Proteomics in Patients with Fibromyalgia Syndrome: A Systematic Review of Observational Studies

Purpose of Review Fibromyalgia syndrome (FMS) is a disease of unknown pathophysiology, with the diagnosis being based on a set of clinical criteria. Proteomic analysis can provide significant biological information for the pathophysiology of the disease but may also reveal biomarkers for diagnosis or therapeutic targets. The present systematic review aims to synthesize the evidence regarding the proteome of adult patients with FMS using data from observational studies. Recent Findings An extensive literature search was conducted in MEDLINE/PubMed, CENTRAL, and clinicaltrials.gov from inception until November 2022. The study protocol was published in OSF. Two independent reviewers evaluated the studies and extracted data. The quality of studies was assessed using the modified Newcastle–Ottawa scale adjusted for proteomic research. Ten studies fulfilled the protocol criteria, identifying 3328 proteins, 145 of which were differentially expressed among patients with FMS against controls. The proteins were identified in plasma, serum, cerebrospinal fluid, and saliva samples. The control groups included healthy individuals and patients with pain (inflammatory and non-inflammatory). Summary The most important proteins identified involved transferrin, α-, β-, and γ-fibrinogen chains, profilin-1, transaldolase, PGAM1, apolipoprotein-C3, complement C4A and C1QC, immunoglobin parts, and acute phase reactants. Weak correlations were observed between proteins and pain sensation, or quality of life scales, apart from the association of transferrin and a2-macroglobulin with moderate-to-severe pain sensation. The quality of included studies was moderate-to-good. FMS appears to be related to protein dysregulation in the complement and coagulation cascades and the metabolism of iron. Several proteins may be dysregulated due to the excessive oxidative stress response. Supplementary Information The online version contains supplementary material available at 10.1007/s11916-024-01244-4.


Introduction
Fibromyalgia syndrome (FMS) is a rheumatic disease (ICD-10 M79.7) of unknown etiology, characterized by chronic widespread pain accompanied by potential neuroinflammation [1], fatigue, stress [2], memory loss, sleep disturbance, and multiple physical symptoms [3].The prevalence of FMS in the general population ranges between 2 and 3% [4], but higher ratios have been reported in specific population groups [5].For example, 14.8% of patients with type 2 diabetes mellitus and 80% of those with Behçet's disease are diagnosed with FMS [4,5].There is however, no gold standard for the diagnostic procedure.Individuals are diagnosed based on clinical criteria suggested by the American College of Rheumatology (ACR), conceived in 1990, and revised in 2010, 2011, and 2016 [6][7][8][9].The ACR 1990 [7] criteria rely on a clinical examination and the existence of tender points, while the 2010 [6] criteria focus on other disease parameters, including fatigue and sleep disturbances.Aside from the diagnosis, no objective biomarkers or tests have been identified to facilitate a more accurate diagnostic process or mediate the development of a precise prognostic model for FMS.
In medicine, biological markers can be used for disease detection and the discovery of drugs, as well as for monitoring the progress of patients [10].Although numerous studies suggest plausible mechanisms driving disease development, definite evidence has been relatively scarce.FMS seems to be associated with altered central nervous system (CNS) processing, enhanced excitability, and decreased inhibition [11].Oxidative stress, vitamin dysregulation, inflammation, autonomic dysfunction, and genetic factors may provide an insight into the pathophysiology [12][13][14][15][16][17].Proteomics, identifying protein markers in biological fluids, can provide critical information in such complex conditions/diseases [10,18], like FMS [19].
The present systematic review aimed to provide a comprehensive summary of the proteome of adult patients with FMS, in an effort to shed light on the pathophysiology of the condition, identify diagnostic and prognostic protein markers, and establish some potential therapeutic targets.

Systematic Review Protocol and PEO
The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [20] and the Synthesis Without Meta-analysis (SWiM) extension [21] were used.By November 2022, the study's protocol was published at the Center for Open Science Framework (OSF) (https:// short url.at/ rHN45).The Population-Exposure-Outcome (PEO) of the research question is detailed in Supplementary Table 1.

