Tandem Mass Tag-Based Proteomic Analysis of Potential Biomarkers for Hepatocellular Carcinoma Differentiation

Figures & data

Figure 1 Volcano plots and hierarchical clustering of differentially expressed proteins (DEPs) among poorly- (G3), moderate- (G2) and well-differentiated (G1) hepatocellular carcinoma (HCC). Volcano plots showing the distribution of significance fold change of DEPs between (A) G1 and G2, (B) G1 and G3, and (C) G2 and G3 HCC. The red spots and green spots indicate significantly up-regulated and down-regulated proteins, respectively. (D) Hierarchical clustering representing of 21 DEPs. Red indicates upregulation and green represents downregulation (color bar next to the figure).

Table 1 List of Differentially Expressed Proteins as Potential Biomarker Candidates for Well- and Poorly-Differentiated Hepatocellular Carcinoma

Confidence	Accession	Gene Symbol	q-value	Coverage [%]	# PSMs	# Peptides	# Unique Peptides	Score
High	P00966	ASS1	0	7	5	4	4	16.47
High	P31327	CPS1	0	25	65	32	32	321.12
High	P06132	UROD	0	11	4	3	3	19.98
High	P68871	HBB	0	88	126	11	6	602.36
High	P00167	CYB5A	0	16	9	2	2	39.38
High	H0Y449	YBX1	0	5	2	1	1	10.88
High	Q9NYL4	FKBP11	0.002	3	2	1	1	0

Table 2 Statistical Information on the Differentially Expressed Proteins

Gene Symbol	G1/G2	G1/G3	G2/G3	G1/G1 Non-Tumor	G2/G2 Non-Tumor	G3/G3 Non-Tumor	HCC/Non-Tumor
ASS1	0.928	0.354 **	0.381 **	0.188**	0.436*	0.979	0.373 **
CPS1	0.831	0.337 **	0.406 **	0.169**	0.586	0.800	0.344 **
UROD	0.835	1.739 *	2.082 **	1.459	2.229**	0.778	1.495 *
HBB	1.479	3.722 **	2.517 **	0.368**	0.632	0.398**	0.616 **
CYB5A	2.866 *	4.257 *	1.485	0.679	0.308*	0.459	0.383 *
YBX1	1.817 **	1.575 *	0.867	1.1	0.69*	1.339	0.805
FKBP11	2.694 *	4.085 **	1.516	5.902**	2.429	0.625	2.048 *

Notes: *P<0.05; **P < 0.01.

Figure 2 Abundance ratio of differentially expressed proteins ASS1, CPS1, UROD, HBB, CYB5A, YBX1, and FKBP11 between hepatocellular carcinoma (HCC) tumor and non-tumorous tissues, and among well- (G1), moderate- (G2), and poorly- (G3) differentiated HCC. ASS1 and CPSI were up-regulated, while UROD and HBB were down-regulated in G3 compared with G1 and G2 tissues; CYB5A, YBX1, and FKBP11 were up-regulated in G1 compared with G2 and G3 tissues.

Figure 3 Representative immunohistochemical staining for seven candidate proteins (ASS1, CPS1, UROD, HBB, CYB5A, YBX1, and FKBP11) between hepatocellular carcinoma (HCC) and adjacent non-tumorous tissues. The scale bar is 500 μm.

Figure 4 Representative immunohistochemical staining for seven candidate proteins (ASS1, CPS1, UROD, HBB, CYB5A, YBX1, and FKBP11) among well- (G1), moderate- (G2), and poorly (G3) differentiated hepatocellular carcinoma (HCC) tissues. The original scale bar is 500 μm.

Figure 5 Gene Ontology enrichment analysis of the 7 DEPs (ASS1, CPS1, UROD, HBB, CYB5A, YBX1, and FKBP11) potentially for identifying poorly- and well-differentiated hepatocellular carcinoma.

Table 3 KEGG Pathway Enrichment Analysis of Differentially Expressed Proteins Between Well- and Poorly-Differentiated Hepatocellular Carcinoma

ID	Description	Count	FDR	Matching Proteins
map00220	Arginine biosynthesis	2	0.000	ASS1, CPS1
map00250	Alanine, aspartate and glutamate metabolism	2	0.000	ASS1, CPS1
map01230	Biosynthesis of amino acids	2	0.001	ASS1, CPS1
map01100	Metabolic pathways	3	0.019	ASS1, CPS1, UROD

Table 4 Protein–Protein Interaction Analysis of the Differentially Expressed Proteins Between Well- and Poorly-Differentiated Hepatocellular Carcinoma

Protein 1	Protein 2	Neighbourhood	Gene Fusion	Coexpression	Experiment	Databases	Text Mining	Score
CPS1	ASS1	0.185	0.627	0.522	0.35	0.8	0.803	0.995
CPS1	YBX1	0	0	0.064	0	0	0.441	0.454
ASS1	UROD	0.147	0	0	0	0	0.353	0.424

Figure 6 STRING networks of the 7 DEPs (ASS1, CPS1, UROD, HBB, CYB5A, YBX1, and FKBP11) potentially for identifying poorly- and well-differentiated hepatocellular carcinoma. Different line colours represent the types of evidence for the association. Green colour depicts neighbourhood; Red colour: Gene fusion; Pink colour: Experiments; Light green colour: Text mining; Blue colour: Cooccurrence; Dark blue colour: Coexpression; Purple colour: Homology; and circle nodes indicated different proteins. Interaction networks are shown in evidence view.