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m/z250 500 750 1000 1250 1500 1750

%

0

100

m/z250 500 750 1000 1250 1500 1750

%

0

1001194.043

204.087341.019

1118.806

621.267

1195.038

1295.578 1678.210

1119.477

1118.470853.091

852.582429.093523.111

853.334

853.844

1119.813

1120.1351137.441 1677.709

m/z500 1000 1500

%

0

100

m/z500 1000 1500

%

0

100204.078

1193.982366.123 621.235 1223.482 1677.618

1137.753

1118.416

445.093

1138.431

1139.093

INTRODUCTION

Glycosylation is one of the most common post-

translational modifications involved in many biological processes such as cell-cell

recognition, cell signaling and regulatory

functions. The use of mass spectrometry for

glycopeptide studies is a challenging area and

often requires sample enrichment prior to

analysis, using techniques such as lectin affinity

chromatography and HILIC.

The focus of this work is to highlight a

combination of enrichment techniques (HILIC

and TiO2) in combination with MS acquisition

strategies, incorporating ion mobility for the

identification and profiling of N-linked

glycopeptides.

MULTI-ACQUISITION ION MOBILITY STRATERGIES UTILIZING LC/MS AND MALDI FOR THE CHARACTERIZATION OF ENRICHED GLYCOPEPTIDES

Lee A. Gethings1, Mark W. Towers1, Chen-Chun Chen2, Pei-Yi Lin2, Yu Ju Chen2 1Waters Corporation, Manchester, UK, 2Department of Chemistry, Academia Sinica, Taipei, Taiwan

RESULTS

Initially results were acquired for proof of principle studies, using standard glycoproteins which had been enriched using HILIC and TiO2 spin columns. Data were collected using LC-DIA and LC-IM-DIA workflows (Figure 2) in addition to complimentary MALDI

data (Figure 3). The experimental design was interrogated further using membrane protein extracts from HeLa cells.

METHODS

Sample preparation

Standard glycoproteins (ovalbumin, fetuin, horseradish

peroxidase & asialofetuin) were denatured, reduced (DTT) and alkylated (IAM) prior to gel assisted digestion with trypsin1.

HILIC or TiO2 spin columns were then used to enrich the

glycopeptides.

Membrane proteins from a HeLa cell line were also prepared

using the same methodology as described for the standard

glycoproteins, with the exception of using TCEP and MMTS for

reduction and alkylation respectively. LC/MS samples were

prepared with 0.1% formic acid at 100 ng/µL.

For the MALDI experiments, a standard glycoprotein mix at

250 ng/µL was spotted onto a MALDI target plate with 10 mg/

mL DHB (20% ACN/0.1% TFA) with mixing on target.

LC-MS conditions

Experiments were conducted using a 40 min gradient from 5 to

40% acetonitrile (0.1% formic acid) at 300 nL/min using a

nanoACQUITY system and a HSS T3 1.8 µm C18 reversed phase 75 µm x 15 cm nanoscale LC column.

Data were acquired using data independent analysis (DIA),

utilizing nanoscale LC nanoACQUITY, directly interfaced to a hybrid IM-oaToF Synapt G2-Si mass spectrometer. Ion mobility

(IM) was used in conjunction with the DIA acquisition schema.2

MALDI conditions

Data were collected using MALDI Synapt G2-Si in sensitivity

mode with ion mobility (parameters provided in Table 1).

Figure 1. Experimental design study for the enrichment and

analysis of N-linked glycopeptides

Figure 2. Data independent analysis of glycopeptides with and without the application of ion mobility. Example HRP data for the

peptide SFANSTQTFFNAFVEAMDR is shown; (a) LC-DIA data showing low and elevated energy spectra; (b) LC-IM-DIA data pro-

viding full peptide and glycan sequence coverage within the same spectrum. Magnification from the Y1 ion yields full glycan se-quence information for both glycoforms which are known to exist (m/z 1604.4 & 1678.2).

