Differential Expression of Serum Proteins in Chronic Obstructive Pulmonary Disease Assessed Using Label-Free Proteomics and Bioinformatics Analyses

Figures & data

Table 1 Clinical Diagnoses and Demographic Characteristics of Participants

Case ID No.	Diagnosis	Sex	Age (Years)	Group ID No.
Case 1	Healthy control	Male	73	1
Case 2	Healthy control	Male	69	2
Case 3	Healthy control	Female	65	3
Case 4	Acute exacerbation of COPD	Female	66	4
Case 5	Acute exacerbation of COPD	Female	71	5
Case 6	Acute exacerbation of COPD	Male	65	6
Case 7	Acute exacerbation of COPD	Male	70	7
Case 8	Acute exacerbation of COPD	Male	67	8
Case 9	Stable COPD	Female	74	9
Case 10	Stable COPD	Male	68	10
Case 11	Stable COPD	Male	67	11

Abbreviations: COPD, chronic obstructive pulmonary disease; ID, identification.

Figure 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lane M contains markers. Lanes 1–3 are proteins without the highly abundant proteins obtained from serum samples of healthy controls. Lanes 4–8 are proteins without the highly abundant proteins obtained from serum samples of patients with acute COPD, whereas lanes 9–11 are proteins without the highly abundant proteins obtained from serum samples of patients with stable COPD.

Table 2 Serum Proteins Differentially Expressed Between Patients with Acute Exacerbation of COPD and Healthy Control Participants

Change	Protein Accession Number	Protein-Coding Gene Name	Protein Description	FC
UP	P08670	VIM	Vimentin	19.45
UP	P08238	HSP90AB1	Heat shock protein HSP 90-beta	8.81
UP	Q01995	TAGLN	Transgelin	8.44
UP	P62979	RPS27A	Ubiquitin-40S ribosomal proteinS27a	5.74
UP	P04040	CAT	Catalase	3.75
UP	P02750	LRG1	Leucine-rich alpha-2-glycoprotein	2.54
UP	P18065	IGFBP2	Insulin-like growth factor binding protein 2	2.51
UP	P05362	ICAM1	Intercellular adhesion molecule 1	1.92
UP	Q8NBJ4	GOLM1	Golgi membrane protein 1	1.91
UP	P35916	FLT4	Vascular endothelial growth factor receptor 3	1.85
UP	Q5T5S1	CCDC183	Coiled-coil domain-containing protein 183	1.72
UP	A0A0C4DH26	IGKV6D-41	Probable non-functional immunoglobulin kappa variable 6D-41	1.66
UP	Q86U17	SERPINA11	Serpin A11	1.56
DOWN	Q16610	ECM1	Extracellular matrix protein 1	0.66
DOWN	P08887	IL6R	Interleukin-6 receptor subunit alpha	0.65
DOWN	P05556	ITGB1	Integrin beta-1	0.64
DOWN	P55103	INHBC	Inhibin beta C chain	0.63
DOWN	P22891	PROZ	Vitamin K-dependent protein Z	0.62
DOWN	P58335	ANTXR2	Anthrax toxin receptor 2	0.61
DOWN	P02787	TF	Serotransferrin	0.60
DOWN	P11362	FGFR1	Fibroblast growth factor receptor 1	0.59
DOWN	O95428	PAPLN	Papilin	0.58
DOWN	P04066	FUCA1	Tissue alpha-L-fucosidase	0.51
DOWN	P02751	FN1	Fibronectin	0.51
DOWN	P02753	RBP4	Retinol-binding protein 4	0.50
DOWN	P08195	SLC3A2	4F2 cell-surface antigen heavy chain	0.49
DOWN	P05154	SERPINA5	Plasma serine protease inhibitor	0.48
DOWN	P35590	TIE1	Tyrosine-protein kinase receptor Tie-1	0.46
DOWN	P00533	EGFR	Epidermal growth factor receptor	0.41
DOWN	P16930	FAH	Fumarylacetoacetase	0.38
DOWN	P28799	GRN	Progranulin	0.35
DOWN	P00488	F13A1	Coagulation factor XIII A chain	0.33
DOWN	O76061	STC2	Stanniocalcin-2	0.33
DOWN	P02768	ALB	Albumin	0.32
DOWN	Q9BWX5	GATA5	Transcription factor GATA-5	0.29
DOWN	Q66K66	TMEM198	Transmembrane protein 198	0.27
DOWN	Q9HCB6	SPON1	Spondin-1	0.26
DOWN	Q07075	ENPEP	Glutamyl aminopeptidase	0.24
DOWN	Q5D862	FLG2	Filaggrin-2	0.23
DOWN	P19440	GGT1	Glutathione hydrolase 1 proenzyme	0.22
DOWN	O43895	XPNPEP2	Xaa-Pro aminopeptidase 2	0.15
DOWN	P23280	CA6	Carbonic anhydrase 6	0.15
DOWN	A0A075B6Q5	IGHV3-64	Immunoglobulin heavy variable 3–64	0.09
DOWN	A0A0C4DH35	IGHV3-35	Probable non-functional immunoglobulin heavy variable 3–35	0.09
DOWN	Q86YZ3	HRNR	Hornerin	0.03

