iTRAQ-based proteomics reveals novel biomarkers of osteoarthritis

Figures & data

Figure 1. Result of shotgun proteomics based on iTRAQ method. (A) Experimental scheme. (B) Venn diagram showing the ratio of proteins that were included in OA and control cartilages based on the results of iTRAQ method. The diagram defines increased proteins for which OA/control ratio was more than 1.25 and decreased proteins for which the OA/control ratio was less than 0.75.

Table 1. The articular cartilage proteome.

No.	Accession	Name	Ratio of OA to control
Top 30 of increased protein in OA cartilage
1	gi|179665	Complement component C3	2.681
2	gi|119605141	Cartilage oligomeric matrix protein, isoform CRA_a	2.391
3	gi|158257468	Unnamed protein product	1.911
4	gi|158255148	Unnamed protein product	1.821
5	gi|1000745	Pro-a2(XI)	1.791
6	gi|62205353	Matrix Gla protein	1.758
7	gi|193787240	Unnamed protein product	1.756
8	gi|118573093	Ovochymase-1	1.656
9	gi|4220894	Transcriptional co-activator CRSP70	1.652
10	gi|50295448	Platelet phospholipase A2	1.633
11	gi|194377958	Unnamed protein product	1.632
12	gi|119220552	Protein sidekick-1 precursor	1.623
13	gi|119604277	Pleiotrophin	1.596
14	gi|119587667	hCG2045237	1.573
15	gi|940106	T-cell receptor delta chain	1.571
16	gi|34364882	Hypothetical protein	1.527
17	gi|49113217	HHIP-like 2	1.524
18	gi|348041283	C-type lectin domain family 3 member A isoform 1	1.514
19	gi|62420929	Actin-like protein	1.503
20	gi|118763987	PRSS3 protein	1.471
21	gi|193786062	Unnamed protein product	1.435
22	gi|18307851	Angiogenin	1.433
23	gi|62897225	Transforming growth factor, beta-induced, 68 kDa variant	1.425
24	gi|68563369	Solute carrier family 4, anion exchanger, member 1	1.414
25	gi|21757045	Unnamed protein product	1.411
26	gi|59806345	Leukocyte cell-derived chemotaxin-2 precursor	1.389
27	gi|1017427	Elastic titin	1.381
28	gi|119609838	Phosphatidylserine receptor, isoform CRA_d	1.376
29	gi|609342	Nucleophosmin-anaplastic lymphoma kinase fusion protein	1.369
30	gi|21755876	Unnamed protein product	1.341
Top 30 of decreased protein in OA cartilage
1	gi|1340142	Alpha1-antichymotrypsin	0.402
2	gi|13171812	Immunoglobulin heavy chain variable region	0.415
3	gi|189054134	Unnamed protein product	0.554
4	gi|67190163	Proteoglycan 4 isoform A	0.598
5	gi|332164775	Pyruvate kinase isozymes M1/M2 isoform c	0.608
6	gi|56205585	S100 calcium binding protein A1	0.629
7	gi|51094573	Procollagen C-endopeptidase enhancer	0.632
8	gi|53690154	Small nuclear ribonucleoprotein polypeptide B	0.649
9	gi|114676256	Hyaluronan and proteoglycan link protein 4	0.661
10	gi|296011023	Stromal cell-derived factor 1 isoform delta	0.685
11	gi|114656687	Protein disulfide-isomerase A3 isoform 1	0.685
12	gi|14595132	Raichu404X	0.695
13	gi|312005	Small nuclear ribonucleoprotein E	0.702
14	gi|194374101	Unnamed protein product	0.702
15	gi|119622424	Aggrecan 1	0.722
16	gi|189069324	Unnamed protein product	0.728
17	gi|134254698	Peroxiredoxin 6	0.741
18	gi|54303904	Aging-associated gene 6 protein	0.754
19	gi|62898165	Biglycan preproprotein variant	0.759
20	gi|119586853	hCG2044782, isoform CRA_b	0.761
21	gi|32891795	Clusterin	0.761
22	gi|48146501	IL17B	0.762
23	gi|662841	Heat shock protein 27	0.771
24	gi|194382308	Unnamed protein product	0.772
25	gi|95115683	Hemoglobin alpha 1-2 hybrid	0.774
26	gi|215274249	Fibulin-1	0.784
27	gi|30354619	YWHAZ protein	0.787
28	gi|2204207	Glutathione S-transferase	0.791
29	gi|74761482	Epithelial splicing regulatory protein 2	0.793
30	gi|21264602	Laminin subunit alpha-5 precursor	0.801

The top 30 proteins that were increased or decreased in OA cartilage on iTRAQ method were extracted.

