Functional proteomics based on protein microarray technology for biomedical research.

This chapter traces a route through Proteomics from its origins to the present day. The different proteomics applications are discussed with a focus on microarray technology. Analytical microarrays, functional microarrays and reverse phase microarrays and their different applications are discussed. Several studies are mentioned where the great versatility of this approach is shown. Finally, the advantages and future challenges of microarray technology are outlined.

Autoantibody Profiling Using Human Autoantigen Protein Array and AlphaScreen.

Autoantibodies that recognize self-antigens are believed to have a close relationship with diseases such as autoimmune diseases, cancer, and lifestyle diseases. Analysis of autoantibodies is essential for investigating pathology mechanisms, diagnosis, and therapeutics of these diseases. We developed an autoantibody profiling assay using a cell-free synthesized protein array and high-throughput screening technology. Our assay system can sensitively detect interaction between recombinant antigen protein and autoantibody and efficiently analyze autoantibody profiling in patients' sera.

Direct competitive assay for HER2 detection in human plasma using Bloch surface wave-based biosensors.

The overexpression and/or amplification of the HER2/neu oncogene has been proposed as a prognostic marker in breast cancer. The detection of the related peptide HER2 remains a grand challenge in cancer diagnosis and for therapeutic decision-making. Here, we used a biosensing device based on Bloch Surface Waves excited on a one-dimensional photonic crystal (1DPC) as valid alternative to standard techniques. The 1DPC was optimized to operate in the visible spectrum and the biosensor optics has been designed to combine label-free and fluorescence operation modes. This feature enables a real-time monitoring of a direct competitive assay using detection mAbs conjugated with quantum dots for an accurate discrimination in fluorescence mode between HER2-positive/negative human plasma samples. Such a competitive assay was implemented using patterned alternating areas where HER2-Fc chimera and reference molecules were bio-conjugated and monitored in a multiplexed way. By combining Label-Free and fluorescence detection analysis, we were able to tune the parameters of the assay and provide an HER2 detection in human plasma in less than 20 min, allowing for a cost-effective assay and rapid turnaround time. The proposed approach offers a promising technique capable of performing combined label-free and fluorescence detection for both diagnosis and therapeutic monitoring of diseases.

Antibody Profiling of Dengue Severities Using Flavivirus Protein Microarrays.

Dengue is a viral disease transmitted by Aedes aegypti mosquitoes. According to the World Health Organization, about half of the world's population is at risk of dengue. There are four serotypes of the dengue virus. After infection with one serotype, it will be immune to such a serotype. However, subsequent infection with other serotypes will increase the risk of severe outcomes, e.g., dengue hemorrhagic fever, dengue shock syndrome, and even death. Since severe dengue is challenging to predict and lacks molecular markers, we aim to build a multiplexed Flavivirus protein microarray (Flaviarray) that includes all of the common Flaviviruses to profile the humoral immunity and cross-reactivity in the dengue patients with different outcomes. The Flaviarrays we fabricated contained 17 Flavivirus antigens with high reproducibility (R-square = 0.96) and low detection limits (172-214 pg). We collected serums from healthy subjects (n = 36) and dengue patients within 7 days after symptom onset (mild dengue (n = 21), hospitalized nonsevere dengue (n = 29), and severe dengue (n = 36)). After profiling the serum antibodies using Flaviarrays, we found that patients with severe dengue showed higher IgG levels against multiple Flavivirus antigens. With logistic regression, we found groups of markers with high performance in distinguishing dengue patients from healthy controls as well as hospitalized from mild cases (AUC > 0.9). We further reported some single markers that were suitable to separate dengue patients from healthy controls (AUC > 0.9) and hospitalized from mild outcomes (AUC > 0.8). Together, Flaviarray is a valuable tool to profile antibody specificities, uncover novel markers for decision-making, and shed some light on early preventions and treatments.

Comprehensive autoantibody profiling in systemic autoimmunity by a highly-sensitive multiplex protein array.

