bims-prodis Biomed News
on Proteomics in disease
Issue of 2019–04–28
twenty papers selected by
Nancy Gough, Bioserendipity



  1. Clin Proteomics. 2019 ;16 16
       Background: Blood-based protein measurement is a routine practice for detecting biomarkers in human disease. Comprehensive profiling of blood/plasma/serum proteome is a challenge due to an extremely large dynamic range, as exemplified by a small subset of highly abundant proteins. Antibody-based depletion of these abundant proteins alleviates the problem but introduces experimental variations. We aimed to establish a method for direct profiling of undepleted human serum and apply the method toward biomarker discovery for Alzheimer's disease (AD), as AD is the most common form of dementia without available blood-based biomarkers in clinic.
    Methods: We present an ultra-deep analysis of undepleted human serum proteome by combining the latest 11-plex tandem-mass-tag (TMT) labeling, exhaustive two-dimensional liquid chromatography (LC/LC) fractionation (the 1st LC: 3 h for 180 fractions, and the 2nd LC: 3 h gradient per fraction), coupled with high resolution tandem mass spectrometry (MS/MS). AD (n = 6) and control (n = 5) sera were analyzed in this pilot study. In addition, we implemented a multiplexed targeted LC-MS3 method (TOMAHAQ) for the validation of selected target proteins.
    Results: The TMT-LC/LC-MS/MS platform is capable of analyzing 4826 protein components (4368 genes), covering at least 6 orders of magnitude in dynamic range, representing one of the deepest serum proteome analysis. We defined intra- and inter- group variability in the AD and control groups. Statistical analysis revealed differentially expressed proteins in AD (26 decreased and 4 increased). Notably, these altered proteins are enriched in the known pathways of mitochondria, fatty acid beta oxidation, and AGE/RAGE. Finally, we set up a TOMAHAQ method to confirm the decrease of PCK2 and AK2 in our AD samples.
    Conclusions: Our results show an ultra-deep serum discovery study by TMT-LC/LC-MS/MS, and a validation experiment by TOMAHAQ targeted LC-MS3. The MS-based discovery and validation methods are of general use for biomarker discovery from complex biofluids (e.g. serum proteome). This pilot study also identified deregulated proteins, in particular proteins associated with mitochondrial function in the AD serum samples. These proteins may serve as novel AD candidate biomarkers.
    Keywords:  Alzheimer’s disease; Biomarker; Human blood; Mass spectrometry; Plasma; Proteome; Proteomics; Serum; Tandem mass tag
    DOI:  https://doi.org/10.1186/s12014-019-9237-1
  2. Asian J Androl. 2019 Apr 16.
      Seminal plasma is a rich source of proteins and serves as an ideal sample for proteomic analysis of male infertility. In varicocele-associated infertility, the contributory role of seminal plasma proteins specific to unilateral and bilateral varicocele is not clear. Furthermore, there is a lack of specific protein biomarker to differentiate bilateral from unilateral varicocele. The main objective is to identify the differentially regulated molecular and cellular pathways in bilateral varicocele. Furthermore, we intend to identify seminal plasma biomarkers to differentiate bilateral and unilateral varicocele patients in comparison with fertile healthy men. Global proteomic analysis of seminal plasma proteins has identified the functionality of differentially expressed proteins (DEPs) in varicocele patients. Bioinformatic analysis has revealed response to reactive oxygen species and oxidative stress, and tissue homeostasis as top process pathways that are affected in bilateral varicocele patients compared to fertile healthy men. In comparison with unilateral varicocele patients, inflammatory response pathways were dysregulated, especially interleukin 6 (IL-6) signaling and Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathways, in bilateral varicocele patients, owing to the involvement of underexpressed DEPs. Key DEPs associated with oxidative stress (peroxiredoxin 2; PRDX2), DNA fragmentation (fatty acid synthase; FASN), and inflammatory response (fibronectin 1; FN1) validated by western blot analysis revealed differential expression of these proteins in unilateral and bilateral varicocele groups. Altered expression of DEPs and its association with key processes show that the seminal plasma homeostasis is compromised in bilateral varicocele patients. Furthermore, we propose PRDX2, FASN, and FN1 as potential noninvasive seminal plasma markers for the differentiation of unilateral and bilateral varicocele patients.
