidation (+15.995 Da). (See Scheme 1 for description of glycan nomenclature.) Mascot Search Parameters. Ribonuclease B data were searched against the SwissProt database (533,049 entries, version 56.3) Monoisotopic precursor mass tolerance was 10 ppm, and monoisotopic fragment mass tolerance was 0.8 Da. Number of missed cleavages was set to a maximum of 2. Fixed modification was cysteine carbamidomethylation (+57.021 Da). Variable modifications were Hex5HexNAc2 (+1216.4228 Da), Hex6HexNAc2 (+1378.4756 Da), Hex7HexNAc2 (+1540.5284 Da), Hex 8 HecNAc 2 (+1702.5812 Da), Hex 9 HexNAc 2 (+1864.6340 Da), and methionine oxidation (+15.995 Da). Z-Core Search Parameters. Ribonuclease B data were searched against the bovine database (36,638 entries) created from the NCBI non-redundant database (updated June 2, 2011). Precursor mass tolerance was 10 ppm. Number of missed cleavages was set to a maximum of 2. Fixed modification was cysteine carbamidomethylation (+57.021 Da). Z-core

allows a maximum of 3 dynamic modifications to be searched at a time. Variable modifications were Hex 5 HexNAc 2 (+1216.4228 Da), Hex 6 HexNAc 2 (+1378.4756 Da), Hex7HexNAc2 (+1540.5284 Da), Hex8HexNAc2 (+1702.5812 Da), Hex9HexNAc2 (+1864.6340 Da), and methionine oxidation (+15.995 Da).

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RESULTS AND DISCUSSION

Ribonuclease B

Bovine pancreatic ribonuclease B (RNase B) is well-studied and is widely used as a standard material for glycoprotein method development. It is a high mannose-type glycoprotein with a single site of glycosylation at Asn-60. The known glycan composition varies from Man5GlcNAc2 to Man9GlcNAc2; see Scheme 2. Endoproteinase Lys-C digestion of RNase B Scheme 2. Known Glycan Structures Present in Ribonuclease B

produces glycopeptides with the sequence SRNLTK. The unenriched glycoprotein digest was analyzed using online ZICHILIC chromatography in combination with HCD product ion-triggered ETD. As the peptides elute into the mass spectrometer, a survey scan full mass spectrum is recorded. The most abundant multiply charged ion in that spectrum is subjected to HCD, and the fragments are detected in the orbitrap. If a peak is observed at m/z 204.09 or m/z 366.14 (corresponding to the HexNAc and HexHexNAc oxonium ions, respectively), an ETD event of the (intact glycopeptide) precursor ion is triggered. Note, this is not MS3. Figure 1 illustrates the HCD product ion-triggered (PI) ETD approach; Figure 1a shows the survey scan recorded in the orbitrap at 4519



