bims-prodis 2019-03-31 papers

bims-prodis

Biomed News

on Proteomics in disease

Issue of 2019–03–31
29 papers selected by
Nancy Gough, Bioserendipity

Postoperative recurrence of epithelial ovarian cancer patients and chemoresistance related protein analyses.
Proteomic alterations of HDL in youth with type 1 diabetes and their associations with glycemic control: a case-control study.
First-trimester proteomic profiling identifies novel predictors of gestational diabetes mellitus.
A Novel Proteomics Approach to Identify Serum and Urinary Biomarkers and Pathways that Associate with Lower Urinary Tract Symptoms in Men and Women: Pilot Results of the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) Study.
Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.
Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment.
Molecular insights into cancer drug resistance from a proteomics perspective.
A quantitative global proteomics approach to understanding the functional pathways dysregulated in the spermatozoa of asthenozoospermic testicular cancer patients.
Cytokines and Chemokines in Pediatric Appendicitis: A Multiplex Analysis of Inflammatory Protein Mediators.
Identification and Verification of Two Novel Differentially Expressed Proteins from Non-neoplastic Mucosa and Colorectal Carcinoma Via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.
Comparative proteomic analysis in serum of former uranium miners with and without radon induced squamous lung cancer.
Proteomic and bioinformatic analysis of condyloma acuminata: mild hyperthermia treatment reveals compromised HPV infectivity of keratinocytes via regulation of metabolism, differentiation and anti-viral responses.
Identification of Urinary CD44 and Prosaposin as Specific Biomarkers of Urinary Tract Infections in Children With Neurogenic Bladders.
Identification of diagnostic upper gastrointestinal cancer tissue type-specific urinary biomarkers.
Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data.
A proteomic signature for dementia with Lewy bodies.
Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.
Quantitative proteomics suggest a potential link between early embryonic death and trisomy 16.
Combined Transcriptomics and Proteomics in Frontal Cortex Area 8 in Frontotemporal Lobar Degeneration Linked to C9ORF72 Expansion.
Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.
Distinct Antibody Signatures Associated with Different Malaria Transmission Intensities in Zambia and Zimbabwe.
Pro-inflammatory, IL-17 pathways dominate the architecture of the immunome in pediatric refractory epilepsy.
MS1 ion current-based quantitative proteomics: A promising solution for reliable analysis of large biological cohorts.
Validation of a multiplexed LC-MS/MS clinical assay to quantify insulin growth factor binding proteins in human serum and its application in a clinical study.
Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry.
Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.
Proteomic test for anti-PD-1 checkpoint blockade treatment of metastatic melanoma with and without BRAF mutations.
Advances in prognostic markers for colorectal cancer.
Lentiviral gene therapy corrects platelet phenotype and function in Wiskott-Aldrich patients.

J Ovarian Res. 2019 Mar 27. 12(1): 29

Postoperative recurrence of epithelial ovarian cancer patients and chemoresistance related protein analyses.

Zhengmao Zhang, Kaiyun Qin, Wenzhe Zhang, Botao Yang, Chen Zhao, Xiaojing Zhang, Fenghua Zhang, Lianmei Zhao, Baoen Shan.