Search Strategy and Algorithm
Two independent reviewers (A.G. and S.G.T.) identified studies through PubMed, the Cochrane Central Register of Controlled Trials (CENTRAL), clinicaltrials.govdatabases, and the grey literature from inception until November 2022.A senior reviewer (M.G.G.) resolved any discrepancies.
To identify studies in databases, we used a combination of keywords using medical subject headings (MeSH) and free text.The search was conducted in English.The keywords and search syntaxes are listed in Supplementary Tables 2 and 3, respectively.
The Rayyan [22] software was used to identify all studies fulfilling inclusion criteria and to remove duplicates.After all identified studies were imported to the software, titles and abstracts were screened to examine whether inclusion criteria were met.The remaining studies were assessed in full text.

Inclusion and Exclusion Criteria
Studies were included in the systematic review when (1) they were observational studies on FMS, (2) of any duration, (3) in adult patients, (4) by assessing the proteome in biological fluids, (5) published until November 2022, (6) and written in the English language.
Studies were excluded when (1) they were published in another language, (2) pooling patients with FMS together with other chronic pain disorders, (3) including pediatric patients, and (4) animal or preclinical studies.

Outcomes of Interest
Any protein identified in body fluids through proteomics in patients with FMS compared to controls was considered as an outcome of interest.Correlations of such proteins with any specific disease score or scale, like the visual analog scale (VAS) [23] for pain or the Fibromyalgia Impact Questionnaire (FIQ), were also recorded.

Quality of Studies
The modified Newcastle-Ottawa scale (NOS) was used for assessing the quality of the included studies by two independent reviewers [24].The maximum score a study can collect is 9 points.The scale was further adapted according to Nguyen et al. [25] and the Molecular & Cellular Proteomics (MCP) initiative [26] to fit the design and methodology of proteomic studies.

Data Synthesis
Since a meta-analysis was not feasible due to the existing heterogeneity between biological samples, a systematic synthesis was conducted.

Search Results
Of the 1099 studies screened, 37 duplicates were removed, and 1062 were reviewed at the title and abstract level.From these, sixty-four studies were reviewed also in fulltext form.Four additional studies were identified through citation searching.In total, 10 studies fulfilled the criteria (Table 1) and were included in the present systematic review [37-44, 45•, 46].The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 [20] flowchart is presented in Fig. 1.A list of excluded studies is detailed in Supplementary Table 4.
One study was excluded for examining gut microbiome and serum metabolome in patients with FMS, combined with custom multiplex cytokine and miRNA analysis (Fire-Plex™ technology) in serum, thus not having relevant results to the present research question [47].

Samples
Patients were recruited through hospitals, Rheumatology and outpatient clinics, and FMS patients' associations.The sample size ranged from 12 to 39 patients and between 12 to 90 controls, for each study.The present systematic review includes a total of 242 patients with FMS and 297 controls, the latter being either healthy, or pain controls (with inflammatory or non-inflammatory pain).
The studies used different exclusion criteria for the selection of participants, including the diagnosis of psychiatric diseases, dementia, epilepsy, alcohol or substance abuse, hypertension, osteoarthritis, use of analgesics, autoimmune diseases, neurological diseases, diabetes mellitus, cardiovascular diseases, pregnancy or childbirth, infectious diseases, active malignant disease, use of immunosuppressants or cortisone, and history of injury.All exclusion criteria used by the studies during sample recruitment are presented in Supplementary Table 5.

Pain and Quality of Life Scales
Most studies used a scale to quantify patient symptomatology; however, as no common recommendations exist, the selection of such scales was arbitrary.The most frequently used scales included the VAS, which quantifies pain on a level of 1 to 10 and the FIQ, for the assessment of quality of life (QoL) [30,31,48,49].Patients' VAS ranged from 5.0 ± 2.2 to 7.9 ± 1.9 (mean ± standard deviation), indicating that they experienced severe pain.Other scales (e.g., PPT, WPI, tender points) were used to quantify pain, while the FIQ, FACIT, PCS-12, MCS-12, and SSS were applied for the QoL [27,33,50].Two research items [38,46] studied sleep quality using the PSQI scale, with results converging on poor sleep quality, frequent sleep interruptions, and fatigue [32].Finally, one study [46] focused on quantifying anxiety and depression using the BAI and BDI scales [35,51], revealing an increased incidence of anxiety and depression in patients with FMS.