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the Y1 fragment according to the Domon and Costello nomenclature.31 In addition, there are a number of peaks corresponding to fragments resulting from cleavage within the glycan. These peaks may be used to decipher the composition of the glycan moiety. Figure 1c shows the ETD mass spectrum, recorded in the ion trap, of precursor ion m/z 645.62, triggered by the presence of peaks at m/z 204.09 and m/z 366.14 in the previous HCD spectrum. All N-Cα bonds were cleaved, and the site of glycosylation is confirmed. It is well-established that thermally driven and radical-driven tandem mass spectrometry approaches provide complementary information about glycopeptides16 and that is also demonstrated here: the ETD mass spectrum of SRN(Hex5HexNAc2)LTK, Figure 1c, confirms the peptide sequence. Structural information about the glycan can be determined from the corresponding HCD mass spectrum, Figure 1b. The ETD data were searched as described above using the Mascot, SEQUEST, and Zcore algorithms in Proteome Discoverer 1.3. The data were also interrogated manually. The HCD PI ETD analysis resulted in identification of all the known RNase B glycopeptides. See Table 1 for examples of glycopeptide identifications by SEQUEST. Triply charged ions w e r e o b s e r v e d f o r gl y c o p e p t i d e s m o d ified with Hex5−9HexNAc2 and doubly charged ions were observed for those with Hex5−6HexNAc2. Examples of the HCD and corresponding ETD mass spectra for the known glycopeptides are shown in Supplemental Figures 1 and 2 respectively. In total, 139 ETD mass spectra were triggered; see Supplemental Table 1. Thirty-three of these corresponded to the known glycopeptides, i.e., SRN(Hex5−9HexNAc2)LTK as determined by manual analysis of the ETD mass spectra: fourteen were correctly identified by SEQUEST and Zcore, six were correctly identified by SEQUEST but incorrectly identified by Zcore, seven were correctly identified by Zcore but incorrectly assigned by SEQUEST, one was incorrectly assigned by both, one was correctly assigned by SEQUEST with no assignment by Zcore, one was incorrectly identified by Zcore with no assignment by SEQUEST, and two were not assigned by either SEQUEST or Zcore. In the final case, the precursor m/z was assigned to the incorrect isotopomer. Mascot searching of the data resulted in no identifications. These results highlight the issues associated with the algorithms available for searching ETD data.32 For all of the ETD events triggered, the corresponding HCD mass spectra were interrogated manually for peaks corresponding to HexNAc (m/z 204.09), fragments of HexNAc (m/z 126.06, 138.06, 144.07, 168.07, and 186.08),25 HexNAc+Hex (m/z 366.14), [SRNLTK+HexNAc] (m/z 921.50 (1+) and 461.25 (2+)), and [SRNLTK+HexNAc+HexNAc] (m/z 1124.58 (1+) and 562.79 (2+)) and grouped according to the peaks observed. This analysis is summarized in Supplemental Table 1. The majority of the HCD spectra (130/139) contained peaks corresponding to either [SRNLTK +HexNAc] or [SRNLTK+HexNAc+HexNAc], suggesting that the precursor ions were the known (in 33 cases) or alternative glycoforms of SRNLTK. Forty-five of the HCD spectra contained all of the peaks listed above, 23 of which corresponded to the known glycopeptides. Thirty-seven HCD spectra contained all but the HexHexNAc peak at m/z 366.14, eight of which corresponded to the known glycopeptides, four to the previously unreported SRN(Hex4HexNAc2)LTK, and one to SRN(HexNAc2)LTK. (It is possible that these species are the result of in-source fragmentation.) Nine HCD spectra

Figure 1. Online ZIC-HILIC liquid chromatography HCD product ion-triggered ETD MS/MS of Lys-C digest of ribonuclease B. (a) Survey scan mass spectrum recorded in the orbitrap at retention time (RT) 25.16 min. (b) HCD MS/MS spectrum of precursor ions with m/z 645.6194. (c) Supplemental activation ETD MS/MS of precursor ions with m/z 645.62.

retention time (RT) 25.16 min. The subsequent HCD spectrum, recorded at RT 25.17 min, is shown in Figure 1b. The precursor m/z 645.6194 corresponds to the triply charged glycopeptide SRN(Hex5HexNAc2)LTK (m/zcalc 645.6193, Δ 0.2 ppm). The base peak in the HCD mass spectrum corresponds to the singly charged [peptide+HexNAc] ion, 4520



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Table 1. Examples of Glycopeptide Identifications by SEQUEST Searching of HCD PI ETD MS/MS Data from Lys-C Digest of Ribonuclease Ba

a

sequence

modification

XCorr

charge

m/zmeas

m/zcalc

RT (min)

SRnLTK SRnLTK SRnLTK SRnLTK SRnLTK SRnLTK SRnLTK

Hex5HexNAc2 Hex5HexNAc2 Hex6HexNAc2 Hex6HexNAc2 Hex7HexNAc2 Hex8HexNAc2 Hex9HexNAc2

1.39 0.88 2.10 0.64 1.82 2.50 1.30

3 2 3 2 3 3 3

645.6194 967.9254 699.6368 1048.9536 753.6545 807.6718 861.6896

645.6193 967.9253 699.6369 1048.9517 753.6545 807.6721 861.6897

16.79 28.72 28.37 28.45 28.67 29.05 28.96

Multiple ETD events were triggered for each glycopeptide; see text for details.