Comprehensive autoantibody evaluation is essential for the management of autoimmune disorders. However, conventional methods suffer from poor sensitivity, low throughput, or limited availability. Here, using a proteome-wide human cDNA library, we developed a novel multiplex protein assay (autoantibody array assay; A-Cube) covering 65 antigens of 43 autoantibodies that are associated with systemic sclerosis (SSc) and polymyositis/dermatomyositis (PM/DM). The performance of A-Cube was validated against immunoprecipitation and established enzyme-linked immunosorbent assay. Further, through an evaluation of serum samples from 357 SSc and 172 PM/DM patients, A-Cube meticulously illustrated a diverse autoantibody landscape in these diseases. The wide coverage and high sensitivity of A-Cube also allowed the overlap and correlation analysis between multiple autoantibodies. Lastly, reviewing the cases with distinct autoantibody profiles by A-Cube underscored the importance of thorough autoantibody detection. Together, these data highlighted the utility of A-Cube as well as the clinical relevance of autoantibody profiles in SSc and PM/DM.

Role of Serum Inflammatory Cytokines in Sepsis Rats Following BMSCs Transplantation: Protein Microarray Analysis.

Bone marrow stromal cells (BMSCs) have emerged as a potential therapy for sepsis, yet the underlying mechanisms remain unclear. In this study, we investigated the effects of BMSCs on serum inflammatory cytokines in a rat model of lipopolysaccharide (LPS)-induced sepsis. Sepsis was induced by intravenous injection of LPS, followed by transplantation of BMSCs. We monitored survival rates for 72 h and evaluated organ functions, histopathological changes, and cytokines expression. Sepsis rats showed decreased levels of white blood cells, platelets, lymphocyte ratio, and oxygen partial pressure, along with increased levels of neutrophil ratio, carbon dioxide partial pressure, lactic acid, alanine aminotransferase, and aspartate aminotransferase. Histologically, lung, intestine, and liver tissues exhibited congestion, edema, and infiltration of inflammatory cells. However, after BMSCs treatment, there was improvement in organ functions, histopathological injuries, and survival rates. Protein microarray analysis revealed significant changes in the expression of 12 out of 34 inflammatory cytokines. These findings were confirmed by enzyme-linked immunosorbent assay. Pro-inflammatory factors, such as interleukin-1ß (IL-1ß), IL-1α, tumor necrosis factor-α (TNF-α), tissue inhibitor of metal protease 1 (TIMP-1), matrix metalloproteinase 8 (MMP-8), Leptin, and L-selectin were upregulated in sepsis, whereas anti-inflammatory and growth factors, including IL-4, ß-nerve growth factor (ß-NGF), ciliary neurotrophic factor (CNTF), interferon γ (IFN-γ), and Activin A were downregulated. BMSCs transplantation led to a decrease in pro-inflammatory cytokines and an increase in anti-inflammatory and growth factors. We summarized relevant molecular signaling pathways that resulted from cytokines in BMSCs for treating sepsis. Our results illustrated that BMSCs could promote tissue repair and improve organ functions and survival rates in sepsis through modulating cytokine networks.

Two Decades of Arrayed Imaging Reflectometry for Sensitive, High-Throughput Biosensing.

Arrayed imaging reflectometry (AIR), first introduced in 2004, is a thin-film interference sensor technique that optimizes optical properties (angle of incidence, polarization, substrate refractive index, and thickness) to create a condition of total destructive interference at the surface of a silicon substrate. The advantages of AIR are its sensitivity, dynamic range, multiplex capability, and high-throughput compatibility. AIR has been used for the detection of antibodies against coronaviruses, influenza viruses, Staphylococcus aureus, and human autoantigens. It has also shown utility in detection of cytokines, with sensitivity comparable to bead-based and ELISA assays. Not limited to antibodies or antigens, mixed aptamer and protein arrays as well as glycan arrays have been employed in AIR for differentiating influenza strains. Mixed arrays using direct and competitive inhibition assays have enabled simultaneous measurement of cytokines and small molecules. Finally, AIR has also been used to measure affinity constants, kinetic and at equilibrium. In this review, we give an overview of AIR biosensing technologies and present the latest AIR advances.

Profiling humoral responses to COVID-19 immunization in Kawasaki disease using SARS-CoV-2 variant protein microarrays.