Proteomic Methodology and Database
All identified studies involved discovery proteomics, with the majority applying liquid chromatography-mass spectrometry technique for protein analysis.Liquid chromatographymass spectrometry (LC-MS) consists of a powerful analytical technique that combines the power of LC with the highly sensitive and selective mass resolution capability of mass spectrometry (MS) [52,53].This technique was utilized by five of the research groups [40-42, 45•, 46].Four research groups applied 2D electrophoresis (2-DE) techniques.Specifically, two used 2-DE coupled to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [39,43], one used 2-DE/surfaceenhanced laser desorption ionization-time of flight mass spectrometry (SELDI-TOF MS) [38], and one applied 2-DE/ MS [44] techniques.One research item analyzed the proteins based on a targeted proteomics platform called Olink [37] which enables the analysis of some hundreds of proteins by multiplex assays.

Quality of Included Studies
The summary results regarding the NOS (modified for proteomics studies) score in each included study are presented in Fig. 2. One study achieved the maximum quality score (9) [44], four studies received a total NOS score of 8 [40, 41,  45 •, 46], and the remaining scored between 5 and 7 [37-39, 42, 43].The domains with quality concerns for most studies included quantification and the representativeness of controls.

Proteins Identified
A total of 3328 proteins were identified, including peptides and amino acids.The level of 145 proteins differed significantly between patients with FMS and controls.Based on these studies, higher number of proteins could be identified in CSF (1721 proteins from two studies), followed by plasma (647 proteins, two studies), serum (480 proteins, four studies), and saliva (480 proteins, two studies) samples.

Proteins Identified in Saliva and Their Role
Two studies [38,39] assessed proteomics in saliva samples and isolated 23 differently expressed proteins in patients with FMS versus controls.One of them [39] used healthy controls, while the other [38] used a mixed sample of a healthy population, patients with rheumatoid arthritis (RA) and patients with migraine.The isolated proteins, the study in which they were identified, the UniProt ID (as the direction of change in their levels, increased or decreased relative to the healthy population) are presented in Table 2.The two studies are in agreement as they both identify transaldolase (TALDO), phosphoglycerate mutase-1 (PGAM1), and calgranulin A (S100-A8) overexpressed in patients with FMS.Additional data regarding fold change and statistical significance are provided in Supplementary Table 6.TALDO mediates cell shape changes and cell motility and is part of the pentose pathway, associated with NADPH production.As a result, its high levels could be interpreted as the body's attempt to increase NADPH production to deal with oxidative stress [54].PGAM1 is an enzyme of the glycolysis pathway, and its elevated levels link FMS to glucose disorders.Additionally, anti-PGAM1 antibodies have been Fig. 2 Summary results regarding studies' quality using the modified Newcastle-Ottawa scale [24] observed in various autoimmune diseases of the CNS [55].Finally, calgranulin A is a low molecular weight protein that binds calcium and belongs to the S100 protein family, regulating various intracellular processes.An increase in the concentration of calgranulin A is probably associated with oxidative stress [56].

Proteins Identified in the CSF and Their Role
Two different studies published by the same author group performed CSF proteomics [41,42].A total of 14 different proteins were identified as being over-or under-expressed in patients with FMS, compared to controls, as shown in Table 3.Additional details are presented in Supplementary Table 7. Proteins identified in more than one study included apolipoprotein C-III, which was also isolated in plasma at low concentrations [44], and complement C4-A, which was observed in high levels at the serum of patients with FMS [46].
Apolipoprotein C-III is an atherogenic protein inhibiting lipoprotein lipase (LPL) activity, displacing LPL from lipid particles.Furthermore, it acts in an inhibitory manner at the triglyceride cycle, leading to higher triglyceride concentrations, which result in arterial stiffness and an increased risk of cardiovascular events [57].Isolation of apolipoprotein C-III in the CSF has been described in the literature; however, its role in the CNS remains unclear [58].Complement C4-A is part of the classical complement pathway and is involved in forming the membrane attack complex (MAC), activated by immune complexes and leading to cell lysis [59].
Three additional proteins may play a role in the pathogenesis of FMS: malate dehydrogenase, galectin-binding protein-3 (LG3BP), and ProSAAS (PCKS1).Malate dehydrogenase is involved in gluconeogenesis and energy production for muscle contraction.Changes in protein levels related to glycolysis and gluconeogenesis were also observed in the saliva of patients with FMS [60].Decreased malate dehydrogenase levels result in low malate concentration; thus, a "Super Malic" dietary supplement has been suggested to have beneficial effects in tampering down FMS symptomatology [61].It is also worth mentioning that lower levels of malate dehydrogenase have been observed in the synovial fluid of patients with RA, reflecting disturbances in its metabolism [61].
LG3BP is a regulator of pro-inflammatory signaling and is elevated in patients with RA, affecting the balance between pro-inflammatory and anti-inflammatory processes [62].Finally, PCKS1 is present in the neurons, including processing fetal neuropeptides and inhibiting the enzyme that converts prohormones to their active form [63,64]. PCKS1 is proteolytically processed and produces various neuropeptides [65].Thus, elevated PCKS1 concentrations may reflect endocrine disturbances already reported in the literature among patients with FMS [66].

