Exploring Site-Specific N‑Glycosylation Microheterogeneity of Haptoglobin Using Glycopeptide CID Tandem Mass Spectra and Glycan Database Search

Glycosylation is a common protein
modification with a significant
role in many vital cellular processes and human diseases, making the
characterization of protein-attached glycan structures important for
understanding cell biology and disease processes. Direct analysis
of protein N-glycosylation by tandem mass spectrometry of glycopeptides
promises site-specific elucidation of N-glycan microheterogeneity,
something that detached N-glycan and deglycosylated peptide analyses
cannot provide. However, successful implementation of direct N-glycopeptide
analysis by tandem mass spectrometry remains a challenge. In this
work, we consider algorithmic techniques for the analysis of LC–MS/MS
data acquired from glycopeptide-enriched fractions of enzymatic digests
of purified proteins. We implement a computational strategy that takes
advantage of the properties of CID fragmentation spectra of N-glycopeptides,
matching the MS/MS spectra to peptide-glycan pairs from protein sequences
and glycan structure databases. Significantly, we also propose a novel
false discovery rate estimation technique to estimate and manage the
number of false identifications. We use a human glycoprotein standard,
haptoglobin, digested with trypsin and GluC, enriched for glycopeptides
using HILIC chromatography, and analyzed by LC–MS/MS to demonstrate
our algorithmic strategy and evaluate its performance. Our software,
GlycoPeptideSearch (GPS), assigned glycopeptide identifications to
246 of the spectra at a false discovery rate of 5.58%, identifying
42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin
N-linked glycosylation sites. We further demonstrate the effectiveness
of this approach by analyzing plasma-derived haptoglobin, identifying
136 N-linked glycopeptide spectra at a false discovery rate of 0.4%,
representing 15 distinct glycopeptides on at least three of the four
N-linked glycosylation sites. The software, GlycoPeptideSearch, is
available for download from http://edwardslab.bmcb.georgetown.edu/GPS.