Kawasaki disease (KD) is a form of acute systemic vasculitis syndrome that predominantly occurs in children under the age of 5 years. Its etiology has been postulated due to not only genetic factors but also the presence of foreign antigens or infectious agents. To evaluate possible associations between Kawasaki disease (KD) and COVID-19, we investigated humoral responses of KD patients against S-protein variants with SARS-CoV-2 variant protein microarrays. In this study, plasma from a cohort of KD (N = 90) and non-KD control (non-KD) (N = 69) subjects in categories of unvaccinated-uninfected (pre-pandemic), SARS-CoV-2 infected (10-100 days after infection), and 1-dose, 2-dose, and 3-dose BNT162b2 vaccinated (10-100 days after vaccination) was collected. The principal outcomes were non-KD-KD differences for each category in terms of anti-human/anti-His for binding antibodies and neutralizing percentage for surrogate neutralizing antibodies. Binding antibodies against spikes were lower in the KD subjects with 1-dose of BNT162b2, and mean differences were significant for the P.1 S-protein (non-KD-KD, 3401; 95% CI, 289.0 to 6512; P = 0.0252), B.1.617.2 S-protein (non-KD-KD, 4652; 95% CI, 215.8 to 9087; P = 0.0351) and B.1.617.3 S-protein (non-KD-KD, 4874; 95% CI, 31.41 to 9716; P = 0.0477). Neutralizing antibodies against spikes were higher in the KD subjects with 1-dose of BNT162b2, and mean percentage differences were significant for the 1-dose BNT162b2 B.1.617.3 S-protein (non-KD-KD, -22.89%; 95% CI, -45.08 to -0.6965; P = 0.0399), B.1.1.529 S-protein (non-KD-KD, -25.96%; 95% CI, -50.53 to -1.376; P = 0.0333), BA.2.12.1 S-protein (non-KD-KD, -27.83%; 95% CI, -52.55 to -3.115; P = 0.0195), BA.4 S-protein (non-KD-KD, -28.47%; 95% CI, -53.59 to -3.342; P = 0.0184), and BA.5 S-protein (non-KD-KD, -30.42%; 95% CI, -54.98 to -5.869; P = 0.0077). In conclusion, we have found that KD patients have a comparable immunization response to healthy individuals to SARS-CoV-2 infection and COVID-19 immunization.

Utility of Protein Microarrays for Detection of Classified and Novel Antibodies in Autoimmune Neurologic Disease.

BACKGROUND AND OBJECTIVES: Neural antibodies are detected by tissue-based indirect immunofluorescence assay (IFA) in Mayo Clinic's Neuroimmunology Laboratory practice, but the process of characterizing and validating novel antibodies is lengthy. We report our assessment of human protein arrays. METHODS: Assessment of arrays (81% human proteome coverage) was undertaken using diverse known positive samples (17 serum and 14 CSF). Samples from patients with novel neural antibodies were reflexed from IFA to arrays. Confirmatory assays were cell-based (CBA) or line blot. Epitope mapping was undertaken using phage display immunoprecipitation sequencing (PhiPSeq). RESULTS: Control positive samples known to be reactive with linear epitopes of intracellular antigens (e.g., ANNA-1 [anti-Hu]) were readily identified by arrays in 20 of 21 samples. By contrast, 10 positive controls known to be enriched with antibodies against cell surface protein conformational epitopes (e.g., GluN1 subunit of NMDA-R) were indistinguishable from background signal. Three antibodies, previously characterized by other investigators (but unclassified in our laboratory), were unmasked in 4 patients using arrays (July-December 2022): Neurexin-3α, 1 patient; regulator of gene protein signaling (RGS)8, 1 patient; and seizure-related homolog like 2 (SEZ6L2), 2 patients. All were accompanied by previously reported phenotypes (encephalitis, 1; cerebellar ataxia, 3). Patient 1 had subacute onset of seizures and encephalopathy. Neurexin-3α ranked high in CSF (second ranked neural protein) but low in serum (660th overall). Neurexin-3α CBA was positive in both samples. Patient 2 presented with rapidly progressive cerebellar ataxia. RGS8 ranked the highest neural protein in available CSF sample by array (third overall). RGS8-specific line blot was positive. Patients 3 and 4 had rapidly progressive cerebellar ataxia. SEZ6L2 was the highest ranked neural antigen by arrays in all samples (CSF, 1, serum, 2; Patient 3, ranked 9th overall in CSF, 11th in serum; Patient 4, 6th overall in serum]). By PhIPSeq, diverse neurexin-3α epitopes (including cell surface) were detected in CSF from patient 1, but no SEZ6L2 peptides were detected for serum or CSF samples from Patient 3. DISCUSSION: Individualized autoimmune neurologic diagnoses may be accelerated using protein arrays. They are optimal for detection of intracellular antigen-reactive antibodies, though certain cell surface-directed antibodies (neurexin-3α and SEZ6L2) may also be detected.