Proteins Identified in Plasma and Their Role
Serum and plasma were studied separately, as they are different biological materials.Plasma is the blood derivative obtained by adding anticoagulant and centrifugation, while serum is the blood derivative that remains after coagulation for 30 min and centrifugation.As a result, differences arise in proteomic and metabolomic studies depending on the biological fluid studied [67].Two research groups assessed plasma proteomics and reported identifying four common proteins at different levels compared to controls, namely transferrin (TRFE), haptoglobin, α2-macroglobulin, and fibrinogen β chain.TRFE has also been observed at elevated levels in the saliva [38] of patients with FMS.The proteins isolated in plasma and their role are shown in Table 4, while additional details can be found in Supplementary Table 8.TRFE is the main iron-binding protein.In the inflammatory context of chronic lung disease, the increase in TRFE may reflect an attempt to avoid the deleterious effects of free iron [68].By binding the free iron, the body is protected from potential damage.Haptoglobin is an acute phase marker and binds to hemoglobin during hemolysis to limit the oxidative properties of heme and allow the complex to be recognized by the macrophage receptor CD163 [69].It protects the organism from oxidative stress.On the other hand, α2-macroglobulin consists of a protease inhibitor, participating in the immune response by trapping proteases, while it is also involved in the coagulation cascade by inhibiting the anticoagulant effect of protein S [70].Fibrinogen α, β, and γ chains polymerize and form fibrin, the basic component of the the remaining are involved in forming the membrane attack complex.This pathway is triggered by IgM/IgG-immunocomplexes, leading to chemotaxis and plasma protein secretion, facilitating opsonization.

Proteins Identified in the Serum and Their Role
Serum was studied by four research teams (Table 5, Supplementary Table 9) [37, 43, 45•, 46].The proteins identified in the serum of patients with FMS that differed from controls and were reported by most studies included fibrinogen A chain and profilin-1 (Table 5).Proteins identified in the serum and plasma involved A, B, and C chain of fibrinogen, serum amyloid (P-component, protein A4), C1qC, and thrombospondin-1.Calgranulin C and the immunoglobin Ig lambda-2 chain C region were identified in both the serum and saliva of patients with FMS.Finally, a common protein between serum and CSF was complement C4-A.
Profilin-1 is a protein that regulates actin dynamics in cells by promoting actin filament assembly and turnover, essential for processes like cell motility, shape maintenance, and endocytosis.It is crucial in facilitating cellular movement and maintaining cell structure [72,73].The serum amyloid p-component reduces neutrophil adhesion to proteins, inhibits the differentiation of monocytes into fibrocytes, attenuates profibrotic macrophages, activates the complement pathway, and promotes phagocytosis of cell debris.These effects regulate key aspects of inflammation and set a threshold for immune cell activation [74][75][76].Serum amyloid A4 is an acute phase reactant with a procoagulant role [77].Finally, thrombospondins have diverse tissue-specific actions that include effects on angiogenesis, platelet activation, inflammation, and cell death that directly impact wound healing and tumorigenesis [78,79].

