Application Note LCMS-76 Identification of a Second N-glycosylation Site in a Human PSA Sample by Combined CID/ETD Fragmentation

Introduction

The analysis of glycosylation patterns in therapeutic antibodies and changes in the glycosylation patterns of proteins associated with human diseases — for example, congenital disorders of glycosylation (CDG) or various kinds of cancer — are important areas in glycoprotein research.

Therapeutic IgG antibodies generally contain only one N-glycosylation site, therefore quality control is normally performed by mass spectrometric analysis of enzymatically released glycans (ref. LCMS-70). However, since glycopro-teins usually contain more than one N-glycosylation site or more than one glycoprotein is present, this approach is not well-suited to the characterization of glycoprotein biomark-ers in biological samples.

A workflow based on ion trap mass spectrometry is avail-able for structural elucidation of N-glycans attached to a known glycosylation site (ref. LCMS-66). This workflow provides information about the glycan and peptide mass of a fragmented glycopeptide and the glycan structure. The workflow is based on LC-MS/MS analysis using CID as a fragmentation method, automatic post-processing, and classification of potential glycopeptide MS/MS spectra and identification of glycan structures in ProteinScape 3.0.

Because ion trap CID spectra of N-glycopeptides mainly comprise glycan fragments (B- and Y-type ions) and pep-tide fragments are rarely seen, little information about the peptide backbone is obtained. Therefore, in the case of unknown glycoproteins, localization of glycosylation sites is difficult. For such analyses, ETD is the fragmentation method of choice.

In contrast to CID, ETD cleaves the N-Cαα bond of the pep-tide backbone; resulting in c- and z-ion series. This means that post-translational modifications — for example glycans — remain attached to amino acid residues. Typically, high quality ETD spectra are obtained from charge states > 2. To increase the average charge states of glycopeptide ions and improve overall signal intensities, we used the CaptiveSpray™ source (Bruker-Michrom) for all experiments with acetonitrile-enriched nitrogen as sheath gas (ref. ASMS Poster 2012, ThP25).

The results presented here were generated during the 2012 gPRG study of the ABRF.1 The main objective of the study was the quantitation of human prostate specific antigen (PSA), which is a biological biomarker for prostate cancer. This application note focuses on the identification of a new N-glycosylation site in the PSA study sample.

Experimental

Sample Preparation and LC ESI-MS

The PSA reference sample was carbamidomethylated and digested with trypsin according to the Sigma-Aldrich proto-col (Trypsin, Technical Bulletin). The tryptic peptides were separated on an UltiMate 3000 nanoRSLC system with an Acclaim® PepMap C18 column (see Table 1 for more details). MS experiments were carried out using the amaZon speed ETD ion trap system equipped with a CaptiveSpray™ ioniza-tion source (CSI, Bruker-Michrom). In an adaptation to CSI standard settings, acetonitrile-enriched nitrogen was used as sheath gas. CID and ETD fragmentation experiments were performed in autoMSn mode using Enhanced resolution mode for MS and UltraScan mode for MS/MS acquisition. Experi-mental details are given in Table 2.

Table 1. LC settings for separation of N-glycopeptides and non-glycosylated peptides.

LC Settings

System Dionex RSLCnano

Trap column Dionex Acclaim® PepMap RSLC, 75 µm x 2 cm, C18, 3 µm, 100 Å

Analytical column Dionex Acclaim® PepMap RSLC, 75 µm x 25 cm, C18, 2 µm, 100 Å

Flow rate 300 nL/min

Gradient 5-30% acetonitrile in 0.1% formic acid in 50 min

[1] Glycoprotein Research Group (gPRG) of The Association of Bio-molecular Resource Facilities (ABRF)

Figure 1: Amino acid sequence of human PSA showing an N-glycosylation site at position 69.

Table 2. MS and MS/MS settings used for acquisition with the amaZon speed ETD.

Table 3. GlycoQuest search parameters.

Data Processing, Glycan Identification and Peptide Assignment

MS data were processed with DataAnalysis 4.1 using default settings for glycopeptide analysis. The resulting peak lists were clas-sified in ProteinScape 3.0. Glycopeptide spectra were then searched against the GlycomeDB database using the integrated search engine GlycoQuest. The used parameters are listed in Table 3. ETD spectra of glycopeptides with an identified glycan structure but unknown peptide backbone were partly de novo sequenced using DataAnalysis 4.1 and the BioTools RapiDeNovo™ tool. The resulting sequence tags were searched using MS-BLAST against the SwissProt database and suggested peptide sequences were validated using BioTools 3.2.