Abbreviations: COPD, chronic obstructive pulmonary disease; FC, fold change (the logarithmic value of the quantitative ratio in the protein concentration between the two groups of samples).

Table 3 Serum Proteins Differentially Expressed Between Patients with Stable COPD and Healthy Control Participants

Change	Protein Accession Number	Protein-Coding Gene Name	Protein Description	FC
UP	P49913	CAMP	Cathelicidin antimicrobial peptide	10.07
UP	Q66K66	TMEM198	Transmembrane protein 198	9.27
UP	P13611	VCAN	Versican core protein	7.61
UP	Q96EE4	CCDC126	Coiled-coil domain-containing protein 126	7.46
UP	P08238	HSP90AB1	Heat shock protein HSP 90-beta	6.20
UP	P0DOX3	N/A	Immunoglobulin delta heavy chain	6.10
UP	P02768	ALB	Albumin	3.87
UP	Q7Z7M9	GALNT5	Polypeptide N-acetylgalactosaminyltransferase 5	2.92
UP	P33151	CDH5	Cadherin-5	2.41
UP	P0DOX5	N/A	Immunoglobulin gamma-1 heavy chain	2.26
UP	P07998	RNASE1	Ribonuclease pancreatic	2.17
UP	P05090	APOD	Apolipoprotein D	2.17
UP	P18428	LBP	Lipopolysaccharide-binding protein	1.82
UP	P08253	MMP2	72 kDa type IV collagenase	1.78
UP	P04003	C4BPA	C4b-binding protein alpha chain	1.78
UP	P09382	LGALS1	Galectin-1	1.71
UP	P02749	APOH	Beta-2-glycoprotein 1	1.67
UP	P48740	MASP1	Mannan-binding lectin serine protease 1	1.54
DOWN	P02753	RBP4	Retinol-binding protein 4	0.66
DOWN	Q9Y4G8	RAPGEF2	Rap guanine nucleotide exchange factor 2	0.65
DOWN	P00734	F2	Prothrombin	0.65
DOWN	P16930	FAH	Fumarylacetoacetase	0.65
DOWN	Q92820	GGH	Gamma-glutamyl hydrolase	0.64
DOWN	P12955	PEPD	Xaa-Pro dipeptidase	0.63
DOWN	P15144	ANPEP	Aminopeptidase N	0.62
DOWN	P80108	GPLD1	Phosphatidylinositol-glycan-specific phospholipase D	0.62
DOWN	P11279	LAMP1	Lysosome-associated membrane glycoprotein 1	0.61
DOWN	P00742	F10	Coagulation factor X	0.59
DOWN	O95445	APOM	Apolipoprotein M	0.59
DOWN	P12821	ACE	Angiotensin-converting enzyme	0.58
DOWN	P06396	GSN	Gelsolin	0.57
DOWN	Q8TAQ9	SUN3	SUN domain-containing protein 3	0.56
DOWN	P19823	ITIH2	Inter-alpha-trypsin inhibitor heavy chain H2	0.56
DOWN	P26038	MSN	Moesin	0.53
DOWN	P03952	KLKB1	Plasma kallikrein	0.53
DOWN	P15151	PVR	Poliovirus receptor	0.53
DOWN	P07195	LDHB	L-lactate dehydrogenase B chain	0.53
DOWN	P04040	CAT	Catalase	0.52
DOWN	Q16610	ECM1	Extracellular matrix protein 1	0.50
DOWN	P07737	PFN1	Profilin-1	0.49
DOWN	A6NMY6	ANXA2P2	Putative annexin A2-like protein	0.47
DOWN	P00338	LDHA	L-lactate dehydrogenase A chain	0.47
DOWN	Q9C0E2	XPO4	Exportin-4	0.46
DOWN	P00751	CFB	Complement factor B	0.45
DOWN	Q08554	DSC1	Desmocollin-1	0.45
DOWN	P04075	ALDOA	Fructose-bisphosphate aldolase A	0.44
DOWN	P02655	APOC2	Apolipoprotein C-II	0.44
DOWN	A1L4H1	SSC5D	Soluble scavenger receptor cysteine-rich domain-containing protein SSC5D	0.43
DOWN	P00390	GSR	Glutathione reductase, mitochondrial	0.42