Proteins Identified in Several Biological Fluids
More than one study identified 20 proteins differentially expressed in patients with FMS compared to controls.Specifically, fibrinogen α, β, and γ chains were identified in serum and plasma samples.These proteins play a key role in the coagulation cascade, polymerize, and create fibrin.However, their levels varied between studies.TRFE, the major iron-binding protein, was elevated in plasma and saliva samples, while profilin-1 was detected in three studies with decreased concentrations in serum and higher levels in saliva samples of patients with FMS.Interestingly, one investigator [45 •] characterized profilin-1 as a discriminating marker between two subgroups of FMS, experiencing pain and stiffness as prominent symptoms, respectively.
C4-A concentrations were elevated in serum and CSF samples, and C1qC was observed in high levels in the plasma and serum samples of patients with FMS.A2macroglobulin and serum amyloid (A4 and p-component) were isolated in plasma and serum, while haptoglobin was identified in plasma samples.Similarly, thrombospondin-1 was identified in plasma and serum samples.Elevated PGAM1, TALDO, and calgranulin A levels were observed in saliva samples of patients with FMS.Calgranulin C was over-expressed in serum and saliva of patients with FMS, while several immunoglobulin fractions were isolated in plasma, serum, and saliva samples.All proteins identified as being differentially expressed in FMS compared to controls in more than one study are presented in Table 6.

Association of Proteins with Scales of Disease Severity and Quality of Life
Bazzichi et al. [39] assessed the association between elevated saliva TALDO and PGAM1 levels and pain (VAS), QoL (FIQ), and the number of tender points.However, no significant association was detected.Similarly, Ciregia et al. [38] failed to observe any association between isolated proteins in saliva and pain (VAS), QoL (FIQ, FACIT), or tender points count.
Wahlen et al. [44] correlated elevated α2-macroglobulin and plasma TRFE concentrations to moderate-to-severe pain intensity.On the other hand, Ruggiero et al. [43] failed to report any associations between isolated serum proteins, disease duration, and pain (VAS) and QoL (FIQ) scales.Finally, Hsu et al. [45•] attempted to discriminate pain and soreness phenotypes in patients with FMS into two groups, focusing on the predominant reported symptom (pain or stiffness), concluding that this allocation is possible using proteomics, as each group exhibits different concentrations of proteomic markers.

Discussion
The results presented herein reveal that the identified proteins with significant alterations in the biological fluids of patients with FMS are mainly related to the immune system, the complement cascade, coagulation, and fibrinolysis.

Iron Metabolism Biomarkers
The most prevalent protein isolated in the included studies involved TRFE, over-expressed in plasma and saliva in FMS compared to healthy and pain controls.Since TRFE is an iron-binding protein, it reflects the body's need for iron supply [80].The association of FMS and iron metabolism disorders has been reported, with a possible mechanism being the involvement of iron in the production of dopamine and serotonin as a cofactor [81].Elevated TRFE concentrations have been observed in the CSF of patients with restless legs syndrome [82,83].However, in a casecontrol study, no difference was observed between patients with FMS and controls regarding serum iron, TRFE, and ferritin levels [84].Recently, a Taiwan nationwide study [85] revealed that adults with iron deficiency anemia had increased chances of developing FMS.An additional piece in the puzzle was provided by an intervention study [86] conducted on women with FMS and low ferritin levels, who had previously failed to improve iron status with oral supplements.When intravenous iron (ferric carboxymaltose) treatment was initiated, a reduction in pain sensation and an improvement in QoL were noted [86].Similar results were also reported from a blind, randomized, placebo-controlled trial [87], indicating a role for iron, in FMS treatment among patients with low iron stores.

Coagulation Biomarkers
Concentrations of fibrinogen α, β, and γ chains differed between patients and controls in serum and plasma samples, but findings regarding the direction of difference varied among investigators.Research is consistent with the elevated fibrinogen levels among patients with FMS, indicative of a prothrombotic state [88].Complement C1QC and C4A were also elevated in various biological fluids (CSF, plasma, serum) in FMS.These proteins are involved in the formation of MAC through the classical complement pathway, which is stimulated by IgM/IgG immune complexes and leads to chemotaxis and mobilization of cell opsonization [59,89].Certain coagulation proteins can also activate the complement cascade [90].These findings are consistent with the involvement of the coagulation cascade and complement in the pathophysiology of FMS.

