Proteomic studies of common chronic pain conditions - a systematic review and associated network analyses

Figures & data

Table 1. Basic characteristics of the identified studies.

Authors	Year	Cohorts	Tissue	Sex	Type of study	Proteomic methods	Comments including pharmacological treatments and wash-out
Muscle
Olausson et al. [81]	2012	TM (n = 37) CWP (n = 18) CON (n = 22)	Muscle dialysate	F	C	2-DE	Pooled samples used. NSAID medication was avoided the week before the study.
Hadrevi et al. [82]	2013	TM (n = 12) CON (n = 12)	Muscle	F	C	2-DE	Excluded subjects with oral steroids or NSAID drugs.
Hadrevi et al. [83]	2016	TM (n = 12) CON (n = 12)	Muscle	F	C	2-DE	Continuation of the previous article. Excluded subjects with oral steroids or NSAID drugs.
Olausson et al. [84]	2015	CWP/FM (n = 18) CON (n = 19)	Muscle	F	C	2-DE	Excluded subjects with anticoagulatory, continuous anti-inflammatory drug, opioid, or steroidal use.
Olausson et al. [96]	2016	CWP/FM (n = 18) CON (n = 19)	Muscle	F	D	2-DE	Excluded subjects with anticoagulatory, continuous anti-inflammatory drug, opioid, or steroidal use.
Saliva
Bazzichi et al. [85]	2009	FM (n = 22) CON (N = 26)	Saliva	F	C + D	2-DE	Proportion of patients using drugs potentially inducing xerostomia reported; no washout.
Ciregia et al. [74]	2019	FM (n = 30) RA (n = 30) Migraine (n = 30) CON (n = 30)	Saliva	F & M	C + D	2-DE	Patients were on different pharmacological treatments; no washout.
Bazzichi et al. [98]	2013	FM (n = 40)	Saliva	F	Mo	2-DE	Pooled samples – 20 for each treatment arm. Patients were on different pharmacological treatments; no washout.
Blood
CWP/FM
Ruggiero et al. [86]	2014	FM (n = 16) CON (n = 12)	Serum	F	C + D	2-DE	Patients were on different pharmacological treatments; no washout.
Wåhlen et al. [87]	2017	CWP/FM (n = 16) CON (n = 23)	Plasma	F	C	2-DE	Excluded subjects with anticoagulatory, continuous anti-inflammatory drug, opioid, or steroidal use.
Ramirez-Tejero et al. [48]	2018	FM (n = 12) CON (n = 12)	Plasma	F	C	LC-MS/MS	None of the subjects used drugs affecting antioxidative status or were under treatment of corticosteroids, estrogens, analgesics, or anti-inflammatory drugs.
Wåhlen et al. [97]	2018	CWP/FM (n = 15) CON (n = 23)	Plasma	F	D	2-DE	Mainly results concerning CWP/FM. Excluded subjects with anticoagulatory, continuous anti-inflammatory drug, opioid, or steroidal use.
Other chronic pain
Ghafouri et al. [88]	2016	CP (n = 13) CON (n = 11)	Plasma	M	C	2-DE	Pharmacological treatment/wash-out not mentioned.
Trigeminal neuralgia
Farajzadeh et al. [77]	2018	Trigeminal neuralgia (n = 13) CON (n = 13)	Plasma	M & F	C + Mo	2-DE	Patients were on different pharmacological treatments; no washout.
CSF
CWP/FM
Olausson et al. [89]	2017	CWP (n = 12) CON (n = 13)	CSF	F	C	2-DE	Pharmacological treatment/wash-out not mentioned.
Khoonsari et al. [90]	2019	FM (n = 13) RA (n = 11) OND (n = 8)	CSF	F	C	LC-MS/MS	Pharmacological treatments mentioned for RA; NSAID not allowed 24 h before sampling. For FM, antidepressants not allowed and NSAID not allowed 24 h before sampling.
Khoonsari et al. [75]	2019	FM (n = 39) CON (n = 38)	CSF	FM: F CON: M & F	C	LC-MS/MS	Pharmacological treatment/wash-out not mentioned.
Lind et al. [76]	2019	FM (n = 40) Healthy CON (n = 11) Minor urology surgery CON (n = 28)	CSF	FM: F CON: M & F	C	LC-MS/MS	Pharmacological treatment/wash-out not mentioned.
Neuropathic pain
Liu et al. [78]	2006	LBP with sciatica (NP) (n = 10) CON (n = 10)	CSF	M & F	C	2-DE	Pharmacological treatment/wash-out not mentioned.
Conti et al. [79]	2005	NP (n = 9) NPN (n = 8) CON (n = 9)	CSF	M & F	C	2-DE	Pharmacological treatment/wash-out not mentioned.
Pattini et al. [92]	2008	NP (n = 8) NPN (n = 8) CON (n = 8)	CSF	M & F	Me	2-DE	Based on data and characteristics of subjects reported in [79]. Pharmacological treatment/wash-out not mentioned.
Cannistraci et al. [93]	2010	NP (n = 7) NPN (n = 8) CON (n = 8)	CSF	M & F	Me	2-DE	Based on data presented in Conti et al. [79] and Pattini et al. [92]. Pharmacological treatment/wash-out not mentioned.
Bäckryd et al. [80]	2015	NP (n = 11) CON (n = 11)	CSF	M & F	C	2-DE	Patients were on different pharmacological treatments; no washout.
Bäckryd et al. [91]	2018	NP (n = 11) CON (n = 11)	CSF	M & F	C + D	2-DE	Patients were on different pharmacological treatments; no washout.
Lind et al. [76]	2019	NP group 1 (n = 14) Minor urology surgery CON (n = 28) NP group 2 (n = 11) Healthy CON (n = 11)	CSF	M & F	C	LC-MS/MS	This study also reported results FM vs. controls – see above Pharmacological treatment/wash-out not mentioned.
Lind et al [99]	2016	NP (n = 14) as own controls	CSF	M & F	Mo	LC-MS/MS	Patients used spinal cord stimulation as treatment. Pharmacological treatment/wash-out not mentioned.
Other pain conditions
Lim et al [95]	2017	LBP with DD (n = 8) CON with DD (n = 8) CON (n = 6)	CSF	M & F	C + D	LC-MS/MS	It was not obvious that these patients had sciatica. Excluded subjects with steroids, narcotics, anti-inflammatory, or algesic drugs. They also excluded subjects using antidepressants not receiving a steady dose for ≥2 months.
Yuan et al [94]	2002	Idiopathic LBP (n = 3)	CSF	Not reported	Me	2-DE	Pharmacological treatment/wash-out not mentioned.