    Keywords:  biomarkers; male infertility; proteomics; seminal plasma; varicocele
    DOI:  https://doi.org/10.4103/aja.aja_121_18
  3. Mol Neuropsychiatry. 2019 Mar;5(1): 52-59
      The field of proteomics is rapidly gaining territory as a promising alternative to genomic approaches in the efforts to unravel the complex molecular mechanisms underlying schizophrenia and other psychiatric disorders. X-aptamer tech-nology has emerged as a novel proteomic approach for high-sensitivity analyses, and we hypothesized that this technology would identify unique molecular signatures in plasma samples from schizophrenia patients (n = 60) compared to controls (n = 20). Using a combinatorial library of X-aptamer beads, we developed a two-color flow cytometer-based approach to identify specific X-aptamers that bound with high specificity to each target group. Based on this, we synthesized two unique X-aptamer sequences, and specific proteins pulled down from the patient and control groups by these X-aptamers were identified by mass spectrometry. We identified two protein biomarkers, complement component C4A and ApoB, upregulated in plasma samples from schizophrenia patients. ELISA validation suggested that the observed differences in C4 levels in patients are likely due to the presence of the illness itself, while ApoB may be a marker of antipsychotic-induced alterations. These studies highlight the utility of the X-aptamer technology in the identification of biomarkers for schizophrenia that will advance our understanding of the pathophysiological mechanisms of this disorder.
    Keywords:  ApoB; C4; Proteomics; Schizophrenia; X-aptamer
    DOI:  https://doi.org/10.1159/000492331
  4. Proteomics Clin Appl. 2019 Apr 26. e1800195
      Due to a lack of early diagnostic markers, pancreatic cancer (PC) remains a lethal disease. Proteomic approaches are now being applied to identify novel PC biomarkers. In this study, we used iTRAQ and LC-MS/MS to perform comparative analyses of serum from PC patients and healthy controls (HC), to identify specific serum biomarkers for PC. Serum levels of candidate proteins were determined using ELISA. Among 869 proteins identified, 55 were potential biomarkers; Vitamin K-dependent protein Z (PROZ) and tumor necrosis factor receptor superfamily member 6b (TNFRSF6B) were selected for further analysis. Serum levels of PROZ and TNFRSF6B were significantly higher in PC patients than in HC or benign controls (BC) (P<0.01). The AUCs ranged from 0.816 to 0.971 for PROZ, TNFRSF6B, and carbohydrate antigen 19-9 (CA19-9), either individually or in combination, in PC vs HC+BC, and from 0.711 to 0.932 in PC Stage I vs. HC+BC. We have demonstrated that PROZ and TNFRSF6B are novel serum biomarkers for detecting early stage PC, and for distinguishing PC from pancreatic benign tumor and healthy individuals. Additional large cohort studies are needed to develop PROZ and TNFRSF6B as clinical PC biomarkers. This article is protected by copyright. All rights reserved.
    Keywords:  PROZ; TNFRSF6B; biomarkers; iTRAQ; pancreatic cancer
    DOI:  https://doi.org/10.1002/prca.201800195
  5. Asian J Androl. 2019 Apr 16.
      Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.
    Keywords:  male fertility; proteomics; seminoma; sperm proteins; sperm quality; testicular cancer
    DOI:  https://doi.org/10.4103/aja.aja_17_19
  6. Clin J Am Soc Nephrol. 2019 Apr 24. pii: CJN.12191018. [Epub ahead of print]
       BACKGROUND AND OBJECTIVES: Microvesicles and exosomes are involved in the pathogenesis of autosomal dominant polycystic kidney disease. However, it is unclear whether they also contribute to medullary sponge kidney, a sporadic kidney malformation featuring cysts, nephrocalcinosis, and recurrent kidney stones. We addressed this knowledge gap by comparative proteomic analysis.
    DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The protein content of microvesicles and exosomes isolated from the urine of 15 patients with medullary sponge kidney and 15 patients with autosomal dominant polycystic kidney disease was determined by mass spectrometry followed by weighted gene coexpression network analysis, support vector machine learning, and partial least squares discriminant analysis to compare the profiles and select the most discriminative proteins. The proteomic data were verified by ELISA.
    RESULTS: A total of 2950 proteins were isolated from microvesicles and exosomes, including 1579 (54%) identified in all samples but only 178 (6%) and 88 (3%) specific for medullary sponge kidney microvesicles and exosomes, and 183 (6%) and 98 (3%) specific for autosomal dominant polycystic kidney disease microvesicles and exosomes, respectively. The weighted gene coexpression network analysis revealed ten modules comprising proteins with similar expression profiles. Support vector machine learning and partial least squares discriminant analysis identified 34 proteins that were highly discriminative between the diseases. Among these, CD133 was upregulated in exosomes from autosomal dominant polycystic kidney disease and validated by ELISA.
