Chemometrics of differentially expressed proteins from colorectal cancer patients

Table 1 Clinicopathological features of 26 colorectal cancer patients involved in study

Patient No.	Age (yr)	Race	Sex	pTNM	Stage	Degree of differentiation	Tumor location
1	62	Malay	Male	pT3N1Mx	IIIB	MD	Sigmoid colon
2	79	Malay	Male	pT2NoM0	I	MD	Descending colon
3	74	Malay	Male	pT3N0M0	IIA	MD	Ascending colon
4	-	Malay	Male	pT3N2Mx	IIIC	MD	Rectum
5	37	Malay	Male	pT3N0M0	IIA	MD	Transverse colon
6	58	Malay	Female	pT3N0Mx	IIA	MD	Recto-sigmoid
7	59	Malay	Female	pT4N2Mx	IIIC	MD	Ileocecal
8	69	Malay	Male	pT3N0Mx	IIA	MD	Sigmoid colon
9	63	Malay	Female	pT3N0Mx	IIA	MD	Recto-sigmoid
10	84	Chinese	Female	pT4N0M0	IIB	MD	Rectum
11	58	Chinese	Male	pT3N0Mx	IIA	MD	Recto-sigmoid

MD: Moderately differentiated adenocarcinoma.

Table 2 List of proteins found in 2D gel of Tris extracts

Spot No.	Protein name	Swissprot No.1	MOWSE score2	MW (Da)	pI	Sequence coverage (%)	GRAVY	Average fold change3
1	Proteasome subunit β type 6	P28072	134	25573	4.80	16	0.034	-2.967
2	14-3-3 protein ζ	P63104	336	35567	6.97	40	-0.744	11.659
3	Tropomyosin α-3C-like protein	A6NL28	127	27407	4.71	31	-0.992	44.183
4	Rho GDP-dissociation inhibitor 1	P52565	167	23120	5.03	29	-0.700	-7.607
5	14-3-3 protein ζ	P63104	282	27919	4.73	16	-0.621	4.127
6	Tubulin β-2C chain	P68371	524	50304	4.83	40	-0.362	-52.184
7	Cathepsin B	P07858	74	22981	5.20	18	-0.433	33.149
8	Rho GDP-dissociation inhibitor 2	P52566	48	22901	5.10	18	-0.799	-10.625
9	SEC13 homolog	P55735	78	36040	5.22	9	-0.372	6.873
10	Hsc70-interacting protein	P50502	164	28464	8.92	21	-0.653	20.959
11	Apolipoprotein A-I	P02647	143	30777	5.56	26	-0.717	-4.478
12	Proteasome subunit α type 3	P25788	201	15958	6.82	41	0.008	4.249
13	Actin, cytoplasmic 2	P63261	105	26169	5.65	14	-0.156	28.601
14	60 kDa heat shock protein	P10809	151	61348	5.70	14	-0.074	131.219
15	Peroxiredoxin-2	P32119	283	21935	5.67	42	-0.210	1.250
16	Guanine nucleotide binding protein subunit β 2	P62879	112	37954	5.60	11	-0.183	-14.442
17	F-actin-capping protein subunit β	P47756	259	34187	6.02	37	-0.574	33.554
18	GST-P	P09211	730	23442	5.44	60	-0.131	4.834
19	Haptoglobin-related protein	P00739	49	39529	6.42	3	-0.308	56.209
20	Cathepsin Z	Q9UBR2	100	27787	5.48	15	-0.545	-60.766
21	F-actin-capping protein subunit β	P47756	245	21280	7.93	34	-0.540	13.278
22	Actin-related protein 3	P61158	148	47704	5.61	27	-0.271	15.881
23	Abhydrolase domain-containing protein 14B	Q96IU4	200	25429	6.82	26	-0.023	0.765
24	Nucleoside diphosphate kinase A	P15531	87	19873	5.42	36	-0.075	73.120
25	L-lactate dehydrogenase B chain	P07195	228	36928	5.71	14	0.056	3.513
26	Fibrinogen β chain	P02675	151	56624	8.54	22	-0.758	41.329
27	Leukocyte elastase inhibitor	P30740	170	42857	5.90	11	-0.249	10.458
28	PDI A3	P30101	674	57202	5.98	35	-0.506	7.579
29	Gelsolin	P06396	238	86103	5.90	20	-0.415	-11.917
30	Heat shock 27 kDa protein	P04792	256	22840	5.98	47	-0.567	-1.508
31	DJ-1 protein	Q99497	122	20079	6.33	54	0.004	4.981
32	Fibrinogen β chain	P02675	75	56624	8.54	22	-0.758	-72.722
33	Selenium-binding protein 1	Q13228	502	52971	5.93	21	-0.254	-26.544
34	Selenium-binding protein 1	Q13228	592	52938	5.93	30	-0.254	27.403
35	Selenium-binding protein 1	Q13228	979	52938	5.93	37	-0.254	-1.887
36	Leukotriene A-4 hydrolase	P09960	215	69792	5.80	22	-0.259	29.759
37	Proteasome subunit α type 6	P60900	71	20988	8.57	39	-0.247	0.768