   OBJECTIVE: To identify the plasma protein biomarkers related to the chemoresistance of postoperative recurrence of epithelial ovarian cancers.
METHODS: Forty plasma samples from patients in chemotherapy-sensitive and chemotherapy-resistant groups (20 for each group) were collected at Gynecology Department in the Fourth Hospital of Hebei Medical University from September 2013 to September 2014. The differentially expressed proteins between two groups were screened with two-dimensional gel electrophoresis (2-DE) and further analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).
RESULTS: Thirty-four differentially expressed spots were identified between the two groups. Compared with the chemo-sensitive group, 21 protein spots were up-regulated and 13 were down-regulated in the chemoresistant group, in which 14 differentially expressed proteins were identified by the Mass spectrometry and Mascot search. Among the 14 proteins, complement C4-A, IgJ chain, clusterin, α-1-antitrypsin and carbonic anhydrase 1 were up-regulated, and transthyretin, haptoglobin, β-2-glycoprotein, Ig γ-2 chain C region, Ig γ-1 chain C region, complement factor I light chain, Igκ chain C region, complement C3 and apolipoprotein E were down-regulated in the chemoresistant group when compared with the chemosensitive group.
CONCLUSION: The up-regulated proteins including transthyretin, apolipoprotein E and haptoglobin proteins and the down-regulated proteins such as clusterin, carbonic anhydrase 1, alpha-1-antitrypsin were differentially expressed in the plasma between the chemo-sensitive group and the chemoresistant group, which may be potential biomarkers for predicting the chemotresistance of epithelial ovarian cancer patients.

Keywords:  Chemotherapy resistance; Differentially expressed proteins; Epithelial ovarian cancer; Proteomics; Time of flight mass spectrometry; Two dimensional gelelectrophoresis

DOI:  https://doi.org/10.1186/s13048-019-0499-z
Cardiovasc Diabetol. 2019 Mar 28. 18(1): 43

Proteomic alterations of HDL in youth with type 1 diabetes and their associations with glycemic control: a case-control study.

Evgenia Gourgari, Junfeng Ma, Martin P Playford, Nehal N Mehta, Radoslav Goldman, Alan T Remaley, Scott M Gordon.

   BACKGROUND: Patients with type 1 diabetes (T1DM) typically have normal or even elevated plasma high density lipoprotein (HDL) cholesterol concentrations; however, HDL protein composition can be altered without a change in cholesterol content. Alteration of the HDL proteome can result in dysfunctional HDL particles with reduced ability to protect against cardiovascular disease (CVD). The objective of this study was to compare the HDL proteomes of youth with T1DM and healthy controls (HC) and to evaluate the influence of glycemic control on HDL protein composition.
METHODS: This was a cross-sectional case-control study. Blood samples were obtained from patients with T1DM and HC. HDL was isolated from plasma by size-exclusion chromatography and further purified using a lipid binding resin. The HDL proteome was analyzed by mass spectrometry using label-free SWATH peptide quantification.
RESULTS: Samples from 26 patients with T1DM and 13 HC were analyzed and 78 HDL-bound proteins were measured. Youth with T1DM had significantly increased amounts of complement factor H related protein 2 (FHR2; adjusted P < 0.05), compared to HC. When patients were analyzed based on glucose control, several trends emerged. Some proteins were altered in T1DM and not influenced by glycemic control (e.g. FHR2) while others were partially or completely corrected with optimal glucose control (e.g. alpha-1-beta glycoprotein, A1BG). In a subgroup of poorly controlled T1DM patients, inter alpha trypsin inhibitor 4 (ITIH4) was dramatically elevated (P < 0.0001) and this was partially reversed in patients with optimal glucose control. Some proteins including complement component C3 (CO3) and albumin (ALB) were significantly different only in T1DM patients with optimal glucose control, suggesting a possible effect of exogenous insulin.
CONCLUSIONS: Youth with T1DM have proteomic alterations of their HDL compared to HC, despite similar concentration of HDL cholesterol. The influence of these compositional changes on HDL function are not yet known. Future efforts should focus on investigating the role of these HDL associated proteins in regard to HDL function and their role in CVD risk in patients with T1DM. Trial registration NCT02275091.

Keywords:  A1BG; Cardiovascular; HDL; ITIH4; Proteomics; Type 1 diabetes

DOI:  https://doi.org/10.1186/s12933-019-0846-9
PLoS One. 2019 ;14(3): e0214457

First-trimester proteomic profiling identifies novel predictors of gestational diabetes mellitus.