Clonal architecture and evolutionary history of Waldenström's macroglobulinemia at the single-cell level.

To provide insight into the subclonal architecture and co-dependency patterns of the alterations in Waldenström's macroglobulinemia (WM), we performed single-cell mutational and protein profiling of eight patients. A custom panel was designed to screen for mutations and copy number alterations at the single-cell level in samples taken from patients at diagnosis (n=5) or at disease progression (n=3). Results showed that in asymptomatic WM at diagnosis, MYD88L265P was the predominant clonal alteration; other events, if present, were secondary and subclonal to MYD88L265P. In symptomatic WM, clonal diversity was more evident, uncovering combinations of alterations that synergized to promote clonal expansion and dominance. At disease progression, a dominant clone was observed, sometimes accompanied by other less complex minor clones, which could be consistent with a clonal selection process. Clonal diversity was also reduced, probably due to the effect of treatment. Finally, we combined protein expression with mutational analysis to map somatic genotype with the immunophenotype. Our findings provide a comprehensive view of the clonality of tumor populations in WM and how clonal complexity can evolve and impact disease progression.

[Conditional optimization and methodological evaluation of liquid protein microarray in detecting cystatin C].

OBJECTIVE: To optimize the detection conditions and evaluate of cystatin C(CysC) by liquid protein microarray. METHODS: CysC was detected by double antibody sandwich method using liquid protein microarray. On the basis of determining the optimal concentration combination of captured antibody and detected antibody, the detection conditions were optimized by determining the biological detection limit and lower detection limit, drawing the S-shaped curve and judging the linear range, and establishing the standard curve and regression equation. Methodsologically evaluate the accuracy, precision, reportable range and analytical specificity of the detection method. RESULTS: The optimal concentration combinations of CycC trapping-detection antibodies were 26.6 µg/mL-1∶800. The lower limits of detection and biologic limits of detection of the CysC were 0.037 and 0.237 ng/mL, respectively. Regression equation were as followes: y=-3.315x~2+283.04x+160.89, R~2=0.9921. The relative bias of CysC which was detected on the liquid protein microarry was 5.81%. The dilution recovery and recovery were 70.35%-84.91%(n=3)and 79.94%-122.41%(n=3)respectively. The correlation coefficient of method ology comparison experiment was r=0.616, P<0.05, and there was no significant difference between the two method(t=0.948, P=0.358); The within-run precision range from 3.54% to 4.03%(n=10); The between-run precision range from 12.07% to 15.05%(D=5, n=3); The reportable range was 0.26-3784.04 ng/mL. The analysis of interference test result showed that the both concentrations of hemoglobin(160.00, 71.11 g/L) had interference to the result of CysC detected on the chip. CONCLUSION: This study completed the optimization of conditions and methodological evaluation of liquid protein microarray in detecting CysC.

Compartmentalized Linker Array: A Scalable and Transferrable Microarray Format for Multiplexed Immunoassays.

Microarrays have been widely used for multiplexed bioassays. Fabrication of a conventional microarray typically requires a complex microarray spotter, using which nanoliter bioreagent (e.g., antibody and cells) droplets are delivered onto a glass slide. However, arraying a delicate bioreagent in nanoliter volumes could cause the loss of bioactivity and needs a complex microarray spotter. Further, mixing of different bioreagents in a multiplexed assay leads to cross-reactions, producing false positive signals that impair assay reproducibility and scalability. In this work, we propose a new microarray format, named "compartmentalized linker array (CLA)", that consists of pre-prepared storable microarrays of chemical linkers in microliter compartments. CLA can be used for binding and patterning bioreagents into microarrays by simply pipetting and incubating bioreagent solutions in compartments. Using commonly used aminosilane linker-based antibody microarray, we developed CLA and demonstrated its application for a multiplexed sandwich immunoassay measuring three cancer-related proteins. A "two-phase" blocking system was established for de-activating background regions on glass where no linker molecules are present. Storage conditions of the CLA chip were explored and demonstrated for long-term storage. In a multiplexed immunoassay, low pg/mL sensitivity was achieved for all the three proteins, comparable to those of conventional assays. Moreover, CLA can be potentially used for other applications beyond protein assays, making microarray technology transferrable and widely available for the biological and biomedical research community.