Table 2. Glycopeptides Identified by SEQUEST Searching of HCD PI ETD Data from Tryptic Digest of Human IgG; All Assignments Were Manually Confirmed sequence EEQYnSTYR EEQYnSTYR TKPREEQYnSTYR TKPREEQYnSTYR TKPREEQYnSTYR EEQFnSTFR EEQFnSTFR TKPREEQFnSTFR TKPREEQFnSTFR TKPREEQFnSTFR TKPREEQFnSTFR

IgG subclass IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG

1 1 1 1 1 2/3 2/3 2/3 2/3 2/3 2/3

modification

XCorr

charge

m/zmeas

m/zcalc

RT min

G0 G2F G0F G1F G2F G0 G1F G0F G1F G2F G0FB

1.30 0.83 2.54 2.30 1.38 0.96 0.96 3.01 2.84 1.30 1.29

2 3 3 3 3 2 3 3 3 3 3

1244.4982 986.7223 1039.4542 1093.4725 1147.4894 1228.505 922.0430 1028.7907 1082.8088 1136.8240 1096.4857

1244.4982 986.7220 1039.4523 1093.4699 1147.4875 1228.50325 922.04167 1028.7890 1082.8066 1136.8242 1096.4821

12.62 13.03 19.57 19.32 19.22 12.04 12.23 18.5 18.7 18.54 18.55

nation with HCD product ion-triggered ETD. The data were searched using the SEQUEST algorithm in Proteome Discoverer 1.3, as described above. SEQUEST allows six variable modifications to be searched simultaneously. In order to search for all previously reported IgG glycans, see Scheme 1, multiple searches were performed. The glycopeptides identified are summarized in Table 2. Many of the glycopeptides were identified several times. The highest scoring assignments are given. Figure 2 shows the HCD and ETD mass spectra obtained for the triply charged IgG2/3 glycopeptide [TKPREEQFnSTFR] with glycan G1F. It is possible to deduce the glycan structure from the HCD spectrum and the peptide sequence from the ETD spectrum. Figure 3 shows the HCD and ETD mass spectra for the doubly charged ions of IgG1 peptide [EEQYnSTYR] with glycoform G0. The HCD spectrum is poor, containing only the HexNAc oxonium, HexHexNAc, and Y1 fragment. Nevertheless, this is sufficient to trigger the ETD spectrum from which the sequence of the peptide can be, at least partially, confirmed. A final example, the IgG2/3 glycopeptide [TKPREEQFnSTFR] with glycan G0FB, is given in figure 4. The remaining HCD and ETD spectra for the identified glycopeptides are given in Supplemental Figures 3 and 4. The quality of the mass spectra obtained from the glycopeptides was variable: The best quality MS/MS spectra derived from peptides containing a missed tryptic cleavage, i.e., TKPREE..., and fucosylated glycans, suggesting empirically that those glycopeptides were more abundant. The potential for isomeric glycopeptides is large: the peptide sequences from the IgG four subclasses combine with the possible structural and positional isomers within the glycan itself. The limitations of the database search become apparent in these circumstances. However, the HCD product iontriggered ETD approach is particularly useful in this regard. For example, one ETD mass spectrum was assigned in one database

contained all the listed peaks except [SRNLTK+HexNAc +HexNAc], and 38 had all but the peak at m/z 366.14 and [SRNLTK+HexNAc+HexNAc]. Of those, one corresponded to the known glycopeptide SRN(Hex7HexNAc2)LTK and one the unreported SRN(Hex4HexNAc2)LTK. One HCD spectrum contained peaks corresponding to HexNAc (and its fragments) and [SRNLTK+HexNAc+HexNAc], but analysis of the ETD spectrum revealed the peptide to be SRN(Hex5HexNAc2)LTK. Six of the remaining nine HCD spectra contained peaks corresponding to HexNAc, suggesting that the precursors were glycopeptides but had a different peptide sequence. The remaining three did not contain any peaks relating to glycans. In those cases, the corresponding ETD event had been triggered as a result of the presence of peaks within the product ion tolerance (±0.05 m/z); however, manual inspection revealed the mass error associated with the peaks to be too large. The false positive rate is therefore calculated as 2%. Human Immunoglobin G (IgG)

The glycoprotein IgG comprises a number of glycoforms:33 The N-linked glycans have complex biantennary structures; see Scheme 1. Heterogeneity arises through the inclusion of zero, one, or two galactose residues in the antenna (G0, G1, G2), and the presence/absence of core fucose, bisecting Nacetylglucosamine (GlcNAc), and sialic acid on the antennary galactose residues. There are four subclasses of human IgG, IgG1, IgG2, IgG3, and IgG4, named for their relative abundance in serum. Among these IgG1 and IgG2 are major subclasses. The four subclasses share >95% sequence homology, with differences in the hinge region and around the N-glycosylation site, Asn297. Tryptic digestion of IgG gives glycopeptides with the sequences EEQYNSTYR (IgG1), EEQFNSTFR (IgG2 and IgG3), and EEQFNSTYR (IgG4), The tryptic peptides from the human IgG sample were analyzed using online ZIC-HILIC chromatography in combi4521



