Protein Biomarkers In Patients Research Articles

Development of a novel microneedle platform for biomarker assessment of atopic dermatitis patients.

Atopic dermatitis (AD) is a chronic inflammatory skin disease whose pathogenesis, cause, and treatment have been extensively studied. The association of AD with Th2 cytokines is well known; therefore, the analysis of this association is crucial for the diagnosis and treatment of AD. This study aimed to present a new method for measuring protein biomarkers in patients with AD, before and after treatment, using minimally invasive microneedles. First, hyaluronic acid-loaded microneedle patches (HA-MNs) for skin sample collection were fabricated. Next, after Institutional Review Board approval, 20 patients with AD were recruited and skin samples were taken before and after treatment using four different sampling techniques: (1) tape stripping, (2) hydrocolloid patches, (3) hollow microneedles, and (4) HA-MNs. Lastly, proteins were isolated from the collected samples, and AD-related biomarkers were analyzed by enzyme-linked immunosorbent assay. Proteins were successfully extracted from the skin samples collected by tape stripping, hydrocolloid patches, and HA-MNs, except hollow microneedles. Interleukin (IL)-4, IL-13, and interferon-γ were detected in the HA-MNs only. By comparing the biomarker level correlation before and after treatment and the improvement score of the patients, we observed a significant negative correlation between IL-4 and IL-13 with an improvement in AD symptoms. Overall, our results verified that HA-MNs can be used to effectively analyze protein levels of biomarkers from skin metabolites of patients with AD and can be applied to monitor the treatment progress of patients with AD in a minimally invasive manner.

Differential proteomic expression in indolent versus transforming oral lichen planus.

Oral lichen planus (OLP) confers an approximately 1% risk of transformation to oral squamous cell carcinoma (OSCC). Early identification of high-risk OLP would be very helpful for optimal patient management. We aimed to discover specific tissue-based protein biomarkers in patients with OLP who developed OSCC compared to those who did not. We used laser capture microdissection- and nanoLC-tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens in patients with indolent OLP (no OSCC after at least 5-year follow-up, n= 6), transforming OLP (non-dysplastic epithelium with lichenoid inflammation marginal to OSCC, n= 6) or normal oral mucosa (NOM, n= 5). Transforming OLP protein profile was enriched for actin cytoskeleton, mitochondrial dysfunction and oxidative phosphorylation pathways. CA1, TNNT3, SYNM and MB were overexpressed, and FBLN1 was underexpressed in transforming OLP compared with indolent OLP. Integrin signalling and antigen presentation pathways were enriched in both indolent and transforming OLP compared with NOM. This proteomic study provides potential biomarkers, such as CA1 overexpression, for higher-risk OLP. While further validation studies are needed, we propose that epithelial-mesenchymal transition may be involved in OLP carcinogenesis.

Proteomics Profiling of Osteoporosis and Osteopenia Patients and Associated Network Analysis.