Inflammation Biomarkers
Inflammation is a known driver of FMS.The increased amounts of skin mast cells [91,92], substance P, and corticotropin-releasing hormone (CRH) have been observed in FMS [93,94], appear to activate the release of IL-8 and monocyte chemoattractant protein-1 (MCP-1, a proinflammatory chemokine, member of a subfamily of the IL-8 supergene family) [95], elevating plasma concentrations [96].As a result, both IL-8 and MCP-1 have been suggested as possible diagnostic biomarkers for FMS, conferring an inflammatory action [97].At the proteomics level, different concentrations of thrombospondin-1 were observed in the serum and plasma of patients with FMS compared to controls.Inflammation in the CNS has been suggested as a mechanism involved in the pathogenesis of FMS, as impaired coagulation and fibrinolysis have been associated with degeneration of the CNS [98].The processing of pain is transmitted from peripheral tissues to the brain and is influenced by a variety of endogenous and exogenous processes [96], including impaired coagulation and fibrinolysis [98].Disturbances in any point in these pathways have been also observed in proteomic studies among patients with multiple sclerosis [99].In parallel, small-fiber neuropathy (SFN) has been an additional consistent finding in FMS [100][101][102], indicative of CNS degeneration.However, more research is required to clarify the association between neuroinflammation and FMS.
As for the possible therapeutic component of antiinflammatory regimes, research has revealed that the consumption of refined olive oil (ROO) among patients with FMS reduced fibrinogen levels, platelet distribution width, neutrophil-to-lymphocyte ratio, and erythrocyte sedimentation rate (ESR) concentrations [103].

Oxidative Stress Biomarkers
Haptoglobin is an acute phase protein with antioxidant activity that binds hemoglobin and prevents the toxic effect of iron.Elevated haptoglobin levels were recorded in the plasma of patients with FMS as a possible coping mechanism to mitigate oxidative stress [104].Conventional analytical methods also verify the existence of higher plasma haptoglobin levels in FMS and associate them with symptoms of depression, hyperalgesia, exhaustion, and sleep disturbances [105].Recent research suggests increased oxygen free radicals among patients with FMS [106].In more detail, prooxidative factors such as nitric oxide, products of free radical lipid peroxidation, including serum malondialdehyde and lipid hydroperoxide demonstrate an increased concentration, whereas xanthine oxidase levels are decreased, a process commonly occurring when oxygen free radicals are produced [106][107][108][109].In parallel, the levels of endogenous antioxidants are reduced, including glutathione, superoxide dismutase, and total antioxidant status in serum, as a response to the elevated oxidative stress [106,110].This phenomena have led to several researchers arguing whether FMS is actually an oxidative stress-driven disorder [107], since greater levels of prooxidative factors and mitophagy appear to augment pain sensitization [109].Interestingly, a recent systematic review concluded that supplementation with antioxidant vitamins and coenzyme Q10 for at least 6 weeks was associated with a reduced pain perception in 80% of the patients with FMS [111], indicating that antioxidants appear to have an analgesic role in FMS management [111,112].
TALDO is an enzyme of the pentose pathway that leads to NADPH production [54,113].Over-expression of TALDO was recorded in the saliva of patients with FMS, and this increase may also reflect the body's attempt to compensate for the increased oxidative stress endured.Although higher TALDO levels were observed in FMS compared with the healthy population, no differences were noted between FMS and patients with RA, or migraine.
Compared to healthy controls, patients with FMS demonstrated increased calgranulin A (S100-A8), but when compared to patients with RA and migraine, this difference ceased to exist.On the other hand, calgranulin C (S100-A12) concentrations were elevated in the saliva and serum of patients with FMS.Calgranulins are homogeneous low molecular weight calcium-binding proteins belonging to the S100 protein family.They are involved in various cellular responses and intracellular pathways that regulate cell differentiation, cytoskeleton, structural organization of membranes, intracellular calcium homeostasis, and protection against oxidative stress [114].It is worth highlighting another study, not included in the present systematic review, published in Italian, which verified elevated calgranulin A and C saliva concentrations in patients with FMS [115].
Research indicates that the consumption of extravirgin olive oil (EVOO) acts protectively in balancing redox homeostasis and tampering down inflammation [116,117].This is mostly due to the phenolic content of EVOO, and in particular hydroxy-tyrosol (HT).Interestingly, a preliminary study [118] assessed the effect of a high-HT nutritional treatment to the proteome of dermal fibroblasts of a single patient with FMS, versus a healthy control.The results revealed that treatment with HT normalized the differential expression in proteins involved in the turnover of extracellular matrix and oxidative metabolism, observed in the patient with FMS, against the healthy control [118].Although greatly underpowered, this study highlighted a possible therapeutic pathway for FMS, in need of more high-quality research.Other research groups have also identified EVOO as an important adjuvant in FMS treatment.In a RCT [119], women with FMS treated with 50 mL of EVOO or other ROO for a period of 3 weeks showed improvements in their antioxidative profiles (protein carbonyls, lipid peroxidation) and pain (FIQ), and QoL scales, exhibiting antithrombotic and anti-inflammatory properties [103].

