Confident assignment of site-specific glycosylation using a LC-MS/MS methodology with online HILIC
enrichment
Kshitij Khatri1, Gregory O. Staples2, Nancy Leymarie1, Deborah R. Leon1, Lilla Turiák1, Yu Huang1, Christian
F. Heckendorf1 and Joseph Zaia1
1
Center for Biomedical Mass Spectrometry, Department of Biochemistry, Boston University School of
Medicine, Boston, MA, USA
2
Agilent Technologies, Santa Clara, CA, USA
Introduction:
Glycans that decorate surface of proteins are critical for their biological functions. These glycans are
highly heterogeneous and any changes in their composition and structure can alter the protein’s
interaction with protein lectin domains that mediate linked biological processes. It is therefore
important to characterize, compare and understand the role of these carbohydrate moieties in a sitespecific manner, to shed light on potential biomolecular interactions and to facilitate therapeutic
interventions. Currently, the field lacks a workflow for accurate and reproducible analysis of site-specific
protein glycosylation. Non-glycosylated peptides in glycoprotein digests, owing to their higher ionization
efficiencies, adversely affect glycopeptide analysis by LC-MS (liquid chromatography-mass
spectrometry). Therefore, glycopeptides need to be enriched prior to any LC-MS analyses. Here, we
present a methodology for confidently assigning glycopeptides by combining online glycopeptide
enrichment and separation with tandem MS.
Methods:
Standard glycoproteins were purchased from Sigma-Aldrich. Purified viral Hemagglutinin samples were
purchased from Immune Technology, New York, NY. Glycoproteins were reduced, alkylated and
subjected to tryptic digestion. Desalted glycoprotein digests were subjected to LC-MS/MS on an Agilent
6550 Q-TOF mass spectrometer coupled with Agilent 1200 series nanoflow HPLC system and Chip-cube
nanospray source. A custom-made HPLC-chip separation device, with HILIC (Hydrophilic interaction
liquid chromatography) enrichment column and reversed-phase analytical column was used for online
enrichment and separation of glycopeptides. Whole tryptic digests were injected onto the HILIC column
in high organic-solvent conditions to allow glycopeptide retention, while non-glycosylated peptides were
washed away. The trapped glycopeptides were eluted from the HILIC column and resolved on the
reversed-phase analytical column using a valve-switch and introduction of high-aqueous mobile phase
followed by a reversed-phase gradient. Glycopeptides eluting were introduced into the mass
spectrometer using an integrated nano-sprayer as they eluted from the analytical column and analyzed
using CID (Collision-induced dissociation) tandem MS. LC-tandem-MS data were analyzed both manually
and using a custom software tool, developed in-house, for analysis of large glycopeptide LC-MS/MS
datasets.
Results:
Because glycosylation presents as a very heterogeneous modification, it can lead to a large search space
when profiling glycopeptide LC-MS data. This large search space arising from different combinations of
glycans and peptides can lead to ambiguous or false assignments if only MS1 data is used. The use of
tandem MS therefore becomes imperative for confident assignment of glycopeptides. Since complexity
in glycopeptide data ranges with number of glycosylation sites present on a glycoprotein, we
demonstrated the usefulness of our method using glycoproteins ranging in number of glycosylation
sites. Human transferrin (2 glycosylation sites), alpha-1-acid glycoprotein (5 glycosylation sites) and
hemagglutinin from influenza-A-virus (9 glycosylation sites) were analyzed using typical C18 and HILICC18 chromatography with tandem-MS.
The HILIC-C18 chromatography takes advantage of HILIC to trap glycosylated peptides by the virtue of
their hydrophilic glycan moiety and depletes any non-glycosylated peptides from the sample. Because
the resolution power of HILIC for glycopeptides is limited, we combined a HILIC enrichment column with
reversed-phase analytical column for efficient separation of enriched glycopeptides. When compared
with a commercially available C18 chip using typical reversed-phase gradient conditions, the HILIC-C18
chip showed significant improvement in glycopeptide signal abundances thereby enhancing the ability
to perform data-dependent tandem-MS on glycopeptides. Tandem-MS allowed unambiguous
assignment of glycopeptides using features like oxonium ions, stub glycopeptides and most importantly,
peptide backbone ions. Each of the listed features from tandem MS, adds confidence to the
glycopeptide assignments. Our prototype software for analysis of glycopeptide tandem MS allowed
rapid interpretation of MS2 spectra and scoring of glycopeptide assignments. Using our improved
chromatography system with online glycopeptide enrichment and separation followed by tandem MS,
we were able to get confident site-specific glycan profiles for each of the three glycoproteins studied.