Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma

Table 1 Commonly used serum tumor markers in clinical practice

Biomarker	Tumor class	Glycosylated protein
Alpha-feto protein (AFP)	Testicular, hepatocellular	Yes
Human chorionic gonadotropin	Testicular	Yes
CA 15-3	Breast	Yes
CA 19-9	Pancreatic	Yes
CA 27-29	Breast	Yes
CA 125	Ovarian	Yes
Carcinoembryonic antigen	Colon	Yes
Human epidermal growth factor receptor 2 (HER-2)	Breast	Yes
Prostate specific antigen (PSA)	Prostate	Yes
Thyroglobulin	Thyroid	Yes

Extracted from references 13, 14, 16, 17, 18, 20.

Table 2 Serum glycoproteins with altered glycosylation identified in pancreatic cancer patients

Techniques	Serum cohort (n)	Serum depletion (Yes/No)	Glycosylation change	Main glycoproteins identified	Ref.
ConA chromatography. Tryptic digestion. N-glycan MS analysis of the N-glycopeptides	2 PaC	No	Increased branching, fucosylation and sialylation		[101]
ConA and WGA chromatography. Glycoprotein separation and array with lectins (SNA, PNA, MAL, AAL).	6 PaC 8 ChrP 10 HC	Yes (IgY 12) (albumin, IgG and major acute-phase proteins)	Increased fucosylation and sialylation	Hemopexin Serum amyloid P component Antithrombin-III Haptoglobin (HPT)-related protein β-2-glycoprotein 1	[110]
Antibody microarray to α1-β-glycoprotein, amyloid P and antithrombin followed by lectin detection (SNA). MS glycoprotein identification.	22 PaC (III/IV) 35 ChrP 89 HC 37 diabetic		Increase affinity to SNA (α2,6-sialic acid) by 69% in PaC patients	α1-β-glycoprotein. ROC curve (cancer vs non cancer samples), 100% sensitivity 98% specificity. AUC = 0.998	[111]
Bead-based antibody-lectin (SNA, Con A) multiplex assay-for determining SNA and Con A reactivity of α1-β-glycoprotein, and amyloid P component.	20 PaC (III/IV) 20 ChrP 20 HC		SNA affinity (α2,6-sialic acid)	α2,6-sialic glycoforms of α1-β-glycoprotein Differentiation of ChrP vs PaC (P = 0.035)	[112]
SNA affinity chromatography to enrich sialylated glycopeptides and compared their relative abundance by ultra performance LC-MS	10 PaC (II-III) 5 Acute Pancreatitis 16 HC	Yes (albumin depleted)	SNA (α2,6-sialic acid)	Sialylated glycopeptides of HPT, α-1-antitrypsin (A1AT), transferrin, ceruloplasmin, α1-acid-glycoprotein (AGP), fetuin A and Igs. Change in acute pancreatitis and PaC	[97]
2DE followed by N-glycan sequencing	9 PaC (I-IV) 3 ChrP 5HC		SLex Fucosylation	Increase in SLex on AGP, HPT and transferrin in advanced PaC and ChrP Increase in core fucosylation of HPT and AGP in PaC vs ChrP and HC	[113]
Electrophoresis (1DE) followed by WB with anti SLex. Immunoprecipitation of ceruloplasmin and SLex detection	20 PaC (IIa-IV) 14 ChrP 13 HC	Yes (IgY 12) (albumin, IgG and major acute-phase proteins	SLex	Ceruloplasmin Tendency to an increase of SLex on ceruloplasmin in PaC vs HC and ChrP	[114]
Lectin (AAL)-antibody ELISA	72 PaC 22 HC 63 pancreatitis		AAL (fucosylation).	Increase of fucosylated HPT in advanced PaC	[115]
AGP purification MS analysis of AGP N-glycans and AAL ELISA	19 PaC (I-IV) 6 ChrP 6 HC		α1,3 fucosylation	Increase of fucosylated AGP in advanced PaC	[116-118]
N-glycan sequencing of human serum ribonuclease (RNase 1).	2 PaC 2 HC		Core fucosylation	Increase of core fucosylation in RNase 1 in PaC	[120]
ELISA to measure N-glycosylation Asn-88 site occupancy of serum RNase 1	91 PaC 60 HC		Asn-88 N-glycosylation	Increase in N-glycosylated Asn-88 of RNase 1 (normalized to RNase 1) in PaC.	[121]
AAL to enrich fucosylated glycoproteins LC-MS/MS analyses ELISA/lectin ELISAs	20 IPMN 10 MCN 37 PaC (I-IV) 30 HC 30 ChrP 22 OJ 30 Type II DM	IgY-14 LC10 columns	Fucosylation (AAL)	ACT trombospondin-1 HPT High diagnostic potential combined with CA 19-9	[119]
nanoLC-MS/MS analysis of iTRAQ labelled glycopeptides.	13 HC 13 ChrP 13 PaC 1 Std	IgY-14 LC10 column	Core-fucosylation	One core fucosylated peptide from ACT different between groups	[108]
PHA-L lectin to enrich complex N-glycoproteins 2D nanoLC-MS/MS analyses Western Blot with biotinylated PHA-L nanoLC-MS/MS of tryptic digested gel bands that corresponded to specific lectin interactions on Western blot	26 HC (include ChrP + pseudo cysts) 76 PaC	Albumin/IgG depletion	Increased fucosylation N211 Novel glycosylation site N64 New N-glycosylated side at N2336 in PaC N-glycosylation N877	HPT Leukemia inhibitory factor receptor LIFR Centrosome-associate protein 350 CE350 Vacuolar protein sorting-associated protein 13A VP13A	[122]
2D-LC-MS/MS	231 serum women samples pooled in groups: time-to-diagnosis	Yes	N-glycosylation occupancy	A1AT HPT AGP	[123]
Lectin (CCL2)-antibody ELISA	109 PaC 91 control (plasma)	No	CCL2 (3’ fucosylation)	MUC5AC	[99]
Antibody lectin sandwitch array	156 PaC (I-IV) 160 control (plasma)	No	SLea related	MUC5AC MUC16	[127]
Antibody microarray capture of proteins. Glycan analysis with lectins (AAL, WGA) and CA 19-9	23 PaC (I-IV) 23 HC	No	CA 19-9	MUC1 CEA	[128]
Antibody lectin sandwich array	23 PaC (I-IV) 23 HC	No	SLea(CA 19-9)	MUC1 MUC5AC	[129]
Antibody array	285 PaC (I-IV) 102 ChrP 144 HC (serum & plasma)	No	CA 19-9	MUC1 MUC5AC MUC16	[130]

