Investigation of ovarian cancer associated sialylation changes in N-linked glycopeptides by quantitative proteomics

Shetty Vivekananda, Hafner Julie, Shah Punit, Nickens Zacharie, Philip, Philip Ramila

Description

In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients’ serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.

Sujets

• Ovarian cancer
• Quantitative proteomics
• Sialic acid
• Lectin
• Mass spectrometry
• Western blot

Informations

• Publié par: biomed
• Publié le: 01 janvier 2012
• Nombre de lectures: 14
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Shetty et al. Clinical Proteomics 2012, 9:10
http://www.clinicalproteomicsjournal.com/content/9/1/10 CLINICAL
PROTEOMICS
RESEARCH Open Access
Investigation of ovarian cancer associated
sialylation changes in N-linked glycopeptides by
quantitative proteomics
*Vivekananda Shetty, Julie Hafner, Punit Shah, Zacharie Nickens and Ramila Philip
Abstract
Background: In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the
advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks
specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific
routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease
at its earliest and treatable stages.
Results: In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and
ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods.
We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated
glycopeptides were significantly up-regulated in ovarian cancer patients’ serum samples. LC-MS/MS analysis of the
non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various
ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer
samples.
Conclusion: Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in
ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve
as biomarkers for ovarian cancer.
Keywords: Ovarian cancer, Quantitative proteomics, Sialylation, Lectin, N-linked glycopeptides, Mass spectrometry,
Western blot
Background It has been shown that in the cancer transformation
The American Cancer Society estimates that in 2011, process, changed expression and post translational
about 21,990 new cases of ovarian cancer will be diag- modification of proteins occurs, resulting in a change in
nosed and 15,460 women will die of ovarian cancer in the protein structure and function. Investigating these
the United States (ovariancancer.org) [1-3]. When ovar- modifications specific for cancer may provide vital infor-
ian cancer is detected early, the five year survival rate is mation and serve as biomarkers for the diseased state.
over 90% [4]. Serum measurement of CA125, the Glycosylation is a common and essential form of post
current standard, has an early stage detection rate of translational modification of proteins. Among all the gly-
only about 28% and when combined with ultrasound cosylation forms, sialylation has received much attention
still only identifies 48% [5,6]. Development of improved owing to the strong correlation between the sialylation
diagnostic tools for early detection of ovarian cancer, in- aberration and cancer [7]. Sialic acid residues are known
cluding the discovery of new ovarian cancer biomarkers, to be linked via an R-2,3 or an R-2,6 bond to Gal/GalNAc
has the potential to significantly improve the survival in proteins. SNA lectin binds to peptides carrying a sialic
rate. acid residue connected to the underlying sugar chains
through an R-2,6 linkage. It has been suggested that there
is an increased branching of glycan structures in cancer
* Correspondence: [e-mail]
along with the increased expression both at RNA andImmunotope, Inc., 3805 Old Easton Road, Doylestown, PA 18902, USA
© 2012 Shetty et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.Shetty et al. Clinical Proteomics 2012, 9:10 Page 2 of 19
http://www.clinicalproteomicsjournal.com/content/9/1/10
1 2
protein level of sialyltransferase [8-10], which leads to a acetyl ( H / D ) labeling at the N-terminus in combin-3 3
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global increase in sialyation of the proteins [11]. Increased ation with O labeling during PNGase F digestion for
activity of sialyltransferase is also shown to be accom- glycosylation site mapping. Further, iTRAQ quantitative
panied by an increase in the level of enzymes, such as analysis of non-glycosylated peptides from the same
ST6Gal-1, which is responsible for linking sialic acid to samples revealed that the observed sialylation changes in
galactose in colorectal, ovarian and breast cancers cancer serum samples are independent of the glycopro-
[10,12-14]. ST6Gal-1 has been implicated in cell-cell tein concentrations. The quantitative proteomics results