    CONCLUSIONS: Our data indicate a different proteomic profile of urinary microvesicles and exosomes in patients with medullary sponge kidney compared with patients with autosomal dominant polycystic kidney disease. The urine proteomic profile of patients with autosomal dominant polycystic kidney disease was enriched of proteins involved in cell proliferation and matrix remodeling. Instead, proteins identified in patients with medullary sponge kidney were associated with parenchymal calcium deposition/nephrolithiasis and systemic metabolic derangements associated with stones formation and bone mineralization defects.
    PODCAST: This article contains a podcast at https://www.asn-online.org/media/podcast/CJASN/2019_04_24_CJASNPodcast_19_06_.mp3.
    Keywords:  Calcification, Physiologic; Cell Proliferation; Cell-Derived Microparticles; Cysts; Discriminant Analysis; Enzyme-Linked Immunosorbent Assay; Exosomes; Flow Cytometry; Kidney Calculi; Least-Squares Analysis; Mass Spectrometry; Medullary Sponge Kidney; Nephrocalcinosis; Polycystic Kidney, Autosomal Dominant; Proteomics; Support Vector Machine; autosomal dominant polycystic kidney disease; calcium; proteomics
    DOI:  https://doi.org/10.2215/CJN.12191018
  7. Clin Chim Acta. 2019 Apr 23. pii: S0009-8981(19)31826-1. [Epub ahead of print]
       BACKGROUND: Certain candidate biomarkers for periodontal diseases in saliva, gingival crevicular fluid (GCF), and serum were reported by some previous studies, but little evidence was obtained in their potentiality for screening patients with periodontal diseases.
    METHODS: Unstimulated whole saliva, GCF, and serum samples, which were collected from 17 patients with chronic periodontitis, 17 with gingivitis, and 16 periodontally healthy persons as control, were analysed by MALDI-TOF MS. Cluster analysis and receiver operating characteristic (ROC) curve analysis were carried out to evaluate the ability of candidate peptides to distinguish patients with periodontal diseases from healthy subjects. Nano-LC/ESI-MS/MS was performed to identify possible proteins that these peptides might derive from.
    RESULTS: Most of the differentially expressed peptides exhibited an increase in participants with chronic periodontitis and gingivitis compared with healthy controls. Cluster analysis showed a good clustering capacity between chronic periodontitis and healthy controls. Most AUCs for differentially expressed peptides were >0.7, whereas some peptides from GCF and serum even exhibited AUCs of 0.9-1.0.
    CONCLUSIONS: Some peptides in saliva, GCF, and serum act as biomarkers for chronic periodontitis and gingivitis, which have certain potentiality for screening patients with periodontal diseases and distinguishing them from healthy individuals in a comparatively large population by mass spectrometry.
    Keywords:  Gingivitis; Mass spectrometry; Periodontitis; Salivary biomarkers
    DOI:  https://doi.org/10.1016/j.cca.2019.04.076
  8. Prostate. 2019 Apr 24.
       BACKGROUND: Proteomic profiling of extracellular vesicles (EVs) from prostate cancer (PCa) and normal prostate cell lines, led to the identification of new candidate PCa markers. These proteins included the nuclear exportin proteins XPO1 (also known as CRM1), the EV-associated PDCD6IP (also known as ALIX), and the previously published fatty acid synthase FASN. In this study, we investigated differences in expression of XPO1 and PDCD6IP on well-characterized prostate cancer cohorts using mass spectrometry and tissue microarray (TMA) immunohistochemistry to determine their diagnostic and prognostic value.
    METHODS: Protein fractions from 67 tissue samples (n = 33 normal adjacent prostate [NAP] and n = 34 PCa) were analyzed by mass spectrometry (nano-LC-MS-MS). Label-free quantification of EVs was performed to identify differentially expressed proteins between PCa and NAP. Prognostic evaluation of the candidate markers was performed with a TMA, containing 481 radical prostatectomy samples. Samples were stained for the candidate markers and correlated with patient information and clinicopathological outcome.
    RESULTS: XPO1 was higher expressed in PCa compared to NAP in the MS data analysis (P > 0.0001). PDCD6IP was not significantly higher expressed (P = 0.0501). High cytoplasmic XPO1 staining in the TMA immunohistochemistry, correlated in a multivariable model with high Gleason scores (P = 0.002) and PCa-related death (P = 0.009).