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods.

OBJECTIVES: To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. METHODS: Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. RESULTS: The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. CONCLUSIONS: The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Screening of novel serum biomarkers for gastric cancer in coastal populations using a protein microarray.

Gastric cancer (GC) has high rates of morbidity and mortality, and this phenomenon is particularly evident in coastal regions where local dietary habits favor the consumption of pickled foods such as salted fish and vegetables. In addition, the diagnosis rate of GC remains low due to the lack of diagnostic serum biomarkers. Therefore, in this study, we aimed to identify potential serum GC biomarkers for use in clinical practice. To identify candidate biomarkers of GC, 88 serum samples were first screened using a high-throughput protein microarray to measure the levels of 640 proteins. Then, 333 samples were used to validate the potential biomarkers using a custom antibody chip. ELISA, western blot, and immunohistochemistry were then used to verify the expression of the target proteins. Finally, logistic regression was performed to select serum proteins for the diagnostic model. As a result, five specific differentially expressed proteins, TGFß RIII, LAG-3, carboxypeptidase A2, Decorin and ANGPTL3, were found to have the ability to distinguish GC. Logistic regression analysis showed that the combination of carboxypeptidase A2 and TGFß RIII had superior potential for diagnosing GC (area under the ROC curve [AUC] = 0.801). The results suggested that these five proteins alone and the combination of carboxypeptidase A2 and TGFß RIII may be used as serum markers for the diagnosis of GC.

Antibody reliability influences observed mRNA-protein correlations in tumour samples.

Reverse phase protein arrays (RPPA) have been used to quantify the abundance of hundreds of proteins across thousands of tumour samples in the Cancer Genome Atlas. By number of samples, this is the largest tumour proteomic dataset available and it provides an opportunity to systematically assess the correlation between mRNA and protein abundances. However, the RPPA approach is highly dependent on antibody reliability and approximately one-quarter of the antibodies used in the the Cancer Genome Atlas are deemed to be somewhat less reliable. Here, we assess the impact of antibody reliability on observed mRNA-protein correlations. We find that, in general, proteins measured with less reliable antibodies have lower observed mRNA-protein correlations. This is not true of the same proteins when measured using mass spectrometry. Furthermore, in cell lines, we find that when the same protein is quantified by both mass spectrometry and RPPA, the overall correlation between the two measurements is lower for proteins measured with less reliable antibodies. Overall our results reinforce the need for caution in using RPPA measurements from less reliable antibodies.

Chemically cross-linked hydrogels from repetitive protein arrays.

Biomaterials for tissue regeneration must mimic the biophysical properties of the native physiological environment. A protein engineering approach allows the generation of protein hydrogels with specific and customised biophysical properties designed to suit a particular physiological environment. Herein, repetitive engineered proteins were successfully designed to form covalent molecular networks with defined physical characteristics able to sustain cell phenotype. Our hydrogel design was made possible by the incorporation of the SpyTag (ST) peptide and multiple repetitive units of the SpyCatcher (SC) protein that spontaneously formed covalent crosslinks upon mixing. Changing the ratios of the protein building blocks (ST:SC), allowed the viscoelastic properties and gelation speeds of the hydrogels to be altered and controlled. The physical properties of the hydrogels could readily be altered further to suit different environments by tuning the key features in the repetitive protein sequence. The resulting hydrogels were designed with a view to allow cell attachment and encapsulation of liver derived cells. Biocompatibility of the hydrogels was assayed using a HepG2 cell line constitutively expressing GFP. The cells remained viable and continued to express GFP whilst attached or encapsulated within the hydrogel. Our results demonstrate how this genetically encoded approach using repetitive proteins could be applied to bridge engineering biology with nanotechnology creating a level of biomaterial customisation previously inaccessible.