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Figure 2. Online ZIC-HILIC LC HCD PI ETD MS/MS of tryptic digest of human IgG. (a) HCD MS/MS spectrum of precursor ions with m/z 1082.8088 recorded at retention time 18.69 min. (b) Product ion-triggered ETD MS/MS spectrum of precursor ions with m/z 1082.8 recorded at retention time 18.70 min.


search to TKPREEQYnSTYR, i.e., IgG1, (n is the modified Asn) with glycan G0F (Xcorr = 1.47), whereas the same ETD mass spectrum was assigned to TKPREEQFnSTYR, i.e., IgG4, with glycan G1 (Xcorr = 1.5) in another database search. The masses of these two glycopeptides are identical. Manual inspection of the ETD mass spectrum (Figure 5a) reveals backbone fragments corresponding to c3 though c7, c10, z3, and z6, all of which have identical masses regardless of the original glycopeptide. The peak observed at m/z 2079 is the result of Y cleavage within the glycan and corresponds to [IgG1+HexNAc2]+. Note that despite its confirmatory nature, this peak would not be matched in a database search as it does not derive from peptide backbone cleavage. Manual inspection of the corresponding HCD mass spectrum (Figure 5b) reveals a peak corresponding to [TKPREEQYNSTYR+HexNAc+Fuc] 2+ (m/zmeas 1010.9775, m/zcalc 1010.9765), and no peaks corresponding to fragments of the IgG4+G1 peptide. Thus, despite the low Xcorr value, it is possible to confidently assign the glycopeptide as IgG1+G0F by combining information from the ETD and HCD spectra. In total, 273 ETD mass spectra were triggered. Twenty seven of these corresponded to the known glycopeptides, i.e., peptides with known sequences from the four subclasses and glycans listed in Scheme 1. For all of the ETD events triggered, the corresponding HCD spectra were interrogated manually for peaks corresponding to HexNAc (m/z 204.09) and its fragments (m/z 126.06, 138.06, 144.07, 168.07 and 186.08),

HexNAc+Hex (m/z 366.14) and [Peptide+HexNAc] (IgG 1: EEQYnSTYR m/z 1392.5922 (1+), 696.8000 (2+), 464.8693 (3+); TKPREEQYNSTYR: m/z 1874.8882 (1+), 937.9480 (2+), 625.6346 (3+)) (IgG 2/3: EEQFNSTFR m/z 1360.6022 (1+), 680.8050 (2+), 454.2059 (3+); TKPREEQFNSTFR m/z 1842.8982 (1+), 921.9530 (2+), 614.9713 (3+)) (IgG 4: EEQFNSTYR m/z 1376.5972 (1+), 688.8025 (2+), 459.5376 (3+); TKPREEQFNSTYR m/z 1858.8932 (1+), 929.9505 (2+), 620.3029 (3+)); and [Peptide+HexNAc+HexNAc] (IgG1: EEQYnSTYR m/z 1595.6712 (1+), 798.3395 (2+), 532.5623 (3+); TKPREEQYNSTYR m/z 2077.9676(1+), 1039.4877 (2+), 693.3277 (3+); IgG 2/3: EEQFNSTFR m/z 1563.6855 (1+), 782.3467 (2+), 521.9004 (3+); TKPREEQFNSTFR m/z 2045.9776 (1+), 1023.4927 (2+), 682.6644 (3+); IgG 4; EEQFNSTYR m/z 1579.6764 (1+), 790.3421 (2+), 527.2307 (3+), for peptide TKPREEQFNSTYR m/z 2061.9726 (1+), 1031.4902 (2+), 687.9961 (3+)) and grouped according to the peaks observed; see Supplemental Table 2. Forty-one of the spectra contained peaks corresponding to HexNAc, HexNAc+Hex, and [Peptide +HexNAc], seven of which also contained peaks corresponding to [Peptide+HexNAc+HexNAc]. Twenty-eight had fragments corresponding to HexNAc and either [Peptide+HexNAc] or [Peptide+HexNAc+HexNac], with 20 containing both. Thirtyfive spectra contained peaks corresponding to HexNAc and 4522



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Figure 5. (a) Product ion-triggered ETD mass spectrum recorded at retention time 18.79 min. The spectrum can be matched to both the IgG1+G0F tryptic peptide and the IgG4+G1 tryptic peptide. (b) The corresponding HCD mass spectrum, recorded at RT 18.79 min, confirms the identity as IgG1+G0F.