    CONCLUSION: High expression of cytoplasmic XPO1 shows correlation with prostate cancer and has added clinical value in tissue samples. Furthermore, as an extracellular vesicles-associated protein, it might be a novel relevant liquid biomarker.
    Keywords:  PDCD6IP; XPO1; biomarker; extracellular vesicles; prostate cancer; tissue microarray
    DOI:  https://doi.org/10.1002/pros.23813
  9. Inhal Toxicol. 2019 Apr 23. 1-9
      Sulfur mustard (SM) is a mutagenic compound that targets various organs. Although it causes a wide range of abnormalities, cellular and molecular mechanisms of its action are not-well-understood. Oxidation of DNA, proteins, lipids, as well as depletion of cellular nicotinamide adenine dinucleotide (NAD), antioxidants and increase of intracellular calcium are the hypothesized mechanisms of its action at the acute phase of injury. In this review, the proteome analysis of SM toxicity has been considered. We selected articles that considered proteomics analysis of SM toxicity with two-dimensional gel electrophoresis (2DE) followed by mass spectrometry. Our search yielded nine related articles, four original in vitro and five human studies. The results of these studies have revealed a change in expression pattern of various proteins such as haptoglobin, amyloid A1, surfactant proteins, S100 proteins, apolipoprotein, Vit D binding protein, transferrin, alpha 1 antitrypsin, protein disulfide isomerase and antioxidant enzymes in patients who were exposed to SM about 30 years ago. Most of these proteins are up- or down-regulated in response to excessive production of reactive oxygen species (ROS) and oxidative stress (OS). There is a tight link between the expression pattern of these proteins with accumulation of leukocytes, inflammatory conditions, antioxidant depletion, mitochondrial deficiency, as well as increased expression or activity of several proteases such as caspases and matrix metalloproteinases (MMPs). Therefore, excessive production of ROS and OS along with chronic inflammatory may be the long-term toxic effects of SM following acute exposure.
    Keywords:  Sulfur mustard; inflammation; oxidative stress; proteomics; reactive oxygen species
    DOI:  https://doi.org/10.1080/08958378.2018.1558316
  10. Ann Rheum Dis. 2019 Apr 20. pii: annrheumdis-2019-215051. [Epub ahead of print]
       OBJECTIVES: The International League of Associations for Rheumatology classification criteria define systemic juvenile idiopathic arthritis (SJIA) by the presence of fever, rash and chronic arthritis. Recent initiatives to revise current criteria recognise that a lack of arthritis complicates making the diagnosis early, while later a subgroup of patients develops aggressive joint disease. The proposed biphasic model of SJIA also implies a 'window of opportunity' to abrogate the development of chronic arthritis. We aimed to identify novel SJIA biomarkers during different disease phases.
    METHODS: Children with active SJIA were subgrouped clinically as systemic autoinflammatory disease with fever (SJIA syst ) or polyarticular disease (SJIA poly ). A discovery cohort of n=10 patients per SJIA group, plus n=10 with infection, was subjected to unbiased label-free liquid chromatography mass spectrometry (LC-MS/MS) and immunoassay screens. In a separate verification cohort (SJIA syst , n=45; SJIA poly , n=29; infection, n=32), candidate biomarkers were measured by multiple reaction monitoring MS (MRM-MS) and targeted immunoassays.
    RESULTS: Signatures differentiating the two phenotypes of SJIA could be identified. LC-MS/MS in the discovery cohort differentiated SJIA syst from SJIA poly well, but less effectively from infection. Targeted MRM verified the discovery data and, combined with targeted immunoassays, correctly identified 91% (SJIA syst vs SJIA poly ) and 77% (SJIA syst vs infection) of all cases.
    CONCLUSIONS: Molecular signatures differentiating two phenotypes of SJIA were identified suggesting shifts in underlying immunological processes in this biphasic disease. Biomarker signatures separating SJIA in its initial autoinflammatory phase from the main differential diagnosis (ie, infection) could aid early-stage diagnostic decisions, while markers of a phenotype switch could inform treat-to-target strategies.