DOWN	P55056	APOC4	Apolipoprotein C-IV	0.41
DOWN	Q13867	BLMH	Bleomycin hydrolase	0.39
DOWN	Q16270	IGFBP7	Insulin-like growth factor-binding protein 7	0.37
DOWN	P07711	CTSL	Procathepsin L	0.34
DOWN	Q9BUN1	MENT	Protein MENT	0.34
DOWN	Q13103	SPP2	Secreted phosphoprotein 24	0.32
DOWN	Q92859	NEO1	Neogenin	0.31
DOWN	Q9UBQ6	EXTL2	Exostosin-like 2	0.31
DOWN	P29401	TKT	Transketolase	0.30
DOWN	A0A0C4DH26	IGKV6D-41	Probable non-functional immunoglobulin kappa variable 6D-41	0.28
DOWN	Q8NG48	LINS1	Protein Lines homolog 1	0.27
DOWN	P61626	LYZ	Lysozyme C	0.25
DOWN	O94966	USP19	Ubiquitin carboxyl-terminal hydrolase 19	0.24
DOWN	Q8NBJ4	GOLM1	Golgi membrane protein 1	0.23
DOWN	Q12841	FSTL1	Follistatin-related protein 1	0.20
DOWN	Q9BWX5	GATA5	Transcription factor GATA-5	0.18
DOWN	O95497	VNN1	Pantetheinase	0.18
DOWN	P02751	FN1	Fibronectin	0.15
DOWN	P04275	VWF	von Willebrand factor	0.15
DOWN	P32004	L1CAM	Neural cell adhesion molecule L1	0.12
DOWN	Q86UN3	RTN4RL2	Reticulon-4 receptor-like 2	0.12
DOWN	Q9NY97	B3GNT2	N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase 2	0.12
DOWN	Q12907	LMAN2	Vesicular integral-membrane protein VIP36	0.12
DOWN	Q13449	LSAMP	Limbic system-associated membrane protein	0.10
DOWN	O76061	STC2	Stanniocalcin-2	0.09
DOWN	Q9H7Z3	NRDE2	Nuclear exosome regulator NRDE2	0.08
DOWN	Q6Q788	APOA5	Apolipoprotein A-V	0.07
DOWN	O60279	SUSD5	Sushi domain-containing protein 5	0.07
DOWN	Q9HBW9	ADGRL4	Adhesion G protein-coupled receptor L4	0.05
DOWN	Q13508	ART3	Ecto-ADP-ribosyltransferase 3	0.03

Abbreviations: COPD, chronic obstructive pulmonary disease; FC, fold change (the logarithmic value of the quantitative ratio in the protein concentration between the two groups of samples).

Figure 2 Differentially expressed proteins (DEPs). (A and C) Volcano plots showing upregulated proteins represented by red dots, downregulated proteins represented by green dots, proteins with no significant represented by black dots (absolute log₁₀ fold change and P < 0.05 as the standard) between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (C). (B and D) Heat map showing the clustergram of the upregulated and downregulated DEPs between the acute COPD group ((B): a, 4–8) and the healthy control group ((B): b, 1–3), and between the stable COPD group ((D): a, 9–11) and the healthy control group ((D): b, 1–3). Each row represents a protein, each column represents a sample, and colors represent different levels of expression (log₂ values of quantitative values are median-corrected during heat mapping).

Figure 3 Gene ontology (GO) functional annotation. Upregulated and downregulated proteins in the GO categories of biological processes, cellular components, and molecular functions between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (B).