Immune Response Biomarkers
Serum amyloid (P component) was elevated in plasma and serum, while serum amyloid (SA4) was high in plasma and low in serum.These proteins belong to the acute phase proteins and regulate the immune response.Different saliva and plasma concentrations of Ig kappa chain C region and Ig alpha-1 chain C region immunoglobulin segments were observed in patients with FMS.In parallel, Ig lambda-2 chain C region segments were high in saliva and low in serum samples.To date, the exact role of the immune system in pain development and sensation remains unclear [120].Nonetheless, an increased incidence of immunodeficiency has been observed in patients with FMS, and conversely, FMS tends to be more common in patients with primary immunodeficiency [121,122].Furthermore, immune aberrations have been reported in FMS, which were considered partially responsible for the sensation of pain [123 •].PGAM1 is an enzyme of glycolysis, and serum autoantibodies against PGAM1 have been reported in autoimmune hepatitis and various neurological diseases, including multiple sclerosis and neuromyelitis optica [55,124].As seen in the present results, PGAM1 concentrations were also increased in the saliva of patients with FMS.As the salivary gland innervates directly from the trigeminovascular system, saliva contains several neuropeptides, potentially providing information regarding CNS pathology and related disorders [125,126].In this manner, the elevated PGAM1 saliva levels could be of particular interest, as FMS has been associated with several neurological disorders [127].More research is required to delineate this association.

Cardiovascular Risk Biomarkers
Profilin-1 is an actin-binding protein that regulates DNA damage response and repair mechanisms [73].Saliva samples of patients with FMS exhibited higher profilin-1 concentrations than controls, whereas lower profilin-1 concentrations were observed in serum samples.In parallel, it has been suggested [45•] that profilin-1 could identify patients with FMS, depending on the main reported symptom (pain or stiffness).Recently, profilin-1 emerged as a new player in the field of atherosclerosis; it is accumulated in high concentrations in stable atherosclerotic plaques and thrombi from infarct-related arteries in cases of acute myocardial infarction [128].Furthermore, several studies have reported histological abnormalities in the muscle tissue of patients with FMS, indicative of microvascular dysfunction, including capillary dysfunction and myocyte mitochondric abnormalities [106,[129][130][131].
Finally, different apolipoprotein C-III concentrations were reported in the plasma and CSF of patients with FMS.Apolipoprotein C-III is an atherogenic protein that leads to higher triglyceride levels, resulting in augmented risk of cardiovascular events.Its role in the CNS remains unclear.Higher serum total cholesterol, LDL, and triglyceride levels have been observed in women with FMS, and these patients are known to have increased cardiovascular risk in parallel to obesity [132][133][134].

Other Biomarkers with Multiple Roles
A common protease inhibitor involved in the coagulation cascade, inflammation, and autoimmunity phenomena is α2-macroglobulin [135].Plasma levels of α2-macroglobulin were elevated in patients with FMS compared with healthy controls.A2-macroglobulin has been detected in the proteome of patients with chronic fatigue syndrome [136] and multiple sclerosis [137].Interestingly, new research and advances in pain management have suggested using α2-macroglobulin for treating neuropathic pain [138•, 139].As a result, α2-macroglobulin injections are frequently applied for managing knee osteoarthritis [140], as they prevent cartilage degeneration by inhibiting catabolic enzymes and cytokines [141].

Associations Between Proteomics and QoL of Patients
The present systematic review aimed to identify potential relationships between specific proteomic markers in FMS and QoL scales; however, for the most part, the results were inconclusive.One study using plasma samples [44] associated elevated α2-macroglobulin and TRFE with moderate and severe pain intensity.Finally, another study using serum samples [45•] concluded that different clinical profiles may be associated with different proteomic markers.