Results

PSA is known to have an N-glycosylation site at position Asn69 (Figure 1). The aim of the gPRG study was the relative quantita-tion of different glycans present at this site. However, during this study, an additional N-glycosylation site was discovered in the PSA sample.

The GlycoQuest search results showed two high-mannose glycan structures (Man5 and Man6) assigned to a peptide with an unexpected Mr of 1545.83 Da (Figure 2). To identify the peptide backbone, ETD fragmen-tation was performed on the same sample. The workflow for complete N-glycopeptide elucidation is illustrated in Figure 3.

Parameter Value

Submitted to search Only classified spectra

Glycan type N-glycan

Taxonomy No restriction

Database Glycome DB

Composition restriction Hex ≤ 9; HexNAc ≤ 7; NeuAc ≤ 3; Fuc ≤ 1

Derivatization Underivatized

Ions H+ up to 4, charge permutation 1 to 4

MS tolerance 0.3 Da

MS/MS tolerance 0.35 Da

#13C 1

Fragmentation A, B, Y; max. 4 cleavages; max. 3 cross ring

MS Settings

Source CaptiveSpray

MS conditions Enhanced resolution mode (8,100 m/z s -1)

400-2,000 m/z scan range

200,000 ICC target

5 Spectra Averages (2 Rolling Averaging)

Target Mass 1,200 m/z (CID) and 900 m/z (ETD)

MS/MS conditions UltraScan mode (32,500 m/z s -1)

Scan begin 100 m/z – scan end 3 x precursor

500,000 ICC target

Smart isolation

5 precursor (Active Exclusion after 2 spectra)

3 Spectra Averages

CID Fragmentation amplitude: 70% (SmartFrag active)

ETD ICC target ETD 500,000, reaction time 100 ms

Human PSA Sequence

Figure 2: GlycoQuest search result of PSA tryptic digest in ProteinScape, showing two N-glycopeptides of high-mannose type in the glycan list with an unknown peptide sequence (Mr = 1545.8 Da).

Figure 3: General workflow for complete N-glycopeptide characterization by mass spectrometry using the amaZon speed ETD.

GlycoQuest Search Results in ProteinScape

Fragment Ion List

Annotated Spectrum

Glycan List

Project Navigator

Glycan Structure View

Peptide + GlcNAc

N-Glycopeptide Identification Workflow

Figure 5: MS Blast search results for the amino acid sequence tag generated from the ETD spectrum of the triply charged precursor at m/z 921.75 via de novo sequencing. a) OS = Homo sapiens; b) OS = Macaca fascicularis

De novo sequencing of the ETD spectrum of the triply charged precursor with m/z 921.75 in DataAnalysis resulted in the amino acid sequence LYNMSLLK (no differentiation between L and I or Q and K, Figure 4). The same sequence was obtained using RapiDeNovo™, a BioTools software module for de novo sequencing. The generated sequence tag matches the theoretical PSA sequence except for one amino acid substitution. The aspartic acid (Asp, D) residue at position 102 is substituted for an asparagine (Asn, N), creating a new N-glycosylation site with the typical motif NXS (Figure 5a).

When this sequence tag was submitted to an MS-BLAST search, one of the results was human PSA (Figure 5a). The MS-BLAST search also returned PSA from macaca fascicu-laris, which contains this potential glycosylation site (Figure 5b). This is a strong indication that a second sequence vari-ant of PSA is present in the human PSA reference sample.

To determine the complete sequence of the tryptic peptide, the tag LYNMSLLK was substituted into the human PSA sequence. Because the C-terminus of the tag derives from a tryptic cleavage, the amino acid sequence was extended towards the protein N-terminus until the theoretical mass was in agreement with the experimental Mr of 1545.83 Da. The resulting peptide sequence 95SFPHPLYNMSLLK107 shows an unspecific cleavage at the N-terminus. This cleavage can be explained by a serine-type endopeptidase activity of PSA.