Bone mass reduction due to an imbalance in osteogenesis and osteolysis is characterized by low bone mineral density (LBMD) and is clinically classified as osteopenia (ON) or osteoporosis (OP), which is more severe. Multiple biomarkers for diagnosing OP and its progression have been reported; however, most of these lack specificity. This cohort study aimed to investigate sensitive and specific LBMD-associated protein biomarkers in patients diagnosed with ON and OP. A label-free liquid chromatography-mass spectrometry (LC-MS) proteomics approach was used to analyze serum samples. Patients’ proteomics profiles were filtered for potential confounding effects, such as age, sex, chronic diseases, and medication. A distinctive proteomics profile between the control, ON, and OP groups (Q2 = 0.7295, R2 = 0.9180) was identified, and significant dysregulation in a panel of proteins (n = 20) was common among the three groups. A comparison of these proteins showed that the levels of eight proteins were upregulated in ON, compared to those in the control and the OP groups, while the levels of eleven proteins were downregulated in the ON group compared to those in the control group. Interestingly, only one protein, myosin heavy chain 14 (MYH14), showed a linear increase from the control to the ON group, with the highest abundance in the OP group. A significant separation in the proteomics profile between the ON and OP groups (Q2 = 0.8760, R2 = 0.991) was also noted. Furthermore, a total of twenty-six proteins were found to be dysregulated between the ON and the OP groups, with fourteen upregulated and twelve downregulated proteins in the OP, compared to that in the ON group. Most of the identified dysregulated proteins were immunoglobulins, complement proteins, cytoskeletal proteins, coagulation factors, and various enzymes. Of these identified proteins, the highest area under the curve (AUC) in the receiver operating characteristic (ROC) analysis was related to three proteins (immunoglobulin Lambda constant 1 (IGLC1), RNA binding protein (MEX3B), and fibulin 1 (FBLN1)). Multiple reaction monitoring (MRM), LC-MS, was used to validate some of the identified proteins. A network pathway analysis of the differentially abundant proteins demonstrated dysregulation of inflammatory signaling pathways in the LBMD patients, including the tumor necrosis factor (TNF), toll-like receptor (TL4), and interferon-γ (IFNG) signaling pathways. These results reveal the existence of potentially sensitive protein biomarkers that could be used in further investigations of bone health and OP progression.

Commentary on: Multi-institutional Validation Study of Cyst Fluid Protein Biomarkers in Patients With Cystic Lesions of the Pancreas.

Commentary on: Multi-institutional Validation Study of Cyst Fluid Protein Biomarkers in Patients With Cystic Lesions of the Pancreas.

Identification of exosome protein biomarkers in patients with advanced hormone receptor-positive breast cancer treated with palbociclib and tamoxifen.

e13014 Background: The combination of a CDK4/6 inhibitor (CDK4/6i) plus endocrine therapy (ET) doubles progression free survival compared with ET alone in hormone receptor (HR)-positive, advanced breast cancer (BC), although not all patients respond and responders eventually develop resistance and disease progression. Exosomes are membrane-bound extracellular vesicles that are released from tumors for cell-to-cell transfer of lipids, proteins, and nucleic acids. Analysis of exosome cargo provides a dynamic and functional read-out of biological pathways that are activated in cancer cells. We performed deep proteomic analysis of plasma exosomes from patients receiving palbociclib/tamoxifen to identify protein networks that predict response to CDK4/6i and ET and that may contribute to drug resistance. Methods: The Big Ten Cancer Research Consortium conducted a phase II trial of palbociclib plus tamoxifen as first line therapy for patients with advanced, HR+/HER2- BC (NCT02668666). Whole blood was collected in Streck tubes from all participants at baseline and at time points during study treatment. Plasma was separated and stored at -80C within 48 hours of collection. Exosome extraction and purification was optimized for maximum proteomic coverage. Proteins were labeled with tandem mass tag 10plex and quantified with ultrasensitive mass spectrometry. Detected proteins were mapped to pathways with the Reactome Pathway Database. An unsupervised machine learning approach with modified graphic neural networks was used to determine whether differential expression of protein networks in plasma exosomes predicts treatment response. Results: We detected more than 700 exosome proteins from100 μl plasma in 16 study participants (responders, n = 11; non-responders, n = 5). Significant enrichment of exosome-specific markers was observed when comparing patient samples with healthy donor samples. Exosomal protein networks in pretreatment samples predicted treatment response with 95% sensitivity and 85% specificity in unsupervised clustering. The top weighted protein networks in the treatment response model are enriched for membrane attack complex, complement activation and lipoprotein receptor binding pathways. Conclusions: Ultrasensitive proteomic analysis combined with deep learning methods provides a detailed picture of the proteome landscape of plasma exosomes in advanced breast cancer patients and is ideally suited for serial analyses to study emergence of resistance mechanisms. This approach also demonstrated unparalleled accuracy as a predictive biomarker to identify patients unlikely to respond to CDK4/6i and ET. If results are confirmed, this novel approach could hold great promise for identifying protein biomarkers and mechanisms of resistance that emerge during anticancer therapy. Clinical trial information: NCT02668666.