Tina Ravnsborg, Sarah Svaneklink, Lise Lotte T Andersen, Martin R Larsen, Dorte M Jensen, Martin Overgaard.

BACKGROUND: Gestational diabetes mellitus (GDM) is a common pregnancy complication associated with adverse outcomes including preeclampsia, caesarean section, macrosomia, neonatal morbidity and future development of type 2 diabetes in both mother and child. Current selective screening strategies rely on clinical risk factors such as age, family history of diabetes, macrosomia or GDM in a previous pregnancy, and they possess a relatively low specificity. Here we hypothesize that novel first trimester protein predictors of GDM can contribute to the current selective screening strategies for early and accurate prediction of GDM, thus allowing for timely interventions.
METHODS: A proteomics discovery approach was applied to first trimester sera from obese (BMI ≥27 kg/m2) women (n = 60) in a nested case-control study design, utilizing tandem mass tag labelling and tandem mass spectrometry. A subset of the identified protein markers was further validated in a second set of serum samples (n = 210) and evaluated for their contribution as predictors of GDM in relation to the maternal risk factors, by use of logistic regression and receiver operating characteristic analysis.
RESULTS: Serum proteomic profiling identified 25 proteins with significantly different levels between cases and controls. Three proteins; afamin, serum amyloid P-component and vitronectin could be further confirmed as predictors of GDM in a validation set. Vitronectin was shown to contribute significantly to the predictive power of the maternal risk factors, indicating it as a novel independent predictor of GDM.
CONCLUSIONS: Current selective screening strategies can potentially be improved by addition of protein predictors.

DOI: https://doi.org/10.1371/journal.pone.0214457
Urology. 2019 Mar 25. pii: S0090-4295(19)30307-3. [Epub ahead of print]

A Novel Proteomics Approach to Identify Serum and Urinary Biomarkers and Pathways that Associate with Lower Urinary Tract Symptoms in Men and Women: Pilot Results of the Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) Study.

LURN Study Group

   OBJECTIVES: To assess the feasibility of a novel proteomics approach to identify biomarkers associated with LUTS within serum and urine, because many clinical factors contribute to lower urinary tract symptoms (LUTS) in men and women. These factors confound clinicians' abilities to reliably evaluate and treat LUTS. Previous studies identified candidate LUTS biomarkers, but none are clinically utilized.
METHODS: Eighteen male and 18 female Symptoms of Lower Urinary Tract Dysfunction Research Network (LURN) Observational Cohort Study participants with LUTS (measured on the LUTS Tool questionnaire) were randomly selected. Twelve male and 12 female controls with minimal or no LUTS were recruited and matched for clinico-demographic characteristics. The SomaScan Assay (SomaLogic) was used to measure the abundance of 1305 proteins contained within urine and serum. Statistical analyses were performed to evaluate reproducibility of assays, compare protein abundances, and estimate effect size.
RESULTS: SomaScan assay results were more reproducible in serum than in urine. Within serum, there were many more differentially abundant proteins between cases and controls in males than in females. An enrichment/pathway analysis of the affected proteins in male and female subjects demonstrated that the enriched Gene Ontology processes were related to prostate morphogenesis in men and growth and inflammation in women.
CONCLUSIONS: The pilot study results support that the etiology and pathophysiologic mechanisms underlying LUTS may be sex-specific. While further studies involving larger numbers of subjects are warranted, our results support the feasibility of a novel proteomic approach to identify biomarkers for diagnostic classification of LUTS.

Keywords:  BPH; Lower urinary tract symptoms; biomarkers; inflammation; proteomics

DOI:  https://doi.org/10.1016/j.urology.2019.03.014
Anal Chem. 2019 Mar 29.

Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

David J Clark, Yingwei Hu, Michael Schnaubelt, Yi Fu, Sean Ponce, Shao-Yung Chen, Yangying Zhou, Punit Shah, Hui Zhang.