    Keywords:  autoinflammation; biomarkers; diagnosis; monitoring; phenotype classification; proteomics
    DOI:  https://doi.org/10.1136/annrheumdis-2019-215051
  11. J Proteomics. 2019 Apr 19. pii: S1874-3919(19)30113-7. [Epub ahead of print]
      Crohn's Disease (CD) and Ulcerative Colitis (UC) are chronic inflammatory bowel diseases (IBD) of the gastrointestinal tract. This study used non-invasive LC-MS/MS to find disease specific microbial and human proteins which might be used later for an easier diagnosis. Therefore, 17 healthy controls, 11 CD patients and 14 UC patients but also 13 Irritable Bowel Disease (IBS) patients, 8 Colon Adenoma (CA) patients, and 8 Gastric Carcinoma (GCA) patients were investigated. The proteins were extracted from the fecal samples with liquid phenol in a ball mill. Subsequently, the proteins were digested tryptically to peptides and analyzed by an Orbitrap LC-MS/MS. For protein identification and interpretation of taxonomic and functional results, the MetaProteomeAnalyzer software was used. Cluster analysis and non-parametric test (analysis of similarities) separated healthy controls from patients with CD and UC as well as from patients with GCA. Among others, CD and UC correlated with an increase of neutrophil extracellular traps and immune globulins G (IgG). In addition, a decrease of human IgA and the transcriptional regulatory protein RprY from Bacillus fragilis was found for CD and UC. A specific marker in feces for CD was an increased amount of the human enzyme sucrose-isomaltase. SIGNIFICANCE: Crohn's Disease and Ulcerative Colitis are chronic inflammatory diseases of the gastrointestinal tract, whose diagnosis required comprehensive medical examinations including colonoscopy. The impact of the microbial communities in the gut on the pathogenesis of these diseases is poorly understood. Therefore, this study investigated the impact of gut microbiome on these diseases by a metaproteome approach, revealing several disease specific marker proteins. Overall, this indicated that fecal metaproteomics has the potential to be useful as non-invasive tool for a better and easier diagnosis of both diseases.
    Keywords:  Crohn's Disease; Fecal samples; Gastrointestinal tract; Inflammatory Bowel Disease; Metaproteomics; Non-invasive diagnosis; Ulcerative Colitis
    DOI:  https://doi.org/10.1016/j.jprot.2019.04.009
  12. Front Physiol. 2019 ;10 379
      Although insulin resistance (IR) is a key pathophysiologic condition underlying various metabolic disorders, impaired cellular glucose uptake is one of many manifestations of metabolic derangements in the human body. To study the systems-wide molecular changes associated with obesity-dependent IR, we integrated information on plasma proteins and microRNAs in eight obese insulin-resistant (OIR, HOMA-IR > 2.5) and nine lean insulin-sensitive (LIS, HOMA-IR < 1.0) normoglycemic males. Of 374 circulating miRNAs we profiled, 65 species increased and 73 species decreased in the OIR compared to the LIS subjects, suggesting that the overall balance of the miRNA secretome is shifted in the OIR subjects. We also observed that 40 plasma proteins increased and 4 plasma proteins decreased in the OIR subjects compared to the LIS subjects, and most proteins are involved in metabolic and endocytic functions. We used an integrative -omics analysis framework called iOmicsPASS to link differentially regulated miRNAs with their target genes on the TargetScan map and the human protein interactome. Combined with tissue of origin information, the integrative analysis allowed us to nominate obesity-dependent and obesity-independent protein markers, along with potential sites of post-transcriptional regulation by some of the miRNAs. We also observed the changes in each -omics platform that are not linked by the TargetScan map, suggesting that proteins and microRNAs provide orthogonal information for the progression of OIR. In summary, our integrative analysis provides a network of elevated plasma markers of OIR and a global shift of microRNA secretome composition in the blood plasma.
    Keywords:  insulin resistance; microRNAs; network analysis; obesity; proteomics
    DOI:  https://doi.org/10.3389/fphys.2019.00379
  13. J Am Acad Dermatol. 2019 Apr 19. pii: S0190-9622(19)30628-0. [Epub ahead of print]
       BACKGROUND: Despite increasing evidence that adults with longstanding atopic dermatitis (AD) have systemic inflammation, little is known about systemic inflammation in recent-onset early pediatric AD.
    OBJECTIVE: To analyze blood inflammatory proteins of early pediatric AD.
    METHODS: Using high-throughput proteomics (proximity extension assay), we assessed 257 inflammatory and cardiovascular risk proteins in blood of 30 children with moderate-to-severe AD <5 years of age (within 6 months of onset), compared to age-matched pediatric controls and adult AD.
