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Targeted Quanti cation of O-Linked Glycosylation Site for Glycan Distribution Analysis
Overview
Purpose:
Develop an automated workflow for identification and quantification of
O
-linked glycopeptides and corresponding glycoforms per targeted protein.
Methods:
Combine IP-MS using MSIA extraction with unbiased HR/AM LC-MS and
MS/MS data acquisition. Perform a novel iterative searching strategy based on
retention time correlation of precursor and product ion accurate mass values creating
a targeted list to perform qualitative and quantitative analysis across samples.
Results:
Incorporation of MSIA IP enrichment strategy resulted in ca. 1,000-fold
increase in measured area under the curve (AUC) values per Apo CIII peptide
compared to the measured AUC values extracted from whole serum digest. The
increase in sensitivity facilitated the detection, characterization, and quantification of
key
O
-linked glycosylation region of Apo CIII and enabled direct comparison to the
unmodified form of the peptides.
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Introduction
The detection of glycoproteins, characterization of various glycoforms, and
identification of the modified region of a protein can be challenging due to the
complexity of the background, dilution of the precursor ion signal, and lack of common
PTM mass shift. Our approach to increase the throughput for identification and
quantification for
O
-linked glycopeptides and corresponding glycoforms is to perform
targeted data extraction from protein/peptide sequences. Multi-level scoring attributes
are implemented to automate data reduction, resulting in a refined list of highly
confident peptides. Incorporation of unbiased data acquisition of high-resolution,
accurate-mass (HR/AM) mass spectrometry (MS) and tandem-MS dramatically
increases the scoring routine.
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The final report provides a score and integrated peak
area across biological samples for modified and unmodified peptides attributed to the
targeted protein(s).
Methods
Sample Preparation
To perform initial testing, serum samples were collected from normal and stroke
patients. Each sample was divided into two equal aliquots. One set of samples were
reduced, alkylated, and digested with trypsin and used without further preparation. The
second set of samples were aspirated into an MS immunoassay (MSIA) direct analysis
in real time (D.A.R.T.) tip
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covalently loaded with an anti-Apo CIII antibody. Following
MSIA extraction, each sample was washed then reduced, alkylated, and digested
using a similar protocol as that for the serum samples with the ratio of analyte:enzyme
held constant. Prior to analysis, a constant amount of the PRTC kit was spiked as an
internal standard.
Liquid Chromatography
LC separation was performed using a Thermo Scientific™ Hypersil GOLD™
100
×
0.075 mm column with 3 µm particle size and a binary solvent system
comprised of: A) 0.1% formic acid and B) 0.1% formic aid in MeCN. A linear gradient of
5–45% B was performed over 40 minutes prior to column washing and re-equilibration.
Mass Spectrometry
All experiments were performed on a Thermo Scientific™ Q Exactive™ mass
spectrometer operated in data-dependent/dynamic-exclusion mode using a Top10
acquisition scheme. Full-scan MS spectra were acquired using a resolution setting of
70K and all HCD product ion spectra were acquired using 15K. All data was acquired
using internal lock mass.
Data Analysis
Initial unbiased data searching was performed using Thermo Scientific™ Proteome
Discoverer™ 1.4 software to identify all proteins and corresponding peptides. The
initial search strategy was performed with typical variable modifications of
phosphorylation, oxidation, and Cys alkylation. The Apo CIII protein sequence and
initial set of identified peptides from step 1 was transferred to prototype version of
Thermo Scientific™ Pinpoint™ 1.4 software to search for
O
-linked glycopeptides. The
screening tool incorporates base peptide sequence information with individual glycan
information to calculate the composite chemical formula facilitating HR/AM MS data
extraction resulting in a list of putative glycopeptides and corresponding glycoforms.
The list was used to perform higher level data extraction, verification/scoring, and
relative quantification using HR/AM MS data. Relative quantification was compared
between the glycopeptides and unmodified peptides originating from Apo CIII.
Results
A common stock of serum sa
equal aliquots. The only differ
using MSIA D.A.R.T. tips load
CIII tryptic peptide is presente
and without MSIA extraction p
The Pinpoint screening tool uti
unmodified peptides (Figure 2
all possible glycan additions (
of peptides and
m/z
values us
sequences and modifications
as retention times, precursor c
location. The results from the
workbook integrated analysis.
FIGURE 1. Representation o
evaluate
O
-linked glycopept
FIGURE 2. Screen capture s
putative modified and unmo
The information generated in t
eliminate peptide candidates i
small protein such as Apo CIII,
N
-terminal residues identified
processing parameters (comm
possible sequences. The scre
than one minute. Enabling the
number of possible sequences
peptides. The list is further red
and MS information to a final li
automated routine. Table 1 list
with non
O
-linked glycans. Ea
elution peak profile. The AUC
those peptides with missed cle
22–40, 59–79, and 61–79 sho
to the “normal.” Each of the mi
than the tryptic peptides.
The incorporation of the PRTC
to the expected times. Figure
the Apo CIII peptides. All of th
peptides with missed cleavage
metrics to each peptide. In ad
peptides provides landmarks f
to elute under similar times.
Serum Samples – Control and Dise
MSIA Extraction
Reduction, Alkylation, and Digesti
HR/AM LC-MS and MS/MS Analy
Whole Seru
Spike PRTC Kit