¹Protein accession number at SwissProt at http://www.expasy.org/uniprot;

²MOWSE score from MASCOT protein database search at http://www.matrixscience.com, where score > 41 is statistically significant (P < 0.05);

³Average ratio of the spot intensity in normal mucosa over tumor tissue (negative variation or decrease) or tumor tissue over normal tissue (positive variation or increase). GST-P: Glutathione S-transferase P; PDI: Protein disulfide isomerase.

Table 3 List of proteins found in 2D gel in thiourea lysis buffer extracts

Spot No.	Protein name	SwissProt No.1	MOWSE score2	MW (Da)	pI	Sequence coverage (%)	GRAVY	Average fold change3
1	Tropomyosin α-4 chain	P67936	139	28506	4.67	33	-1.033	-51.151
2	Putative tropomyosin α-3-chain-like protein	A6NL28	53	27407	4.71	25	-0.992	4.922
3	GC1q-R, mitochondrial	Q07021	123	31768	4.74	20	-0.461	-3.333
4	Calreticulin	P27797	73	47092	4.30	11	-1.191	1.394
5	Prohibitin	P35232	421	29890	5.57	41	0.024	0.032
6	Heat shock 70 kDa protein	P11021	775	72488	5.07	42	-0.487	-32.940
7	Tubulin β-2C chain	P68371	299	48142	4.70	25	-0.347	-9.060
8	PDI	P07237	266	57510	4.82	42	-0.450	-1.515
9	ATP synthase subunit β, mitochondrial	P06576	1096	56559	5.26	43	0.018	-15.661
10	ATP synthase D chain	O75947	117	18406	5.22	32	-0.569	-5.129
11	Chloride intracellular channel protein 1	O00299	299	27123	5.09	30	-0.293	20.288
12	Tubulin α-1 chain	Q71U36	61	50800	4.94	6	-0.229	-30.291
13	Apolipoprotein A-I	P02647	129	28078	5.27	37	-0.840	78.135
14	Actin, cytoplasmic 2	P63261	52	42009	5.31	4	-0.205	-26.716
15	Actin, aortic smooth muscle	P62736	261	42154	5.23	21	-0.233	46.181
16	Stomatin-like protein 2	Q9UJZ1	151	38644	6.88	28	-0.161	-29.709
17	60 kDa heat shock protein, mitochondrial	P10809	451	61386	5.70	28	-0.074	14.023
18	Triosephosphate isomerase	P60174	167	26828	6.51	24	-0.126	16.757
19	Annexin A5	P08758	195	35994	4.94	39	-0.330	-2.019
20	Cytochrome b-c1 complex subunit 1, mitochondrial	P31930	96	53342	5.94	18	-0.141	13.151
21	Annexin A3	P12429	140	36396	5.63	22	-0.430	31.244
22	Annexin A4	P09525	165	35983	5.85	33	-0.447	11.890
23	α-enolase	P06733	143	47385	6.99	12	-0.226	85.960
24	Lamin-A/C	P02545	198	65192	6.40	25	-0.947	-3.378

¹Protein accession number as SwissProt at http://www.expasy.org/uniprot;

²MOWSE score from MASCOT protein database search at http://www.matrixscience.com, where score > 41 is statistically significant (P < 0.05);

³Average ratio of the spot intensity in normal mucosa over tumor tissue (negative variation or decrease) or tumor tissue over normal tissue (positive variation or increase). PDI: Protein disulfide isomerase.