Multiinstitutional Validation Study of Cyst Fluid Protein Biomarkers in Patients With Cystic Lesions of the Pancreas.

Prospective evaluation of 2 clinical-molecular models in patients with unknown pathology who underwent endoscopic ultrasound with fine-needle aspiration (EUS-FNA) for a cystic lesion of the pancreas. Preoperative prediction of histologic subtype (mucinous vs nonmucinous) and grade of dysplasia in patients with pancreatic cystic neoplasms is challenging. Our group has previously published 2 clinical-molecular nomograms for intraductal papillary mucinous neoplasms (IPMN) that incorporated both clinical/radiographic features and cyst fluid protein markers (sFASL, CA72-4, MMP9, IL-4). This multiinstitutional study enrolled patients who underwent EUS-FNA for a cystic lesion of the pancreas. Treatment recommendations regarding resection were based on standard clinical, radiographic, and endoscopic features. Predicted probabilities of high-risk IPMN (high-grade dysplasia/invasive cancer) were calculated using the previously developed clinical-molecular nomograms. Cyst fluid was obtained from 100 patients who underwent diagnostic EUS-FNA. Within this group there were 35 patients who underwent resection, and 65 were monitored radiographically. Within the group that underwent resection, 26 had low-risk IPMN or benign non-IPMN lesions, and 9 had high-risk IPMN. Within the surveillance group, no patient progressed to resection or developed cancer after a median follow-up of 12months (range: 0.5-38). Using the clinical/radiographic nomogram alone, 2 out of 9 patients with high-risk IPMN had a predicted probability >0.5. In the clinical-molecular models, 6 of 9 patients in model 1, and 6 of 9 in model 2, had scores >0.5. This prospective study of patients with unknown cyst pathology further demonstrates the importance of cyst fluid protein analysis in the preoperative identification of patients with high-risk IPMN. Longer follow-up is necessary to determine if this model will be useful in clinical practice.

Combined serum anti-SSA/Ro and salivary TRIM29 reveals promising high diagnostic accuracy in patients with primary Sjögren's syndrome.

ObjectivesTo determine the diagnostic potential of simultaneous presence of serum anti-SSA/Ro and upregulated salivary protein biomarkers in patients with primary Sjögren’s syndrome (pSS).MethodsPrevious proteomics data on the intensity of neutrophil elastase, calreticulin, tripartite motif containing protein 29 (TRIM29), clusterin and vitronectin provided basis for performing extended analysis. Protein data was obtained by liquid chromatography tandem mass spectrometry technique in whole saliva from 24 patients with pSS and 16 patients having symptoms of pSS, but not fulfilling the American College of Rheumatology/European League against Rheumatism classification criteria (non-pSS). Serum anti-SSA/Ro antibody was measured using enzyme-linked immunosorbent assays. Receiver operating characteristic curve (ROC) value was calculated for combined biomarkers.ResultsSimultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided the most optimal combination with an area under curve (AUC) of 0.995 (95% CI 0.98–1.00, p = 2.0E-7 and standard error 0.007) and combinations of sensitivity and specificity within the interval of 91–100%. ROC analysis showed that salivary levels of TRIM29 alone enabled differentiation between pSS and non-pSS with an area under curve (AUC) of 0.88 (95%CI 0.77–1.00). All patients with pSS and 3 non-pSS patients were serum anti-SSA/Ro positive.ConclusionsSimultaneous presence of serum anti-SSA/Ro and upregulated salivary TRIM29 provided a high diagnostic accuracy exceeding that of currently available tools used in pSS diagnostics. This biomarker combination represents a promising less invasive diagnostic tool for pSS. The clinical applicability of TRIM29 needs further testing in independent cohorts using relevant analytical techniques.