Optically Controlled Construction of Three-Dimensional Protein Arrays.

Protein crystallization is an important tool for structural biology and nanostructure preparation. Here, we report on kinetic pathway-dependent protein crystals that are controlled by light. Photo-responsive crystallites are obtained by complexing the model proteins with cationic azobenzene dyes. The crystalline state is readily switched to a dispersed phase under ultraviolet light and restored by subsequent visible-light illumination. The switching can be reversibly repeated for multiple cycles without noticeable structure deterioration. Importantly, the photo-treatment not only significantly increases the crystallinity, but creates crystallites at conditions where no ordered lattices are observed upon directly mixing the components. Further control over the azobenzene isomerization kinetics produces protein single crystals of up to ≈50 µm. This approach offers an intriguing method to fabricate metamaterials and study optically controlled crystallization.

Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique.

We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 (p < 0.05), and the direct bilirubin (DBIL) of low concentration and the indirect bilirubin (IBIL) of high concentration could not interfere with the detected results of nine indicators. The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.

Reverse Phase Protein Array Profiling Identifies Recurrent Protein Expression Patterns of DNA Damage-Related Proteins across Acute and Chronic Leukemia: Samples from Adults and the Children's Oncology Group.

DNA damage response (DNADR) recognition and repair (DDR) pathways affect carcinogenesis and therapy responsiveness in cancers, including leukemia. We measured protein expression levels of 16 DNADR and DDR proteins using the Reverse Phase Protein Array methodology in acute myeloid (AML) (n = 1310), T-cell acute lymphoblastic leukemia (T-ALL) (n = 361) and chronic lymphocytic leukemia (CLL) (n = 795) cases. Clustering analysis identified five protein expression clusters; three were unique compared to normal CD34+ cells. Individual protein expression differed by disease for 14/16 proteins, with five highest in CLL and nine in T-ALL, and by age in T-ALL and AML (six and eleven proteins, respectively), but not CLL (n = 0). Most (96%) of the CLL cases clustered in one cluster; the other 4% were characterized by higher frequencies of deletion 13q and 17p, and fared poorly (p < 0.001). T-ALL predominated in C1 and AML in C5, but both occurred in all four acute-dominated clusters. Protein clusters showed similar implications for survival and remission duration in pediatric and adult T-ALL and AML populations, with C5 doing best in all. In summary, DNADR and DDR protein expression was abnormal in leukemia and formed recurrent clusters that were shared across the leukemias with shared prognostic implications across diseases, and individual proteins showed age- and disease-related differences.

[Screening of apoptosis-related differential proteins in rheumatoid arthritis patients with cold-dampness syndrome based on protein microarray technology].

Objective Screening the differentially expressed proteins related to apoptosis in cold-dampness syndrome of rheumatoid arthritis (RA). Methods Peripheral blood mononuclear cells (PBMCs) were collected from healthy people and RA patients with cold-dampness syndrome. 43 apoptosis-related proteins were detected by antibody chip, which was verified by ELISA. Results In 43 apoptosis-related proteins, 10 of them were up-regulated and 3 were down-regulated. The most differentially expressed were tumor necrosis factor receptor 5 (CD40) and soluble tumor necrosis factor receptor 2 (sTNFR2). Compared with the normal group, the expression of CD40 and sTNFR2 in RA patients with cold-dampness syndrome increased significantly. The results of receiver operating characteristic curve (ROC) showed that CD40(AUC=0.8133) and sTNFR2(AUC=0.8117) could be used as diagnostic markers of RA patients with cold-dampness syndrome. The results of Spearman correlation analysis showed that CD40 was negatively correlated with Fas and Fas ligand (FasL), while sTNFR2 was positively correlated with erythrocyte sedimentation rate and negatively correlated with mental health score (MH). Logistic regression analysis showed that rheumatoid factor (RF), 28 joint disease activity scores (DAS28) and vitality (VT) were risk factors for CD40. ESR, anti-cyclic citrullinated peptide (CCP) antibody, self-rating depression scale (SAS) and MH were the risk factors of sTNFR2. Conclusion CD40 and sTNFR2 are proteins related to apoptosis in RA patients with cold-dampness syndrome, which are closely related to clinical indexes and apoptosis indexes.