Table 2. Characteristics of the OA patients and controls in this study.

No	Sample	Age	Gender	Height	Weight	BMI	K-L Grade	Joint
A Patients list that was used in the iTRAQ analysis
1	Control	46	F	156	55	23	–	–
2	OA	59	F	150	51	23	IV	Hip
B Patients list that was used in the Western Blot analysis
1	Control	70	M	165	32.7	12	–	–
2	Control	70	M	158	52.7	21	–	–
3	Control	31	F	162	46	17	–	–
4	Control	39	M	180	81.5	25	–	–
5	Control	77	M	164	52	19	–	–
6	OA	75	M	159.5	70	27	III	Hip
7	OA	60	F	153	49	20	III	Hip
8	OA	56	F	156.8	56.2	22	IV	Hip
9	OA	52	M	173	73.8	24	III	Hip
10	OA	63	F	159	59	23	IV	Hip
11	OA	67	F	165	62.5	22	III	Knee
12	OA	74	F	147.8	54	24	IV	Knee
13	OA	87	F	151	50	21	IV	Knee
14	OA	82	F	135	49.7	27	IV	Knee
15	OA	79	F	143.7	37.7	18	III	Knee
16	OA	76	M	152	52	22	IV	Knee
17	OA	75	F	153	57.4	24	IV	Knee
18	OA	77	F	146.5	64	29	III	Knee
19	OA	63	M	162	69.1	26	III	Knee
20	OA	77	M	174.3	59.7	19	III	Knee
21	OA	81	F	149	50.3	22	II	Knee

Figure 2. Western blot analysis of LECT2, BAALC and PRDX6. (A) We confirmed the protein expression levels of LECT2, BAALC and PRDX6 in OA and control. Protein gel was stained with SYPRO ruby. (B) Statistical analysis of western blot. Quantities are indicated relative to the average value of each protein level in controls. *p < 0.05, statistically significant differences between OA and control, as determined by Student’s t-test. Error bars indicate SEM. LECT2 (control, n = 5; OA, n = 16; p = 0.005), BAALC (control, n = 5; OA, n = 16; p = 0.051), PRDX6 (control, n = 5; OA, n = 16; p = 0.011). (#) p = 0.051.

Figure 3. Experimental correlation between LECT2 protein levels and Age in OA patients. (A) Age (>65, n = 11; <65, n = 5; LECT2, p = 0.012; BAALC, p = 0.397; PRDX6, p = 0.166). (B) K-L Grade (>IV, n = 8; <III, n = 8; LECT2, p = 0.401; BAALC, p = 0.282; PRDX6, p = 0.131). (C) Height (>160 cm, n = 4, <160 cm, n = 12; LECT2, p = 0.057; BAALC, p = 0.107; PRDX6, p = 0.482). (D) BMI (>25, n = 4; <25, n = 12; LECT2, p = 0.298; BAALC, p = 0.301; PRDX6, p = 0.330). (E) Gender (male, n = 5; female, n = 11; LECT2, p = 0.420; BAALC, p = 0.284; PRDX6, p = 0.304). (F) Hip or Knee (hip, n = 5; knee, n = 11; LECT2, p = 0.338; BAALC, p = 0.217; PRDX6, p = 0.106). *p < 0.05, statistically significant differences between >65 and <65, as determined by Student’s t-test. Error bars indicate SEM. (#) p = 0.057. Quantities are indicated relative to the average value of each protein level in controls.

Figure 4. The level of LECT2 in control and OA patient at over 65 years of age. We recalculated expression level of LECT2 in OA and control cartilage by excluding the case of under 65 years of age. LECT2 was increased in OA with a significant difference compared with control (Control n = 3, OA n = 11). *p < 0.05, as determined by Student’s t-test. Error bars indicate SEM.