Table 4 mean ± SD and percentage coefficient of variation of spot intensities of Tris proteins

Protein spot No.	Intensity of spots(mean ± SD)	% CV of spot intensity
1	2565.84 ± 2247.86	87.60
2	3865.47 ± 3766.11	97.42
3	2424.01 ± 1847.71	76.23
4	4957.17 ± 2923.49	58.97
5	3901.55 ± 3900.52	99.97
6	2105.64 ± 2444.14	116.08
7	2572.91 ± 1765.28	68.61
8	2959.95 ± 2177.86	73.58
9	2478.29 ± 1697.98	68.51
10	1253.48 ± 1472.88	117.50
11	3373.93 ± 2451.35	72.66
12	3247.26 ± 2519.26	77.58
13	9413.58 ± 10 685.11	113.51
14	2735.49 ± 2665.85	97.45
15	8354.35 ± 4824.59	57.75
16	7370.39 ± 7935.67	100.34
17	14 200.72 ± 16 194.91	114.04
18	6254.81 ± 5105.54	81.63
19	14 364.73 ± 10 849.77	75.53
20	10 753.33 ± 14 509.06	134.93
21	5171.49 ± 3304.12	63.89
22	3230.12 ± 1905.24	58.98
23	2114.69 ± 1164.19	55.05
24	2331.41 ± 2122.56	91.04
25	9254.07 ± 4830.01	52.19
26	9118.41 ± 9336.23	102.39
27	3750.45 ± 3869.35	103.17
28	8098.16 ± 5450.79	67.31
29	3984.55 ± 2658.12	66.71
30	4236.70 ± 4229.74	99.84
31	3932.80 ± 2507.88	63.77
32	1681.49 ± 2019.10	120.08
33	6600.04 ± 4860.85	73.65
34	3121.51 ± 2694.58	86.32
35	8587.77 ± 5871.40	68.37
36	939.46 ± 1682.25	179.07
37	3780.67 ± 1967.05	52.03

CV: Coefficient of variation.

Table 5 mean ± SD and percentage coefficient of variation of spot intensities of thiourea lysis buffer proteins

Protein spot No.	Intensity of spots(mean ± SD)	% CV of spot intensity
1	10 918.80 ± 8005.09	73.31
2	8516.42 ± 7898.33	92.74
3	3986.45 ± 3471.51	87.08
4	36 146.18 ± 24 859.84	68.78
5	13 329.50 ± 7123.20	53.44
6	4091.51 ± 4636.51	113.32
7	6512.40 ± 6048.73	92.88
8	13 401.28 ± 8031.43	59.93
9	24 196.99 ± 14 907.64	61.61
10	4861.29 ± 4327.71	89.02
11	4128.52 ± 3764.18	91.18
12	3522.46 ± 2821.84	80.11
13	9624.81 ± 8295.52	86.19
14	5407.19 ± 5270.17	97.47
15	4683.89 ± 6994.94	149.34
16	2633.26 ± 2593.91	98.51
17	10 104.77 ± 10 369.91	102.62
18	16 086.82 ± 19 928.39	123.88
19	6791.99 ± 5063.21	74.55
20	7596.19 ± 4759.49	62.66
21	2685.37 ± 3298.54	122.84
22	5022.01 ± 3735.74	74.39
23	5957.62 ± 7526.42	124.65
24	2323.67 ± 2269.62	97.67

CV: Coefficient of variation.

Table 6 Percentage of correct classification of normal and colorectal cancer tissues in Tris extracts using linear discriminant analysis

Type	Predicted group membership		% correct classification
	Cancer	Normal
Original count
Cancer (26)	21	5	82.7
Normal (26)	4	22
Cross-validated count
Cancer (26)	21	5	82.7
Normal (26)	4	22

Table 7 Percentage of correct classification of normal and colorectal cancer tissues in thiourea lysis buffer extracts using linear discriminant analysis

Type	Predicted group membership		% correct classification
	Cancer	Normal
Original count
Cancer (26)	19	7	78.8
Normal (26)	4	22
Cross-validated count
Cancer (26)	16	10	71.2
Normal (26)	5	21