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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
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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
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2533.1592166.955