Hex+HexNAc, but no known peptide containing peaks. Eightythree spectra had peaks corresponding to HexNAc, but no known peptide containing peaks. Eight had peaks corresponding to Hex+HexNAc only. The remaining 78 HCD spectra did not contain any of the above peaks, and the corresponding ETD spectra were triggered because of the presence of peaks within the product ion tolerance. In summary, 69/273 spectra corresponded to glycoforms of the known IgG1−4 peptides, and 126/273 spectra were confirmed as glycopeptides by manual analysis but the peptide sequence was unassigned. For this analysis, 29% of the ETD events triggered were not glycopeptides.

core, that is, fragments that are common to all N-glycans, HCD PI ETD can be applied to the analysis of glycopeptides without prior knowledge of the glycan structure. In this analysis, highmannose glycans and complex glycans with varying degrees of galactosylation and fucosylation were identified. The product ions used to trigger ETD were HexNAc (m/z 204) and HexHexNAc (m/z 366). Manual analysis of the HCD data (Supplemental Tables 1 and 2) suggests that the HexNAc oxonium ion is the more reliable trigger of the two; in the RNase B analysis, 136/139 HCD spectra contained peaks at m/ z 204, whereas 54/139 contained peaks at m/z 366. For the IgG analysis, the figures are 195/273 and 84/273 respectively. Nevertheless, some ETD events were triggered solely by the presence of the HexHexNAc HCD fragment. Thus, more comprehensive glycoproteome coverage is achieved by employing multiple potential product ion triggers. The results confirm the complementary nature of HCD and ETD: HCD provides information on the glycan structure, whereas ETD provides information on peptide sequence. For the ribonuclease B analysis, ETD events were triggered for all of the known glycopeptides. Although manual analysis of the ETD mass spectra confirmed the identity of the glycopeptides, that was not true for the protein database search algorithms. SEQUEST and Zcore correctly assigned ∼2/3 of the ETD mass spectra, and Mascot failed to correctly assign any. The

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CONCLUSION We have applied HCD product ion-triggered ETD MS/MS to the analysis of proteolytic digests of the glycoproteins ribonuclease B and immunoglobulin G. The results show that the approach allows targeted analysis of N-linked glycopeptides with complex samples without pre-enrichment. A number of enrichment strategies are currently employed in glycoproteomics research, as reviewed by Zhang et al.34 As those authors note, multiple enrichment strategies are required to identify the maximum number of glycoproteins because of the specificities of the individual methods. The HCD PI ETD method circumvents that requirement. Moreover, because the approach relies on the generation of fragments from within the N-glycan 4523