interaction, enhanced motility and increased invasive- were further verified by western blot analysis of SNA
ness of tumor cells [10]. enriched selected glycoproteins that are strongly impli-
In the last decade, with the evolution of proteomics cated in ovarian cancer.
and glycomics technologies, the potential for the identi-
fication of biomarkers has increased tremendously, in Results
spite of the extreme complexity of the serum with a dy- Identification of glycopeptides and determination of
namic range in concentration of several orders of magni- glycosylation sites
tude [15]. In order to identify these low abundant The glycopeptide identification and glycosylation site de-
disease marker proteins in serum, various methods have termination were achieved by following the strategy as
been developed to deplete the abundant proteins such as outlined in Figure 1. First, abundant IgG glycoproteins
albumin and IgG, which constitutes about 90% of the were depleted from normal and ovarian cancer patients’
serum protein concentration [16,17]. Alternatively, sev- serum samples. Then equal amounts (5 mg each) of pro-
eral methods were developed to enrich a specific class of teinfrom normaland cancerserum samplesweredigested
proteins, such as glycoproteins by a lectin affinity en- by trypsin and glycopeptides containing sialic acid were
richment strategy, which can increase the chances for enriched by using SNA lectin. The resulting sialylated gly-
the identification of elusive glycosylation changes in low copeptides were labeled using light and heavy isotopes of
abundant proteins. Recent advances in glycoproteomics acetic anhydride reagents and mixed. Next, N-linked gly-
have made it possible to probe specific glycosylation cans were cleaved by PNGase F in the presence of heavy
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changes [18], in particular sialylation changes [19-21], in ( O) water to introduce a 3 Da mass shift with the aim
proteins between the disease and normal state. The level to unequivocally identify glycosylation sites. Finally, the
of sialic acid was observed to be significantly elevated in N-deglycosylated peptide mixture was analyzed by nano
ovarian cancer patients plasma compared to the healthy LC-MS/MS to identify peptide sequences in addition to
controls [19,20]. Berbec et al. [21], reported that the determining the glycosylation sites. Each glycopeptide
average concentration of sialic acid in total serum in identified in the database search results was inspected for
ovarian cancer patients was significantly higher than in the NXS/Tconsensus sequence as well as for a 3 Da mass
the healthy control group and may reflect the develop- shift. Also, the MS/MS spectrum of each glycopeptide was
ment of malignancy and should be considered as a sup- verified manually and unambiguously characterized the
porting tumor marker in ovarian cancer diagnosis. In complete peptide sequence. For instance, two glycopep-
recent years, several groups have investigated the sialyla- tides (VVLHPNYSQVDIGLIK and NLFLNHSENATAK)
tion aberration in the glycoproteome of cancer serum were identified for haptoglobin and the tandem mass
samples using diverse proteomics strategies including spectrometry data in Figure 2b shows all the signature
lectin affinity, hydrazine chemistry, HPLC and chemical ions (b and y ions) confirming the sequence of the light
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enrichment methods [18,22-27]. isotope of CH CO-VVLHPD( O)YSQVDIGLIK-OCCH3 3
In our previous work, we probed the prostate cancer glycopeptide sequence. Similarly, in the MS/MS spectrum
serum glycoproteome by employing the lectin-directed of Figure 2a, all b and y ions, including a shift of 3 Da
tandem labeling (LTL) quantitative proteomics method modification in b ions (b ,b ,b ) and y ions (y,y,y )13 14 15 2 4 5
[28] and identified several N-linked sialylated glycopep- due to heavy acetyl group at the N-terminus, confirm the
tides that showed significant sialylation aberration be- identity of the heavy isotope of the CD CO-VVLHPD3
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tween normal and prostate cancer serum samples. In the ( O)YSQVDIGLIK-OCCD glycopeptide. It should be3
current study, we report the results of the sialylation ab- noted that the above two peptides possess NXT/S consen-
erration analysis in ovarian cancer-associated N-linked sus sequences and, in both peptides, asparagine is modi-
glycoproteins. We employed the LTL method to identify fied to aspartic acid, as evidenced by 3 Da mass shift for
N-linked sialylation sites and accurately identified the b ion in Figure 2a and 2b. However, the total mass is9
changes in sialylation between normal a