    RESULTS: In pediatric AD blood, Th2 (CCL13, CCL22) and Th17 (PI3/elafin) markers were increased, together with markers of tissue remodelling (MMP3/9/10, uPAR), endothelial activation (E-Selectin), T-cell activation (IL2RA), neutrophil activation (myeloperoxidase), lipid metabolism (FABP4), and growth factors (FGF21, TGF-alpha). Total numbers of dysregulated proteins were smaller (n=22) than in adult AD (n=61). Clinical severity scores were positively correlated with receptors for IL-33 and IL-36, and inversely correlated with some Th1 markers (IFN-γ, CXCL11).
    LIMITATIONS: Different baseline expression levels in healthy pediatric vs. adult samples.
    CONCLUSIONS: Within months of pediatric AD onset, systemic immune activation is present, with Th2/Th17 skewing but otherwise different proteomic patterns from adult AD. Future correlation of proteomic patterns with disease course, comorbidity development, and drug response may yield predictive biomarkers.
    Keywords:  Atopic dermatitis; OLINK; infant; multiplex assay; pediatric; peripheral blood; proximity extension assay
    DOI:  https://doi.org/10.1016/j.jaad.2019.04.036
  14. Clin Proteomics. 2019 ;16 15
       Background: Prostate cancer (PCa) is the most frequently diagnosed non-skin cancer and a leading cause of mortality among males in developed countries. However, our understanding of the global changes of protein complexes within PCa tissue specimens remains very limited, although it has been well recognized that protein complexes carry out essentially all major processes in living organisms and that their deregulation drives the pathogenesis and progression of various diseases.
    Methods: By coupling tandem mass tagging-synchronous precursor selection-mass spectrometry/mass spectrometry/mass spectrometry with differential expression and co-regulation analyses, the present study compared the differences between protein complexes in normal prostate, low-grade PCa, and high-grade PCa tissue specimens.
    Results: Globally, a large downregulated putative protein-protein interaction (PPI) network was detected in both low-grade and high-grade PCa, yet a large upregulated putative PPI network was only detected in high-grade but not low-grade PCa, compared with normal controls. To identify specific protein complexes that are deregulated in PCa, quantified proteins were mapped to protein complexes in CORUM (v3.0), a high-quality collection of 4274 experimentally verified mammalian protein complexes. Differential expression and gene ontology (GO) enrichment analyses suggested that 13 integrin complexes involved in cell adhesion were significantly downregulated in both low- and high-grade PCa compared with normal prostate, and that four Prothymosin alpha (ProTα) complexes were significantly upregulated in high-grade PCa compared with normal prostate. Moreover, differential co-regulation and GO enrichment analyses indicated that the assembly levels of six protein complexes involved in RNA splicing were significantly increased in low-grade PCa, and those of four subcomplexes of mitochondrial complex I were significantly increased in high-grade PCa, compared with normal prostate.
    Conclusions: In summary, to the best of our knowledge, the study represents the first large-scale and quantitative, albeit indirect, comparison of individual protein complexes in human PCa tissue specimens. It may serve as a useful resource for better understanding the deregulation of protein complexes in primary PCa.
    Keywords:  Differential co-regulation analysis; Differential expression analysis; Prostate cancer; Protein complex; Quantitative proteomics; TMT-SPS-MS3; Tissue
    DOI:  https://doi.org/10.1186/s12014-019-9236-2
  15. Ann Clin Transl Neurol. 2019 Apr;6(4): 698-707
       Objective: To identify novel CSF biomarkers in GRN-associated frontotemporal dementia (FTD) by proteomics using mass spectrometry (MS).
    Methods: Unbiased MS was applied to CSF samples from 19 presymptomatic and 9 symptomatic GRN mutation carriers and 24 noncarriers. Protein abundances were compared between these groups. Proteins were then selected for validation if identified by ≥4 peptides and if fold change was ≤0.5 or ≥2.0. Validation and absolute quantification by parallel reaction monitoring (PRM), a high-resolution targeted MS method, was performed on an international cohort (n = 210) of presymptomatic and symptomatic GRN, C9orf72 and MAPT mutation carriers.
    Results: Unbiased MS revealed 20 differentially abundant proteins between symptomatic mutation carriers and noncarriers and nine between symptomatic and presymptomatic carriers. Seven of these proteins fulfilled our criteria for validation. PRM analyses revealed that symptomatic GRN mutation carriers had significantly lower levels of neuronal pentraxin receptor (NPTXR), receptor-type tyrosine-protein phosphatase N2 (PTPRN2), neurosecretory protein VGF, chromogranin-A (CHGA), and V-set and transmembrane domain-containing protein 2B (VSTM2B) than presymptomatic carriers and noncarriers. Symptomatic C9orf72 mutation carriers had lower levels of NPTXR, PTPRN2, CHGA, and VSTM2B than noncarriers, while symptomatic MAPT mutation carriers had lower levels of NPTXR and CHGA than noncarriers.