MS: Mass-spectrometry; PaC: Pancreatic cancer; WGA: Wheat germ agglutinin; SNA: Sambucus nigra agglutinin; AAL: Aleuria aurantia lectin; ChrP: Chronic pancreatitis; HC: Healthy controls.

Table 3 List of proposed glycoproteins as possible biomarkers for pancreatic cancer

Glycoprotein	Glycosylation sites	Reported glycosylation	Serum auto-antibodies	Ref.
Mesothelin (cleaved form)	3 N-glycosylation sites	Reduction of MW after PNGase F digestion of A431 cancer cells	Yes	[133-144]
IGFBP-3	3 N-glycosylation sites O-glycosylation	Increase of N-glycosylation levels in tumor tissues Increase of biantennary complex type N-glycans having more mannose, fucose, bisecting GlcNAc and terminal sialic acid in breast cancer serum	Not described	[109,145]
IGFBP-2	O-glycosylation	Not described	Not described	[141,146]
REG 1A REG 1B REG 3A REG 4	O-glycosylation O-glycosylation 1 potential N-glycosylation site 1 N-glycosylation site	Increase protein glycoform diversity in pancreatic ductal fluid of PaC by western blot	Not described	[147-151]
TIMP-1	2 N-glycosylation sites	Core fucosylated, biantennary N-glycans with Gal or GalNAc in HEK293. Some of the glycans are sialylated, and many have outer arm fucosylation. Aberrant N-glycosylation in colon cancer	Not described	[152-156]
HER-2	7 N-glycosylation sites O-glycosylation	Altered N-glycosylation in breast cancer cells. Altered O-glycans (Tn and T) in PaC cells	Yes	[157-162]

IGFBP: Insulin-like growth factor binding protein; REG: Regenerating protein; PaC: Pancreatic cancer.