Combining Biomarkers for Diagnosis and Treatment
The quest for diagnostic biomarkers for FMS continues, aiming to provide a critical step towards prompt intervention [96].Annemans [22] revealed that establishing an FMS diagnosis decreases the financial costs associated with tests and imaging, referrals, doctor visits, and pharmaceuticals, showing that a diagnosis reduces the use of resources.Thus, efforts to identify and combine diagnostic markers are warranted to reduce the burden and costs associated with FMS.It is important to note, that although several different proteins were identified herein, it is difficult to understand if the same biological pathways act as triggers for the development of FMS, or if different pathways result in the same disease phenotype for different patients.Thus, we are currently unsure which combination of biomarkers can offer improved diagnostic ability for FMS, or whether a combination therapy (antioxidants, iron, antithrombotic, etc.) conferring a comprehensive synergistic action can tamper down FMS symptoms.What is known though from an early study [142], is that approximately 30% of patients with FMS have reported taking dietary supplements or making holistic dietary changes in response to their disease, according to their healthcare professionals, all achieving improved pain relief.As for the diagnosis, one of the studies [46] included herein reported using a decision tree model to differentiate patients with FMS and controls based on the expression levels of histidine protein methyltransferase 1 homolog (HMPT1), Interleukin-1 receptor accessory protein (IL1RAP) and Ig lambda chain V-IV region (IGL3-25), yielding an accuracy of up to 0.97.No other efforts to achieve improved diagnostic accuracy were reported by the authors of the remaining proteomics studies, and as quantitative data were not included in all research, we were unable to conduct this either.

Limitations of the Included Studies
Some of the included studies shared common authors.Specifically, the two studies using CSF samples [41,42] had the same first author and two additional authors in common.Also, the two studies investigating proteomics in saliva [38,39] were conducted by the same research group core, sharing five common authors, with one study essentially being the extension of the other, as it included a healthy population and a group of patients with chronic pain, as controls.This could be considered a confounding factor.
Most research items failed to report the medications taken by the patients or controls.Drugs can affect the proteome, as they have various actions on the metabolism and excretion of proteins from the body.
Finally, there were great differences in the proteomics methodologies among the studies.Distinct methods have varying sensitivity and specificity and may produce different results in the proteins detected and quantified.Each method has its advantages and limitations and may excel in different areas of proteomic research.Finally, the inherent differences in sample preparation, separation, and detection can lead to variations in the total number of proteins detected.

Conclusions
In summary, proteomics consist of a useful tool, providing insight into the processes and signaling pathways that may be involved in the pathogenesis of FMS.In the present systematic review, the proteome of patients with FMS was studied, and specific protein expression patterns were identified.Proteins related to the complement cascade, the coagulation cascade, inflammation, the immune system, iron metabolism, and the oxidative stress process were found to be dysregulated in FMS patients.FMS appears closely related to the oxidative stress pathway, as many proteins protecting the body from oxidative stress appear are dysregulated.However, more primary studies are required to aid our understanding of this association.

Fig. 1
Fig. 1 PRISMA 2020 flowchart of the studies' selection process

Table 1
Characteristics and results of the included studies

Table 3
Differently expressed proteins identified in the CSF of patients with FMS versus controls and their role Proteins appearing in more than one study are presented in bold font.↑ Increased; ↓ decreased α1ACT Α1 antichymothrypsin, ApoC3 apolipoprotein C-III, ApoD apolipoprotein D, C4-A complement C4-A, CAMKIIα calcium/calmodulindependent protein kinase type II subunit α, FMS fibromyalgia syndrome, HDL high-density lipoprotein, LG3BP galectin-3-binding protein, LRP1 low-density lipoprotein receptor-related protein 1, L1CAM L1 cell adhesion molecule, LYSC lysozyme C, MDH malate dehydrogenase, PCSK1 proprotein convertase subtilisin/kexin type 1, PGRN progranulin, PTPRz receptor-type tyrosine-protein phosphatase zeta a Decrease relative to other neurologic diseases, increase relative to rheumatoid arthritis

Table 4
Differently expressed proteins identified in the plasma of patients with FMS versus controls and their role appearing in more than one study are presented in bold font.↑