    Interpretation: We identified and validated five novel CSF biomarkers in GRN-associated FTD. Our results show that synaptic, secretory vesicle, and inflammatory proteins are dysregulated in the symptomatic stage and may provide new insights into the pathophysiology of genetic FTD. Further validation is needed to investigate their clinical applicability as diagnostic or monitoring biomarkers.
    DOI:  https://doi.org/10.1002/acn3.745
  16. Methods Mol Biol. 2019 ;1981 163-173
      Primary biliary cholangitis is a chronic cholestatic liver disease characterized by the presence of serum antimitochondrial antibodies and immune-mediated destruction of the small and medium-sized intrahepatic bile ducts. However, the pathophysiology of primary biliary cholangitis has not yet been completely elucidated. In recent years, proteomics has been comprehensively applied in many research fields, including the pathogenesis, prognosis, and diagnosis of disease. Among multiple methods, isobaric tag for relative and absolute quantitation is a powerful analytic method to characterize complex protein mixtures in combination with liquid chromatography-tandem mass spectrometry. In this chapter, we describe a strategy for using isobaric tag for relative and absolute quantitation to discover those differentially expressed proteins in primary biliary cholangitis. The goal is to identify the differences in protein expression between patients with primary biliary cholangitis and healthy controls for defining biomarkers and elucidating molecular mechanisms underlying disease states.
    Keywords:  Bioinformatics; Differentially expressed proteins; Full-scan MS spectra; High pH reverse phase liquid chromatography (RHPLC); Isobaric tag for relative and absolute quantitation (iTRAQ); Liquid chromatography-tandem mass spectrometry (LC-MS/MS); Plasma; Primary biliary cholangitis; Proteomics
    DOI:  https://doi.org/10.1007/978-1-4939-9420-5_11
  17. Front Immunol. 2019 ;10 540
      Immune responses to citrullinated peptides have been described in autoimmune diseases like rheumatoid arthritis (RA) and multiple sclerosis (MS). We investigated the post-translational modification (PTM), arginine to citrulline, in brain tissue of MS patients and controls (C) by proteomics and subsequently the cellular immune response of cerebrospinal fluid (CSF)-infiltrating T cells to citrullinated and unmodified peptides of myelin basic protein (MBP). Using specifically adapted tissue extraction- and combined data interpretation protocols we could establish a map of citrullinated proteins by identifying more than 80 proteins with two or more citrullinated peptides in human brain tissue. We report many of them for the first time. For the already described citrullinated proteins MBP, GFAP, and vimentin, we could identify additional citrullinated sites. The number of modified proteins in MS white matter was higher than control tissue. Citrullinated peptides are considered neoepitopes that may trigger autoreactivity. We used newly identified epitopes and previously reported immunodominant myelin peptides in their citrullinated and non-citrullinated form to address the recognition of CSF-infiltrating CD4+ T cells from 22 MS patients by measuring proliferation and IFN-γ secretion. We did not detect marked responses to citrullinated peptides, but slightly more strongly to the non-modified version. Based on these data, we conclude that citrullination does not appear to be an important activating factor of a T cell response, but could be the consequence of an immune- or inflammatory response. Our approach allowed us to perform a deep proteome analysis and opens new technical possibilities to analyze complex PTM patterns on minute quantities of rare tissue samples.