For final confirmation, the peptide sequence SFPH-PLYNMSLLK was modified with the identified high-mannose glycan structure Man5 and matched to the ETD spectrum of m/z 921.75 (3+). Figure 6 shows the annotated spectrum in BioTools.

ETD Spectrum of Unexpected N-glycopeptide

MS Blast Search Results

Figure 4: ETD spectrum derived from the triply charged precursor with m/z 921.75. De novo sequencing was performed using the Annotation Tool in DataAnalysis 4.1.

b)

a)

Summary and Conclusion

The approach described here demonstrates the great potential of combining CID and ETD for complete structure elucidation of N-glycopeptides.

Glycan structures are identified from CID spectra using the GlycoQuest search engine in ProteinScape. In addition the molecular weight of the peptide backbone is calculated automatically. In this study, high-mannose type glycan struc-tures attached to an unexpected peptide sequence with Mr 1545.8 Da were detected.

For research use only. Not for use in diagnostic procedures. Bru

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Bruker Daltonik GmbH

Bremen · GermanyPhone +49 (0)421-2205-0 Fax +49 (0)421-2205-103 sales@bdal.de

Bruker Daltonics Inc.

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www.bruker.com/ms

Instrumentation & Software

amaZon speed ETD

ProteinScape

BioTools

RapiDeNovo

Keywords

Glycoproteins

ETD

N-Glycosylation

Authors: Kristina Marx, Anja Resemann, Ulrike Schweiger-Hufnagel, Waltraud Evers, Andrea Kiehne, Markus Meyer

References: Bruker Application Note LCMS-70 Uwe Möginger, Daniel Kolarich, Ulrike Schweiger-Hufnagel, Kristina Neue (2012). Detailed Structural Characterization of IgG N-glycans using Porous Graphitic Carbon LC-ESI Ion Trap MS/MS. #702896

The quality of the glycopeptide ETD spectra allows genera-tion of peptide sequence tags either manually or with the RapiDeNovo option of BioTools. An MS-BLAST search of the tag LYNMSLLK, which represents a potential N-glyco-sylation site, returned a part of the human PSA sequence with the amino acid substitution Asp102Asn.

Combining the results from CID and ETD experiments led to the following observations for the human PSA sample provided for the gPRG 2012 study:

Two PSA sequence variants were observed; the expected sequence with an aspartic acid residue at position 102 (confirmed by database search against the unmodified sequence), and a second variant with an asparagine residue substitution at position 102.

Asn102 represents a new glycosylation site, which to our knowledge, has not been described in human PSA samples before. Two glycopeptides with the sequence SFPHPLYN102MSLLK carrying the high-mannose type structures Man5 and Man6 were identified.

In summary, this study clearly illustrates the importance of analyzing both released glycans and intact N-glycopeptides when characterizing glycoproteins. The great benefit of ETD fragmentation for the identification of unknown glycopep-tide sequences could be demonstrated. Moreover, it should be noted that PNGase F treatment of glycoproteins leads to deamidation of N-glycosylation sites (N→ D), meaning that the observed sequence variant Asp102Asn would not have been discernible by analysis of the PNGase F-treated protein.

Figure 6: Ion trap ETD spectrum of the triply charged N-glycopep-tide with m/z 921.75 annotated in BioTools. The peptide 95SFPH-PLYNMSLLK107 is modified at Asn102 with a Man5 high-mannose glycan structure.

Fully Annotated ETD Spectrum

Bruker Application Note LCMS-66 Kristina Neue, Andrea Kiehne, Markus Meyer, Marcus Macht, Ulrike Schweiger-Hufnagel, Anja Resemann (2012) Straightforward N-glycopep-tide analysis combining fast ion trap data acquisition with new ProteinScape functionalities. #284649 ASMS Poster 2012, ThP25 Varki , R.D. Cummings, J.D. Esko, H.H. Freeze, P. Stanley, C.R. Ber-tozzi, G.W. Hart, M.E. Etler; 2008, Essentials of Glycobiology, 2nd edition, CSH press. B. Domon and C. Costello; A systematic nomenclature for carbohy-drate fragmentations in FAB-MS/MS spectra of glycoconjugates; Glycoconjugate, 5, 397 409 (1988). CFG: http://www.functionalglycomics.org/static/consortium/Nomenclature.shtml