Mass-spectrometry based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder, however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-Tip method, comprising a simple, automated strategy utilizing reversed-phase resin tip-based format and "on-bead" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-Tip using the standard protein bovine fetuin. Next, we applied the C4-Tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS, with identified proteins from the C4-Tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-Tip method, revealing a high Spearman correlation R value for shared proteins identified across all tips. Together, we have shown the C4-Tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.

DOI: https://doi.org/10.1021/acs.analchem.8b05234
PLoS One. 2019 ;14(3): e0214198

Phenotypic subgrouping and multi-omics analyses reveal reduced diazepam-binding inhibitor (DBI) protein levels in autism spectrum disorder with severe language impairment.

Chatravee Pichitpunpong, Surangrat Thongkorn, Songphon Kanlayaprasit, Wasana Yuwattana, Waluga Plaingam, Siriporn Sangsuthum, Wan Mohd Aizat, Syarul Nataqain Baharum, Tewin Tencomnao, Valerie Wailin Hu, Tewarit Sarachana.

BACKGROUND: The mechanisms underlying autism spectrum disorder (ASD) remain unclear, and clinical biomarkers are not yet available for ASD. Differences in dysregulated proteins in ASD have shown little reproducibility, which is partly due to ASD heterogeneity. Recent studies have demonstrated that subgrouping ASD cases based on clinical phenotypes is useful for identifying candidate genes that are dysregulated in ASD subgroups. However, this strategy has not been employed in proteome profiling analyses to identify ASD biomarker proteins for specific subgroups.
METHODS: We therefore conducted a cluster analysis of the Autism Diagnostic Interview-Revised (ADI-R) scores from 85 individuals with ASD to predict subgroups and subsequently identified dysregulated genes by reanalyzing the transcriptome profiles of individuals with ASD and unaffected individuals. Proteome profiling of lymphoblastoid cell lines from these individuals was performed via 2D-gel electrophoresis, and then mass spectrometry. Disrupted proteins were identified and compared to the dysregulated transcripts and reported dysregulated proteins from previous proteome studies. Biological functions were predicted using the Ingenuity Pathway Analysis (IPA) program. Selected proteins were also analyzed by Western blotting.
RESULTS: The cluster analysis of ADI-R data revealed four ASD subgroups, including ASD with severe language impairment, and transcriptome profiling identified dysregulated genes in each subgroup. Screening via proteome analysis revealed 82 altered proteins in the ASD subgroup with severe language impairment. Eighteen of these proteins were further identified by nano-LC-MS/MS. Among these proteins, fourteen were predicted by IPA to be associated with neurological functions and inflammation. Among these proteins, diazepam-binding inhibitor (DBI) protein was confirmed by Western blot analysis to be expressed at significantly decreased levels in the ASD subgroup with severe language impairment, and the DBI expression levels were correlated with the scores of several ADI-R items.
CONCLUSIONS: By subgrouping individuals with ASD based on clinical phenotypes, and then performing an integrated transcriptome-proteome analysis, we identified DBI as a novel candidate protein for ASD with severe language impairment. The mechanisms of this protein and its potential use as an ASD biomarker warrant further study.

DOI: https://doi.org/10.1371/journal.pone.0214198
Expert Rev Proteomics. 2019 Mar 29.

Molecular insights into cancer drug resistance from a proteomics perspective.

Yao An, Li Zhou, Zhao Huang, Edouard C Nice, Haiyuan Zhang, Canhua Huang.