Cohorts: CWP=chronic widespread pain; FM=fibromyalgia; RA=rheumatoid arthritis; TM=chronic trapezius myalgia; NP=chronic neuropathic pain; NPN= Neuropathy without pain; CP=farmers with chronic musculoskeletal pain; LBP=low back pain; OND=Other Neurological Diseases (i.e., patients with noninflammatory neurological symptoms and without pain).

Sex/gender: F=female gender/sex; M=male gender/sex.

Type of study: Me=methodological; C=Comparative; D=Diagnostic, Mo=Monitoring

Proteomic methods: 2-DE= Two-Dimensional gel electrophoresis; LC-MS/MS=the combination of liquid chromatography (LC) with mass spectrometry (MS). MS/MS is the combination of two mass analyzers in one mass spectrometry instrument.

Figure 1. Protein network interaction of altered proteins in muscle from patients with trapezius myalgia compared to healthy controls identified in three studies [81–83]. Nodes denote genes/peptides. The protein-protein Interaction (PPI) enrichment analysis (P < 1.0e-16) separated the identified proteins in 3 clusters. Cluster I is represented by proteins involved in muscle contraction (red – 11 proteins: ACTA2, DES, MYBPC1, MYH2, MYH6, MYH7, MYL1, MYL2, MYL3, MYLPF, and TPM2). Proteins in cluster I are muscle fiber components that affect motor activity and cytoskeletal protein binding (actin, microtubule, or intermediate filament cytoskeleton). Cluster II included proteins involved mainly in cellular metabolic process (blue – 18 proteins: ACTB, ALB, ALDOA, APOA1, B2M, CD38, ENO3, GAPDH, HBB, HSPA1A, MYH6, MYH7, NGF, PGM1, PKM, PYGM, SERPINA1, TF, and TPI1). Proteins in cluster II are part of the cytoplasm component that functions as an enzyme/enzyme inhibitor activity, ion/protein binding, and microfilament motor activity. Cluster III was dominated by proteins involved in ATP metabolic process (green – three proteins: OGDH, ATP5A1, and HSPA1A). Proteins in cluster III are all mitochondrial proteins that function as small molecule binders.

Figure 2. Pathway analysis for altered proteins in muscle from CWP/FM compared to controls [81,84]. The protein-protein interaction (PPI) enrichment analysis had a P-value < 1.0e-16, indicating that the proteins are at least partially biologically connected as a group. Three clusters were identified. Cluster I was dominated by proteins involved in platelet degranulation (yellow – four proteins: SERPINA1, TF, APOA1, and ALB). Cluster I included extracellular proteins that affect enzyme binding and ion binding. Proteins in cluster II (AK1, ATP5B, PKM, GAPDH, ALDOA, and TP11) were involved in small molecule metabolic process (blue) and phosphorylation (green). The four proteins in cluster II are enzymes. The proteins in cluster III were s involved in muscle system processes (red: MYL1, MYL3, DES, and TNNT1). Proteins in cluster III are part of the sarcomere, contractile fiber, myosin binding, and cytoskeletal protein binding.

Figure 3. Pathway analysis of altered proteins in saliva in FM compared to controls [74,85]. The protein-protein interaction (PPI) enrichment analysis (P-value = 7.81e-09) identified three protein clusters. Cluster I includes proteins involved in regulation of actin cytoskeleton organization (green: PFN1, CF1, and GSN). The biological process that proteins in cluster II were involved include the carbohydrate metabolic process (blue: TALDO1, ENO1, and PGAM1). Cluster III was dominated by proteins involved in transport (red: TF, HP, and ALB). Clusters I and II were connected by a transport protein – PPIA. Proteins in cluster I are cytoskeletal proteins that bind actin. Proteins in cluster II are secretory enzymes, and cluster III includes secretory proteins that affect ion and protein binding.