Rapid single-molecule digital detection of protein biomarkers for continuous monitoring of systemic immune disorders

Digital protein assays have great potential to advance immunodiagnostics because of their single-molecule sensitivity, high precision, and robust measurements. However, translating digital protein assays to acute clinical care has been challenging because it requires deployment of these assays with a rapid turnaround. Herein, we present a technology platform for ultrafast digital protein biomarker detection by using single-molecule counting of immune-complex formation events at an early, pre-equilibrium state. This method, which we term “pre-equilibrium digital enzyme-linked immunosorbent assay” (PEdELISA), can quantify a multiplexed panel of protein biomarkers in 10 µL of serum within an unprecedented assay incubation time of 15 to 300 seconds over a 104 dynamic range. PEdELISA allowed us to perform rapid monitoring of protein biomarkers in patients manifesting post-chimeric antigen receptor T-cell therapy cytokine release syndrome, with ∼30-minute sample-to-answer time and a sub–picograms per mL limit of detection. The rapid, sensitive, and low-input volume biomarker quantification enabled by PEdELISA is broadly applicable to timely monitoring of acute disease, potentially enabling more personalized treatment.

Current updates on protein as biomarkers for diabetic kidney disease: a systematic review.

Background:In the past decade, researchers have been focused on discovering protein biomarkers for diabetic kidney disease. This paper aims to search for, analyze, and synthesize current updates regarding the development of these efforts.Methods:We systematically searched the ScienceDirect, SpringerLink, and PubMed databases for observational studies of protein biomarkers in patients with diabetes mellitus. We included studies published between January 2018 and April 2020, that were based on a population of patients with type-1 or type-2 diabetes mellitus aged ⩾18 years, with an observational design such as cross-sectional, case–control, or cohort studies. The dependent variable of the research results was in the form of protein biomarkers from urine, plasma, or serum.Results:Following the screening process, 20 research articles with available full text met the inclusion criteria. These could be categorized as glomerular biomarkers (ANGPTL4, beta-2 microglobulin, Smad1, and glypican-5); inflammatory biomarkers (MCP-1 and adiponectin); and tubular biomarkers (NGAL, VDBP, megalin, sKlotho, and KIM-1). The development of a panel of biomarkers showed more promising results than those for a single biomarker in diagnosing diabetic kidney disease.Conclusion:All the biomarkers discussed in this review showed promising results for predicting diabetic kidney disease because they correlate with albuminuria, eGFR, or both. However, of the 11 protein biomarkers, none have prognostic value beyond albuminuria and eGFR.

Mitochondrial-associated protein biomarkers in patients with attention-deficit/hyperactivity disorder

This study aimed to determine the role of mitochondria-associated proteins (HtrA2, α-synuclein, and Park7) in attention deficit/hyperactivity disorder (ADHD). A total of 125 patients with ADHD (77.6% were males) and 66 healthy controls (66.7% were males) were recruited. We found that girls with ADHD demonstrated higher plasma HtrA2 level than control girls, and their HtrA2 levels were positively correlated with verbal comprehensive ability, and negatively correlated to behavior symptoms. Among boys, we observed no correlations between these mitochondrial proteins, neuropsychological findings, and clinical symptoms. Our findings suggest that an underlying gender-specific mitochondria pathway may influence with the pathophysiology of ADHD.

Pro-inflammatory biomarkers in women with non-obstructive angina pectoris and coronary microvascular dysfunction

BackgroundStudies that evaluate larger numbers of protein biomarkers in patients with coronary microvascular dysfunction (CMD) have not previously been performed, and very little is known concerning the pathogenetic mechanisms leading to CMD.Our objective was to analyze associations between a broad cardiovascular disease (CVD) protein biomarker assay and CMD, and further explore internal biomarker relations in order to identify possible targets for future treatment interventions. MethodsIn 174 women with angina pectoris and no significant obstructive coronary artery disease (<50% stenosis on invasive coronary angiography), CMD was assessed by transthoracic Doppler echocardiography measuring coronary flow velocity reserve (CFVR). Blood samples were analyzed with a CVD proteomic panel encompassing 92 biomarkers. The relation between biomarkers and CFVR was evaluated by regression analysis, and possible interrelations between significant biomarkers were investigated by principal component analysis (PCA). ResultsMedian age (SD) was 64 years (9.8), median CFVR (IQR) was 2.3 (1.9–2.7), and 28% of patients had CFVR < 2.0. Eighteen biomarkers were significantly correlated with CFVR. In PCA, 8 of the biomarkers significantly related to CFVR showed high loadings on principal component 1 (PC1). The component scores of PC1 were significantly related to CFVR (p = 0.002). The majority of the 8 interrelated PC1 biomarkers were related to the pro-inflammatory TNF-α – IL-6 – CRP pathway. ConclusionEighteen protein biomarkers were significantly associated with CMD. Eight biomarkers were interrelated in PCA, and share connection with pro-inflammatory pathways, highlighting a possible important role of inflammation in CMD.