   INTRODUCTION: Resistance to chemotherapy and development of specific and effective molecular targeted therapies are major obstacles facing current cancer treatment. Comparative proteomic approaches have been employed for the discovery of putative biomarkers associated with cancer drug resistance and have yielded a number of candidate proteins, showing great promise for both novel drug target identification and personalized medicine for the treatment of drug-resistant cancer. Areas covered: Herein, we review the recent advances and challenges in proteomics studies on cancer drug resistance with an emphasis on biomarker discovery, as well as understanding the interconnectivity of proteins in disease-related signaling pathways. In addition, we highlight the critical role that post-translational modifications (PTMs) play in the mechanisms of cancer drug resistance. Expert opinion: Revealing changes in proteome profiles and the role of PTMs in drug-resistant cancer is key to deciphering the mechanisms of treatment resistance. With the development of sensitive and specific mass spectrometry (MS)-based proteomics and related technologies, it is now possible to investigate in depth potential biomarkers and the molecular mechanisms of cancer drug resistance, assisting the development of individualized therapeutic strategies for cancer patients.

Keywords:  Biomarker discovery; Cancer drug resistance; Comparative proteomics; CyTOF; Mass spectrometry; PTMs; Personalized medicine

DOI:  https://doi.org/10.1080/14789450.2019.1601561
Andrology. 2019 Mar 29.

A quantitative global proteomics approach to understanding the functional pathways dysregulated in the spermatozoa of asthenozoospermic testicular cancer patients.

M K Panner Selvam, A Agarwal, P N Pushparaj.

   BACKGROUND: Testicular cancer (TC) is the most common cancer diagnosed in men of reproductive age group. Sperm banking is recommended in these patients prior to cancer treatment. There is no literature describing the proteins dysregulated in the spermatozoa of TC patients with poor motility.
OBJECTIVE: The primary objective of this study was to compare the differences in the sperm proteome of normozoospermic (motility > 40%) and asthenozoospermic (motility < 40%) TC patients who had cryopreserved semen samples before initiating cancer therapy.
MATERIALS AND METHODS: Pooled sperm samples from healthy fertile men (n = 8), normozoospermic (n = 20), and asthenozoospermic (n = 11) TC patients were used for quantitative global proteomic profiling by liquid chromatography-tandem mass spectrometry (LC-MS). The functional bioinformatic analysis was done by ingenuity pathway analysis software. Key differentially expressed proteins (DEPs) associated with binding of zona pellucida (CCT3), mitochondrial dysfunction (ATP5A1 and UQCRC2), sperm motility (ATP1A4), and an exosomal protein involved in the metabolic process of the spermatozoa (MMP9) were validated using Western blot analysis by comparing normozoospermic (n = 10) with asthenozoospermic (n = 10) TC patients. Statistical analysis was conducted using Mann-Whitney test.
RESULTS: A total of 813 and 957 proteins were detected in spermatozoa of normozoospermic and asthenozoospermic TC patients, respectively. On the other hand, 1139 proteins were detected in the spermatozoa of healthy fertile men. 198 proteins were identified as DEPs between TC patients with normal and abnormal semen parameters. Validation of DEPs revealed downregulation of key proteins (CCT3, ATP1A4, ATP5A1, and UQCRC2) implicated in the reproductive function in asthenozoospermic TC patients.
DISCUSSION: DEPs involved in the reproductive function pathways suggest defective spermatogenesis and maturation process in asthenozoospermic TC patients. Furthermore, dysfunctional exosomal pathway may be a possible cause of infertility in TC patients.
CONCLUSION: The proteins associated with sperm function and fertilization process are compromised in TC patients irrespective of their semen parameters.

Keywords:  asthenozoospermic; normozoospermic; proteomics; spermatozoa; testicular cancer

DOI:  https://doi.org/10.1111/andr.12620
Mediators Inflamm. 2019 ;2019 2359681

Cytokines and Chemokines in Pediatric Appendicitis: A Multiplex Analysis of Inflammatory Protein Mediators.

S Ali Naqvi, Graham C Thompson, Ari R Joffe, Jaime Blackwood, Dori-Ann Martin, Mary Brindle, Herman W Barkema, Craig N Jenne.