References

1. A multiplexed quantitative strategy for membrane proteomics. Li Han et al., MCP. 2008;7:1983-1997.

2. Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Li GZ, et al. Proteomics. 2009 Mar;9(6):1696-719.

3. Semi-Automated identification of N-Glycopeptides by Hydrophilic Interaction Chromatography, nano-Reverse-Phase LC-MS/MS, and Glycan Database Search. Pompach P et al., J. Proteome Res. 2012; 11:1728-1740.

4. Desaire Research Team, Dept. Chemistry, University of Kansas, USA

CONCLUSIONS

Incorporating ion mobility into the acquisition schema

provides high sequence coverage of both peptide and

glycan moiety within the same spectra.

TiO2 enrichment shows glycoprotein recovery to

increase by 2-fold when compared with HILIC.

Over 200 membrane HeLa proteins have been

identified from HILIC and TiO2 enrichment, with 68

positively identified as glycosylated.

Proteins of low abundance are identified with a high

degree of confidence.

low energy

elevated energy

ion mobility/gas phase

separation

liquid phase

separation

retention time aligned precursor and product ions

drift time aligned precursor and product ions

HeLa cells (membrane proteins)

Gel-assisted DigestionAlkylation (MMTS)

Reduction (TCEP)

Standard Glycoproteins

Tryptic DigestionReduction (DTT)

Alkylation (IAM)

Peptide & Glycopeptides

HILICSpin Column

TiO2

Spin Column

Enriched glycopeptides

LC-MS & MALDI

Bioinformatics

Figure 3 . Example MALDI data: 500fmol HRP

(SFANSTQTFFANAFVEAMDR, m/z 3355). Transfer fragmenta-

tion provides glycan (upper spectrum) and peptide sequence coverage (magnified region). Potential contamination peaks

are reduced with the implementation of ion mobility.

Figure 7. (a) Functional classification of identified HeLa glyco-

proteins from both enrichment strategies; (b) KEGG Pathway

analysis - Identified HeLa proteins such as integrin and CD44 are mapped to the viral carcinogenesis pathway (human papil-

lomavirus).

Figure 4 . Venn diagram (inset) highlights the number of HeLa

membrane proteins identified from the HILIC and TiO2 enrich-

ment methods. Proteins associated as glycoproteins (based on NXS/T motif) are represented predominantly with the NXT mo-

tif (55%). Combining both enrichment techniques reveals 68

glycoprotein identifications.

Table 1 . MALDI Synapt G2-Si system parameters

0

50

100

150

200

250

# enriched proteins

NXS/T motif NXT motif NXS motif

HILIC

TiO

Instrument Parameter Value

IMS velocity (start) 600 m/s

IMS velocity (end) 150 m/s

Trap DC Bias 130

Cooling Gas 50

Trap Gas 2

Collision Energy 200 eV

Bioinformatics

The LC-MS glycopeptide data were processed and searched with ProteinLynx GlobalSERVER. GlycoPeptide Search (GPS)3

was used for determining glycan composition, whilst MALDI

data were processed and searched using GlycoPep ID and Gly-

coPep DB for peptide and glycan interpretation respectively.4

m/z500 750 1000 1250 1500 1750

%

0

100938.438

867.416

720.323

1199.554 1447.666 1576.700

E V F A N F F T Q T S

Elevated Energy

Low Energy

Figure 6. Example glycoprotein identification (Laminin subunit

β-4), providing assigned glycopeptide sequences and potential

glycan moieties (mass filtered to <15 ppm) provided by the GPS software.

Protein ID

47 20034

HILIC TiO2

Figure 5. Normalized protein abundances resulting from HILIC

(black) and TiO2 (blue) enrichment. TiO2 appears to selectively

enrich lower abundant proteins with many being identified as glycosylated.

Elevated Energy

Low Energy

m/z1000 2000 3000 4000

%

0

100 7.85e33354.420

938.438

120.081

867.416

2533.1592166.955