Serum Metabolites Associated with Computed Tomography Findings after Traumatic Brain Injury.

There is a need to rapidly detect patients with traumatic brain injury (TBI) who require head computed tomography (CT). Given the energy crisis in the brain following TBI, we hypothesized that serum metabolomics would be a useful tool for developing a set of biomarkers to determine the need for CT and to distinguish among different types of injuries observed. Logistical regression models using metabolite data from the discovery cohort (n = 144, Turku, Finland) were used to distinguish between patients with traumatic intracranial findings and those with negative findings on head CT. The resultant models were then tested in the validation cohort (n = 66, Cambridge, United Kingdom). The levels of glial fibrillary acidic protein and ubiquitin C-terminal hydrolase-L1 were also quantified in the serum from the same patients. Despite there being significant differences in the protein biomarkers in patients with TBI, the model that determined the need for a CT scan validated poorly (area under the curve [AUC] = 0.64: Cambridge patients). However, using a combination of six metabolites (two amino acids, three sugar derivatives, and one ketoacid) it was possible to discriminate patients with intracranial abnormalities on CT and patients with a normal CT (AUC = 0.77 in Turku patients and AUC = 0.73 in Cambridge patients). Further, a combination of three metabolites could distinguish between diffuse brain injuries and mass lesions (AUC = 0.87 in Turku patients and AUC = 0.68 in Cambridge patients). This study identifies a set of validated serum polar metabolites, which associate with the need for a CT scan. Additionally, serum metabolites can also predict the nature of the brain injury. These metabolite markers may prevent unnecessary CT scans, thus reducing the cost of diagnostics and radiation load.

Identification of circulating protein biomarkers in patients with hepatocellular carcinoma concomitantly infected with chronic hepatitis C virus

Context: The incidence rate of hepatocellular carcinoma (HCC) is higher in developing countries, and most cases are associated with chronic hepatitis C virus (HCV) infection.Objective: To evaluate the circulating proteins as liver biomarkers for the identification of HCC associated with HCV infection in Egyptian patients using LC-MS/MS analysis.Methods: Blood sera were collected from 31 HCC patients and the fractionated proteins were subjected to LC-MS/MS analysis. Protein candidates were validated by enzyme-linked immunosorbent assay (ELISA).Results: Thirty-three proteins were significantly identified in the sera of HCC patients with persistent HCV infection. These proteins are involved in several biological processes including acute phase response, complement activation, hemostasis process and lipid metabolism. The level of lectin galactoside-binding soluble 3 binding protein (LGALS3BP), Kininogen-1 (KNG1), serum amyloid A2 (SAA2) and paraoxonase 1 (PON1) and alpha-fetoprtoein (AFP) were elevated in serum.Conclusion: In HCC patients with chronic HCV infection, we identified a group of differentially expressed circulating proteins involved in regulating different cellular mechanisms.