Figure 4 Gene ontology (GO) functional enrichment. Enriched GO terms and the associated numbers of proteins between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (B). The circle size represents the number of differentially expressed proteins in the GO term. The circle color indicates the -log₁₀ P value representing the significant degree of enrichment.

Figure 5 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. KEGG pathways enriched for the upregulated and downregulated proteins and the associated numbers of proteins between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (B).

Figure 6 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation. KEGG pathway annotation for the upregulated and downregulated proteins and the associated number of protein between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (B). The circle size represents the number of differentially expressed proteins in the GO term. The circle color indicates the −log₁₀ P value representing the significant degree of enrichment.

Figure 7 Clusters of Orthologous Groups (COGs) annotated for differentially expressed proteins. COG annotation for the upregulated and downregulated proteins and their associated frequency between the group with acute exacerbation of COPD and the healthy control group (A), and between the group with stable COPD and the healthy control group (B). The x-axis represents the functional class in the COG database. The y-axis represents the frequency of each functional class.

Table 4 Demographic and Biomarker Data of Patients Used for ELISA

Variables	Non-COPD (n=30)	COPD (n=30)	P
Age (years)	60.50 (55.00–69.50)	66.50 (58.25–71.00)	0.399^a
Female, n (%)	13 (43.3)	14 (46.7)	0.795^b
WBC (10^9/L)	5.79±0.20	11.83±0.97	<0.001^c
Neutrophil (10^9/L)	3.49 (2.89–3.82)	9.11 (6.08–12.89)	<0.001^a
Lymphocyte (10^9/L)	1.65 (1.35–2.24)	1.13 (0.65–2.02)	0.015^a
Eosinophil (10^9/L)	0.11 (0.06–0.24)	0.02 (0.00–0.10)	<0.001^a
Basophil (10^9/L)	0.03 (0.02–0.04)	0.02 (0.01–0.04)	0.067^a
Monocyte (10^9/L)	0.42 (0.35–0.47)	0.65 (0.33–0.90)	0.012^a

Notes: Statistical methods used to calculate p-values: ^amann–Whitney U-test; ^bChi-Square test; ^cIndependent-Samples T test.

Abbreviations: WBC, white blood cell; ELISA, enzyme linked immunosorbent assay; COPD, chronic obstructive pulmonary disease.

Figure 8 Comparison of biomarker levels between patients with acute exacerbation of COPD and healthy control participants. The horizontal axis represents the healthy control group and the group with acute exacerbation of COPD. The y-axis indicates the concentration of biomarker. Values are shown as median with interquartile range; n = 30. *P < 0.005 indicates a significant difference for the healthy control group (Non-COPD) vs the group with acute exacerbation of COPD analyzed by Mann–Whitney U-test.

Abbreviations: IGFBP2, insulin-like growth factor binding protein 2; LRG1, leucine-rich alpha-2-glycoprotein; TAGLN, transgelin.

Table 5 Biomarker Levels in Patients with Acute Exacerbation of COPD and Healthy Control Participants

Biomarkers	Non-COPD (n=30)	COPD (n=30)	Statistical Result	P value	AUROC (95% CI)	COP	SEN	SPE
IGFBP2 (ng/mL)	377.27 (213.26–675.17)	972.45 (662.04–1209.43)	z = −4.78	<0.001	0.86 (0.77,0.95)	660.12	0.80	0.77
LRG1 (µg/mL)	569.62 (528.71–639.54)	738.33 (623.92–848.52)	z = −4.41	<0.001	0.83 (0.73,0.94)	681.21	0.73	0.90
TAGLN (ng/mL)	80.28 (71.90–116.17)	112.99 (102.56–120.63)	z = −2.94	0.03	0.72 (0.58,0.86)	93.34	0.83	0.73

Abbreviations: IGBP2, insulin-like growth factor binding protein 2; LRG1, leucine-rich alpha-2-glycoprotein; TAGLN, transgelin; COPD, chronic obstructive pulmonary disease; AUROC, the area under the receiver operating characteristic; CI, confidence interval; COP, cutoff point; SEN, sensitivity; SPE, specificity.

Figure 9 The receiver operating characteristic curves of these biomarkers.

Abbreviations: IGFBP2, insulin-like growth factor binding protein 2; LRG1, leucine-rich alpha-2-glycoprotein; TAGLN, transgelin; COPD, chronic obstructive pulmonary disease; AUROC, the area under the receiver operating characteristic; Combination, the combination of the three key protein.