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(9) Hochepied, T.; Berger, F. G.; Baumann, H.; Libert, C. α1-acid glycoprotein: An acute phase protein with inflammatory and immunomodulation properties. Cytokine Growth Factor Rev. 2003, 14 (1), 25−34. (10) Kandzia, S.; Grammel, N.; Grabenhorst, E.; Conradt, H. S. Quantitative N-glycan mapping of glycoprotein therapeutics by HPAEC-PAD: Glycosylation chracteristics of different recombinant EPO products. In Cells and Culture, Proceedings of the 20th ESACT Meeting; Springer: Dordrecht, 2010; pp 867−871. (11) Raju, T. S.; Briggs, J. B.; Borge, S. M.; Jones, A. J. S. Speciesspecific variation in glycosylation of IgG: evidence for the speciesspecific sialylation and branch-specific galactosylation and importance for engineering recombinant glycoprotein therapeutics. Glycobiology 2000, 10 (5), 477−486. (12) Imperiali, B.; O’Connor, S. E. Effect of N-linked glycosylation on glycopeptide and glycoprotein structure. Curr. Opin. Chem. Biol. 1999, 3 (6), 643−649. (13) Hagglund, P.; Bunkenborg, J.; Elortza, F.; Jensen, O. N.; Roepstorff, P. A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sutes using HILIC enrichment and partial deglycosylation. J. Proteome Res. 2004, 3 (3), 556−566. (14) Hagglund, P.; Matthiesen, R.; Elortza, F.; Hojrup, P.; Roepstorff, P.; Jensen, O. N.; Bunkenborg, J. An enzymatic deglycosylation scheme enabling idneitification of core fucosylated N-glycans and Oglycosylation site mapping of human plasma proteins. J. Proteome Res. 2007, 6 (8), 3021−3031. (15) Tarentino, A. L.; Gomez, C. M.; Plummer, T. H. Deglycosylation of asparagine-linked glycans by peptide:N-glycosidase F. Biochemistry 1985, 24 (17), 4665−4671. (16) Hakansson, K.; Cooper, H. J.; Emmett, M. R.; Costello, C. E.; Marshall, A. G.; Nilsson, C. L. ECD and IRMPD MS/MS of an Nglycosylated tryptic peptide to yield complementary sequence information. Anal. Chem. 2001, 73 (18), 4530−4536. (17) Mikesh, L. M.; Ueberheide, B.; Chi, A.; Coon, J. J.; Syka, J. E. P.; Shabanowitz, J.; Hunt, D. F. The utility of ETD mass spectrometry in proteomic analysis. Biochim. Biophys. Acta, Proteins Proteomics 2006, 1764 (12), 1811−1822. (18) Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Localization of O-glycosylation sites in peptides by ECD in a Fourier transform mass spectrometer. Anal. Chem. 1999, 71 (20), 4431−4436. (19) Alley, W. R., Jr; Mechref, Y.; Novotny, M. V. Characterisation of glycopeptides by combining collision-induced dissociation and electron transfer dissociation mass spectrometry data. Rapid Commun. Mass Spectrom. 2009, 23 (1), 161−170. (20) Catalina, M. I.; Koeleman, C. A. M.; Deelder, A. M.; Wuhrer, M. Electron transfer dissociation of N-glycopeptides: loss of the entire Nglycosylated asparagine side chain. Rapid Commun. Mass Spectrom. 2007, 21 (6), 1053−1061. (21) Scott, N. E.; Parker, B. L.; Connolly, A. M.; Paulech, J.; Edwards, A. V. G.; Crossett, B.; Falconer, L.; Kolarich, D.; Djordjevic, S. P.; Hojrup, P.; Packer, N. H.; Larsen, M. R.; Cordwell, S. J. Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the Nlinked glycoproteome of Campylobacter jejuni. Mol. Cell. Proteomics 2011, 10 (2), No. M000031. (22) Olsen, J. V.; Macek, B.; Lange, O.; Makarov, A.; Horning, S.; Mann, M. Higher-energy C-trap dissociation for peptide modification analysis. Nat. Methods 2007, 4 (9), 709−712. (23) Segu, Z. M.; Mechref, Y. Characterising protein glycosylation sites through higher-energy C-trap dissociation. Rapid Commun. Mass Spectrom. 2010, 24 (9), 1217−1225. (24) Hart-Smith, G.; Raftery, M. J. Detection and characterisation of low abundance glycopeptides via higher-energy C-trap dissociation and orbitrap mass analysis. J. Am. Soc. Mass Spectrom. 2012, 23, 124− 140. (25) Zhao, P.; Viner, R.; Teo, C. F.; Boons, G.-J.; Horn, D. M.; Wells, L. Combining high-energy C-trap dissociation and electron transfer

limitations of the database search algorithms were further highlighted in the IgG analysis. While the mass spectrometry method does not require prior knowledge of the glycan structure, the search algorithm requires that variable modifications be defined. Additionally, only a maximum of six variable modifications are permitted. In summary, the HCD PI ETD approach shows promise for the targeted analysis of glycopeptides. Particularly exciting is the possibility of analysis of N-linked glycopeptides of unknown structure and heterogeneous O-linked glycopeptides. However, in order for its potential to be fully realized, development of software tools which are capable of (a) assigning glycan structures on the basis of the HCD spectrum and (b) defining those structures as variable modifications in a subsequent search of the ETD data are required.

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary figures and tables. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +44 (0)121 414 7527. Fax: +44 (0)121 414 5925. E-mail [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Ribonuclease B and immunoglobulin G samples were a kind gift of Prof Roy Jefferis. The Advion Triversa Nanomate, Dionex LC and Thermo Fisher Orbitrap mass spectrometer used in this research were funded through the Birmingham Science City Translational Medicine: Experimental Medicine Network of Excellence Project, with support from Advantage West Midlands (AWM).