Evaluation of potential protein biomarkers in patients with high sperm DNA damage

The laboratory evaluation of male infertility remains an essential area of research as 40-60% of infertility cases are attributable to male-related factors. Current sperm analysis methods add only partial information on sperm quality and fertility outcomes. The specific underlying cause of infertility in most cases is unknown, while a proportion of male infertility could be caused by molecular factors such as the absence or abnormal expression of some essential sperm proteins. The objective of this study was to screen for associations between sperm protein profiles and sperm concentration, motility, and DNA fragmentation index in patients undergoing fertility evaluation in a clinical setting. Based on those parameters, semen samples were categorized as either normal or abnormal. We screened 34 semen samples with various abnormal parameters and compared them to 24 normal control samples by using one dimensional (1-D) gel electrophoresis and mass-spectrometry. In this study, we anticipated to establish a normal sperm parameter profile which would be compared to abnormal sperm samples and reveal candidate proteins. Our preliminary results indicate that no normal uniform profile could be established, which affirms the complexity of male fertility and confirms the limitations of standard semen analysis. Four main protein groups were identified in correlation with abnormal DNA fragmentation and/or motility. The first group included sperm nuclear proteins such as the SPANX (sperm protein associated with the nucleus on the X chromosome) isoforms and several types of histones. The second group contained mitochondria-related functions and oxidative stress proteins including Mitochondrial Ferritin, Mitochondrial Single-Stranded DNA Binding Protein, and several isoforms of Peroxiredoxins. Two other protein groups were related to sperm motility such as microtubule-based flagellum and spindle microtubule as well as proteins related to the ubiquitin-proteasome pathway. Further research is required in order to characterize these potential biomarkers of male fertility potential.

Protein biomarkers in patients with subarachnoid hemorrhage, vasospasm, and delayed ischemic neurological deficits.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating neurological disease. It has many sequelae, including vasospasm and delayed ischemic neurological deficits (DINDs). We explored the blood proteome in patients with aSAH using transcranial Doppler (TCD) velocity as a guide to patients who are at risk for symptomatic vasospasm and DIND. Blood was drawn on all days that patients were observed in the neurocritical care unit (NCCU) after aSAH. A team of neurologists and neurosurgeons identified patients with clinical evidence of vasospasm and DIND. Serum was fractionated using protein chips and surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF MS). We detected a pattern of protein expression associated with those at risk for elevated TCD velocities by day 8, compared with blood collected in the presymptomatic stage (days 1-3). We further analyzed serum using pooled samples from study entry to the time of elevated TCD velocities using a protein microarray that analyzed 500 human proteins thematically oriented toward inflammation. After identifying several candidates with elevated concentrations in the pooled samples, we then used reverse protein arrays to quantitate the concentration of potential candidate proteins in the individual samples. Proteins with significantly elevated concentrations included apolipoprotein-E, apolipoprotein-A, serum amyloid protein-4, and serum amyloid protein-P. Future studies in larger sample populations are needed to evaluate these biomarkers further as representative of biosystems involved in vasospasm and DIND or as potential biomarkers predictive of risk associated with disease.

P-776 - Protein biomarkers in psychotic patients

Introduction Psychotic disorders such as schizophrenia and bipolar disorder are complex neuropsychiatric conditions of multifactorial etiology whose diagnosis is currently based solely on clinical interview methodology. Therefore, biomarkers which could improve the current classification and in perspective stratify patients on a biological basis into more homogeneous clinically distinct subgroups, are highly needed. Objectives We describe here the preliminary data on the application of a protein profiling investigation for the identification of peripheral markers for psychotic disorders, based on a proteomic approach. Aims To identify serum protein biomarkers in patients with acute phase psychotic disorder compared with anxiety or depressive disorders with no history of psychosis. Methods Patients were sampled and evaluated by means of SCID-I, PANSS, YMRS, HAM-A, HAM-D. Lymphocytes have been separeted and Two-Dimensional Electrophoresis (2-DE) was carried out on protein extracts. Analysis was performed using Progenesis Same Spot software. Result 2-DE showed significant differences in protein patterns between the two groups, in particular some protein spots are upregulated in a statistically significant manner and are under identification by mass spectrometry. PCA underlies a clear separation between the two groups, with a t1 value for the first component of 54%. Conclusions Preliminary analysis of a small sample shows significant differences in protein pattern between acute psychotic patients compared with non-psychotic psychiatric controls. Further work will widen the sample and will identify the single proteins.