    Keywords:  T cell reactivity; autoimmune; citrullination; human brain; multiple sclerosis; proteomics
    DOI:  https://doi.org/10.3389/fimmu.2019.00540
  18. Int J Mol Sci. 2019 Apr 19. pii: E1932. [Epub ahead of print]20(8):
      Sjögren syndrome (SS) or dry eye disease (DED) is one of the most complicated ocular surface diseases. The goal of this study is to elucidate the relationship of the changes in clinical indices of tear film (TF) homeostasis with respect to tear components to allow for SS-DED monitoring and avoid stably controlled SS-DED patients from re-entering a vicious cycle. This prospective case-control study compared stable SS-DED patients with non-SS-DED control from several aspects, including clinical indices for TF homeostasis, 2 DED diagnostic biomarkers (MMP-9 and lactoferrin), and the proteome of flush tears. Compared with non-SS-DED controls, stably controlled SS-DED subjects had less tear secretion and higher ocular surface inflammation, a higher concentration ratio of tear MMP-9/lactoferrin, a more diverse tear proteome, and lower spectral intensities of lipocalin-1, lacritin, and prolactin-inducible protein among the abundant tear proteins. For stable SS-DED patients, the concentration ratio of tear MMP-9/lactoferrin and the corrected lipocalin-1 signal was positively correlated with ocular inflammation and TF stability, respectively. MMP-9 released from stressed ocular surface epithelium and lipocalin-1 secreted from the energetic lacrimal gland are two tear biomarkers responding well to TF homeostasis. The tear proteomics approach through flush tears is a promising method for monitoring SS-DED patients with a standardized sampling procedure and lactoferrin-corrected analysis.
    Keywords:  Sjögren syndrome; biomarkers; dry eye disease; homeostasis; tears
    DOI:  https://doi.org/10.3390/ijms20081932
  19. Klin Onkol. 2018 ;31(Supplementum 2): 102-107
       BACKGROUND: Although immune responses to &#8220;cancer neoantigens&#8221; have been known for decades, the first neoantigen vaccines emerged only very recently. Current developments in genomics and proteomics have enabled descriptions of tumor mutational landscapes, and the immunogenicity of corresponding neoantigens can now be predicted either in silico or in vitro. Cancer regression could be achieved via a combination of neoantigen vaccination and an appropriate immunology approach. Research in model organisms and the results of initial clinical trials of neoantigen vaccines have shown them to be effective.
    PURPOSE: We aim to emphasize the importance of neoantigen vaccines in personalized cancer treatment and describe their preparation. We summarize mutations leading to expression of an immunogenic antigen necessary for vaccine development. The processes leading to activation of T-cell anticancer immunity in a patient are briefly introduced. We especially focus on the identification of high confidence neoantigens by next-generation sequencing (NGS) and mass spectrometry (MS), which is key element in the process of designing neoantigen vaccines. Briefly, we describe a proteogenomic platform for confident identification of mutant peptides in biological material. We mention the possibility of neoantigen quantification in biological material using mass spectrometry such as SRM (selected reaction monitoring) and SWATH (sequential windowed acquisition of all theoretical fragment ion spectra). Successful clinical studies demonstrating the potential of neoantigen vaccination in personalized cancer treatment are summarized at the end of the paper. Key words: vaccination - mass spectrometry - neoplasms - organ-specific neoantigen This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 9. 7. 2018.
    Keywords:  or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Accepted: 9. 7. 2018; products; vaccination - mass spectrometry - neoplasms - organ-specific neoantigen This work was supported by the project MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs
    DOI:  https://doi.org/10.14735/amko20182S102
  20. Int J Mol Sci. 2019 Apr 25. pii: E2038. [Epub ahead of print]20(8):
      Preeclampsia (PE) has been associated with placental dysfunction, resulting in fetal hypoxia, accelerated erythropoiesis, and increased erythroblast count in the umbilical cord blood (UCB). Although the detailed effects remain unknown, placental dysfunction can also cause inflammation, nutritional, and oxidative stress in the fetus that can affect erythropoiesis. Here, we compared the expression of surface adhesion molecules and the erythroid differentiation capacity of UCB hematopoietic stem/progenitor cells (HSPCs), UCB erythroid profiles along with the transcriptome and proteome of these cells between male and female fetuses from PE and normotensive pregnancies. While no significant differences were observed in UCB HSPC migration/homing and in vitro erythroid colony differentiation, the UCB HSPC transcriptome and the proteomic profile of the in vitro differentiated erythroid cells differed between PE vs. normotensive samples. Accordingly, despite the absence of significant differences in the UCB erythroid populations in male or female fetuses from PE or normotensive pregnancies, transcriptional changes were observed during erythropoiesis, particularly affecting male fetuses. Pathway analysis suggested deregulation in the mammalian target of rapamycin complex 1/AMP-activated protein kinase (mTORC1/AMPK) signaling pathways controlling cell cycle, differentiation, and protein synthesis. These results associate PE with transcriptional and proteomic changes in fetal HSPCs and erythroid cells that may underlie the higher erythroblast count in the UCB in PE.
    Keywords:  erythropoiesis; hematopoietic stem/progenitor cells; preeclampsia; umbilical cord blood
    DOI:  https://doi.org/10.3390/ijms20082038