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REFERENCES

(1) Apweiler, R.; Hermjakob, H.; Sharon, N. On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim. Biophys. Acta, Gen. Subj. 1999, 1473 (1), 4−8. (2) Khoury, G. A.; Baliban, R. C.; Floudas, C. A., Proteome-wide post-translational modification statistics: Frequency analysis and curation of the SWISS-PROT database. Sci. Rep. 2011, 1, article 90, doi: 10.1038/srep00090. (3) Marth, J. D.; Ohtsubo, K. Glycosylation in cellular mechanisms of health and disease. Cell 2006, 126 (5), 855−867. (4) Varki, A. Biological roles of oligosaccharides - All of the theories are correct. Glycobiology 1993, 3 (2), 97−130. (5) Go, E. P.; Chang, Q.; Liao, H.-X.; Sutherland, L. L.; Alam, S. M.; Haynes, B. F.; Desaire, H. Glycosylation site-specific analysis of Clade C HIV-1 envelope proteins. J. Proteome Res. 2009, 8 (9), 4231−4242. (6) Zhang, M.; Gaschen, B.; Blay, W.; Foley, B. L.; Haigwood, N.; Kuiken, C.; Korber, B. Tracking global patterns of N-linked glycosylation site variation in highly variable glycoproteins: HIV, SIV and HCV envelopes and influenza hemagglutinin. Glycobiology 2004, 14 (12), 1229−1246. (7) Dijk, W.; Turner, G. A.; Mackiewicz, A. Changes in glycosylation of acute-phase proteins in health and disease: Occurence, regulation and function. Glycoconjugate J. 1984, 1 (1), 5−14. (8) Higai, K.; Aoki, Y.; Azuma, Y.; Matsumoto, K. Glycosylation of site-specific glycans of α1-acid glycoprotein and alterations in acute and chromic inflammation. Biochim. Biophys. Acta 2005, 1725 (1), 128−135. 4524



Article

dissociation for protein O-GlcNAc modification site assignment. J. Proteome Res. 2011, 10 (9), 4088−4104. (26) Sweet, S. M. M.; Bailey, C. M.; Cunningham, D. L.; Heath, J. K.; Cooper, H. J. Large-scale localization of protein phosphorylation by electron capture dissociation mass spectrometry. Mol. Cell. Proteomics 2009, 8 (5), 904−912. (27) Sweet, S. M. M.; Creese, A. J.; Cooper, H. J. Strategy for the identification of sites of phosphorylation in proteins: Neutral loss triggered electron capture dissociation. Anal. Chem. 2006, 78 (21), 7563−7569. (28) Creese, A. J.; Grant, M. M.; Chapple, I. L. C.; Cooper, H. J. Online liquid chromatography neutral-loss triggered ETD mass spectrometry for the targeted analysis of citrullinated peptides. Anal. Methods 2011, 3 (2), 259−266. (29) Steentoft, C.; Vakhrushev, S. Y.; Vester-Christinasen, M. B.; Schjoldager, K. T.-B. G.; Kong, Y.; Bennett, E. P.; Mandel, U.; Wandall, H.; Levery, S. B.; Clausen, H. Mining the O-glycoproteome using sinc-finger nuclease-glycoengineered SimpleCell lines. Nat. Methods 2011, 8 (11), 977−982. (30) Zhang, Y.; Ficarro, S. B.; Li, S.; Marto, J. A. Optimized orbitrap HCD for quantitative analysis of phosphopeptides. J. Am. Soc. Mass Spectrom. 2009, 20, 1425−1434. (31) Domon, B.; Costello, C. E. A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconjugate J. 1988, 5 (4), 397−409. (32) Sweet, S. M. M.; Jones, A. W.; Cunningham, D. L.; Heath, J. K.; Cooper, H. J. Database search strategies for proteomic datasets generated by ECD mass spectrometry. J. Proteome Res. 2009, 8 (12), 5475−5484. (33) Perdivara, I.; Peddada, S. D.; Miller, F. W.; Tomer, K. B.; Deterding, L. J. Mass spectrometric determination of IgG subclassspecific glycosylation profiles in siblings discordant for myositis syndromes. J. Proteome Res. 2011, 10 (7), 2969−2978. (34) Zhang, L.; Lu, H.; Yang, P. Specific enrichment methods for glycoproteome research. Anal. Bioanal. Chem. 2010, 396 (1), 199− 203.

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