Prognostic value of serum protein biomarkers in patients with epithelial ovarian cancer.

10567 Background: Study was designed to identify biomarker candidate proteins (bcp) in discovery phase and validate them in confirmation phase using serum samples from healthy control and epithelial ovarian cancer (EOC) patients. Then bcp's were examined in EOC patients in context of current disease status; during chemotherapy treatments (c), remission (RM), and recurrence (RC). Methods: We combined mass spectrometry-based Multidimensional Protein Identification Technology (MUDPIT) proteomics strategy with high throughput Multiplex beads-based LUMINEX assay and enzyme-linked immunosorbent assay (ELISA) to identify and validate the bcps in serum. 88 healthy controls and 83 EOC patients were analyzed; 18 during chemotherapy, 28 in remission and 34 with recurrent disease. Results: Expression levels of acute phase protein Serum amyloid A (SAA), C-reactive protein (CRP), alpha 1 antitrypsin (SERPINA1), secreted phosphoprotein 1 (SSP1), and matrix metallopeptidase 1 (MMP1) were increased in cancer patients, while chemokine ligand 2 (CCL2) showed lower expression level in the cancer samples versus controls (Table). Elevated serum levels of CRP (p=0.027)and MMP1(p=0.015) were associated with cancer recurrence when compared to remission. Conclusions: Serum levels of bcps SAA, MMP1, CRP, SSP1, SERPINA1, and CCL2 were significantly changed in cancer and some protein level changes correspond to EOC clinical status. These bcp's may be important in CA125 negative patients and deserve further study. Symbol Study phases RC/control p C/control p RC + C/control p RC/RM p SAA MS discovery 2.62 0.0272 1.89 0.2664 2.24 0.0400 2.93 0.0112 ELISA/LUMINEX confirmation 3.37 0.0212 4.59 0.0956 3.80 0.0055 2.30 0.0748 SERPINA1 MS discovery 2.01 0.1814 3.29 0.0067 2.68 0.0157 0.84 0.6201 ELISA/LUMINEX confirmation 1.18 0.0343 1.31 0.02 1.23 0.0027 0.82 0.0871 CRP MS discovery 4.93 0.0016 6.00 0.0019 5.49 0.0000 2.98 0.0081 ELISA/LUMINEX confirmation 4.09 0.0003 5.38 0.0172 4.54 0.0000 2.03 0.0270 SPP1 ELISA/LUMINEX confirmation 2.51 0.0004 3.12 0.1100 2.72 0.0013 1.48 0.1089 MMP1 ELISA/LUMINEX confirmation 1.90 0.0005 1.80 0.0183 1.86 0.0000 1.56 0.0159 CCL2 ELISA/LUMINEX confirmation 0.80 0.0445 1.17 0.4893 0.93 0.5348 0.87 0.3513 No significant financial relationships to disclose.

Acid hydrolysis followed by matrix‐assisted laser desorption/ionization mass spectrometry for the rapid diagnosis of serum protein biomarkers in patients with major depression

We have developed a technique combining acid hydrolysis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid study of the changes in the levels of positive and negative acute phase protein biomarkers in the sera of patients suffering from major depression. The serum proteins were first precipitated using an organic solvent; after separation, they were subjected to hydrochloric acid hydrolysis (6 M HCl) for 10 min. The resulting peptides were characterized using MALDI-TOF MS. Short-term treatment of the serum proteins with HCl efficiently removed interference from the abundant protein - albumin - and produced abundant peptide ion signals in the range of m/z 4000-10 000. This approach allowed us to rapidly detect the peptide ions originating from transferrin (a common negative acute phase protein) and fibrinogen (a common positive acute phase protein). The average ratios and (standard deviations) of the ion signals derived from transferrin/fibrinogen were 3.58 (+/-1.93) for the healthy control subjects and 1.02 (+/-0.52) for the patients suffering from major depression. The differences in transferrin/fibrinogen ratios between healthy controls and patients suggest that major depression will induce internal inflammation and cause either an increase in the level of fibrinogen or a decrease in the level of transferrin.