Figure 4. Pathway analysis of altered proteins in plasma from CWP/FM compared to controls [48,86,87]. The protein-protein interaction (PPI) enrichment analysis was highly significant (P-value< 1.0e-16), indicating the proteins were at least partially biologically connected as two large groups (clusters I and II). Cluster I was dominated by proteins involved in regulation of cellular protein metabolic process (green: A2M, AHSG, FGB, FGG, GSN, HRG, PROS1, FETUB, SERPINA1, SERPINF2, and THBS1) and proteins involved in PTM (yellow: AHSG, APOA1, APOL1, C3, CP, FGG, SERPINA1, SERPINA10, and TF). The proteins in cluster II were involved in complement activation (blue: C1QC, C1R, C1S, C2, C3, CFB, CFH, CFI, and FCN3). Many proteins involved in the immune system (red: A2M, ACTB, APCS, APOA1, C1QC, C1R, C1S, C2, C3, C7, C9, CD14, CFB, CFH, CFI, F2, FCN3, FGB, FGG, GSN, HRG, ORM1, ORM2, PROS1, RBP4, and THBS1) were identified in the whole network including clusters I and II.

Figure 5. Pathway analysis of altered proteins in CSF from patients with CWP/FM compared to healthy controls [75,76,89]. The protein-protein interaction (PPI) enrichment analysis (P-value = 1.17e-07) identified a group of proteins involved in transport (cluster I, blue: APOC3, CLU, TTR, A2M, and IGF2). The proteins in cluster I are extracellular proteins involved in signaling receptor binding.

Figure 6. Pathway analysis of altered proteins in CSF from patients with neuropathic pain compared to healthy controls [78–80]. The protein-protein interaction (PPI) enrichment analysis was highly significant (P-value < 1.0e-16) and identified three groups of proteins: proteins involved in inflammatory responses (blue), in immune responses (red), and in metabolic processes (yellow). The proteins are extracellular with molecular functions such as enzyme activity, protein/lipid/ion binding, and transporter activity.

Table 2. Studies relating the protein pattern to clinical variables; for details concerning cohorts see Table 1.

Authors	Year	Pain and Tissue	Clinical variables	Statistical analysis	Results and comments
Olausson et al. [96]	2016	CWP/FM – muscle	PI PPT	OPLS	12 proteins correlated significantly with PI in CWP/FM. 16 proteins correlated significantly with PPT in CWP/FM but no significant correlation in CON.
Bazzichi et al. [85]	2009	FM – saliva	PI FIQ TP	Spearman’s rank correlation	No significant correlations between clinical variables and transaldolase and phosphoglycerate mutase-1. No correlations reported for the other proteins identified. Note that proteins that differed between groups were used in the analyses.
Ciregia et al. [74]	2019	FM -saliva	PI FIQ FIQR TP FACIT	Spearman’s rank correlation	No significant correlations between clinical variables and proteins different in FM. Note that proteins that differed between groups were used in the analyses.
Ruggiero et al. [86]	2014	FM -blood	PI Pain duration FIQ	unclear	No correlations were found between the important proteins (alpha1-antitrypsin, transthyretin and retinolbinding protein 4) and clinical characteristics. Note that proteins that differed between groups were used in the analyses.
Wåhlen et al. [97]	2018	FM/CWP – blood	PI HADS-total Age BMI	OPLS	20 proteins correlated significantly with PI in CWP/FM. 18 proteins correlated significantly with HADS-total in CWP/FM. 12 proteins correlated significantly with HADS-total in CON. 12 proteins correlated significantly with age in CWP/FM. 19 proteins correlated significantly with age in CON. 21 proteins correlated significantly with BMI in CWP/FM. 31 proteins correlated significantly with BMI in CON.
Conti et al. [79]	2005	NP – CSF	PI Pain duration	unclear	The article mentions that no correlations existed between Cystatin C and pain intensity or pain duration (data not shown).
Bäckryd et al. [91]	2018	NP-CSF	PI Pain duration	OPLS	21 proteins correlated significantly with PI in NP. 16 proteins correlated significantly with pain duration in NP.
Lim et al. [95]	2017	LBP-CSF	PI MPQ ODI Thompson scale	Spearman’s rank correlation	Cystain C correlated with Thompson scale. Hemopexin correlated with Thompson scale, PI, MPQ, and ODI. Note that they used selected proteins that differed between groups for their analyses.

PI = pain intensity; PPT = pressure pain threshold; OPLS = orthogonal partial least squares regression; FIQ = Fibromyalgia Impact Questionnaire; TP = number of tender points; FIQR = Revised Fibromyalgia Impact Questionnaire; FACIT = Functional Assessment of chronic Illness Therapy-Fatigue; HADS-total = sum of the two subscales of Hospital Anxiety and Depression Scale; BMI = Body mass index, MPQ = McGill pain questionnaire-measures pain experience; ODI = Oswestry Disability. Index: Thompson scale = severity of DD.

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