Objectives: We aimed to demonstrate the potential of precision medicine to describe the inflammatory landscape present in children with suspected appendicitis. Our primary objective was to determine levels of seven inflammatory protein mediators previously associated with intra-abdominal inflammation (C-reactive protein-CRP, procalcitonin-PCT, interleukin-6 (IL), IL-8, IL-10, monocyte chemoattractant protein-1-MCP-1, and serum amyloid A-SAA) in a cohort of children with suspected appendicitis. Subsequently, using a multiplex proteomics approach, we examined an expansive array of novel candidate cytokine and chemokines within this population.
Methods: We performed a secondary analysis of targeted proteomics data from Alberta Sepsis Network studies. Plasma mediator levels, analyzed by Luminex multiplex assays, were evaluated in children aged 5-17 years with nonappendicitis abdominal pain (NAAP), acute appendicitis (AA), and nonappendicitis sepsis (NAS). We used multivariate regression analysis to evaluate the seven target proteins, followed by decision tree and heat mapping analyses for all proteins evaluated.
Results: 185 children were included: 83 with NAAP, 79 AA, and 23 NAS. Plasma levels of IL-6, CRP, MCP-1, PCT, and SAA were significantly different in children with AA compared to those with NAAP (p < 0.001). Expansive proteomic analysis demonstrated 6 patterns in inflammatory mediator profiles based on severity of illness. A decision tree incorporating the proteins CRP, ferritin, SAA, regulated on activation normal T-cell expressed and secreted (RANTES), monokine induced by gamma interferon (MIG), and PCT demonstrated excellent specificity (0.920) and negative predictive value (0.882) for children with appendicitis.
Conclusions: Multiplex proteomic analyses described the inflammatory landscape of children presenting to the ED with suspected appendicitis. We have demonstrated the feasibility of this approach to identify potential novel candidate cytokines/chemokine patterns associated with a specific illness (appendicitis) amongst those with a broad ED presentation (abdominal pain). This approach can be modelled for future research initiatives in pediatric emergency medicine.

DOI: https://doi.org/10.1155/2019/2359681
Pathol Oncol Res. 2019 Mar 29.

Identification and Verification of Two Novel Differentially Expressed Proteins from Non-neoplastic Mucosa and Colorectal Carcinoma Via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry.

Yuru Bai, Jiabao Wang, Zhihua Gao, Erqing Dai.

  Recurrence or metastasis of colorectal cancer (CRC) is common following surgery and/or adjuvant therapy, particularly in patients with an advanced stage of the cancer. Identifying key molecular markers of CRC is beneficial for early diagnosis and early treatment, which may eventually improve the prognosis of patients with CRC. Isobaric mass tags for relative and absolute quantification (iTRAQ) in combination with multidimensional liquid chromatography and tandem mass spectrometry (LC-MS/MS) were used to identify differentially expressed proteins between CRC tissues and paired adjacent normal mucosa. Among the 105 patients, adenocarcinoma was the most common CRC subtype, stage III was the most common Tumor-Node-Metastasis stage and high levels of Ki-67 indicated the rapid proliferation of tumor cells in the samples. The LC-MS/MS-based iTRAQ technology identified 271 differentially expressed proteins, with 130 upregulated proteins and 141 downregulated proteins. Bioinformatics analysis revealed that golgin subfamily A member 2 (GOLGA2) and heterogeneous nuclear ribonucleoprotein D0 (hnRNPD) were located in the center of the upregulated protein network, and were closely associated with the development of CRC. The upregulation of GOLGA2 and hnRNPD was further verified in human tissues using western blotting and immunohistochemistry. GOLGA2 and hnRNPD were identified as two novels differentially expressed proteins in human CRC. Furthermore, the LC-MS/MS-based iTRAQ proteomic approach is a useful tool for searching and identifying differentially expressed proteins, and may be used to provide a comprehensive understanding of the processes that mediate the development of CRC.

Keywords:  Colorectal cancer; Differential expressed proteins; Isobaric mass tags for relative and absolute quantification; Multidimensional liquid chromatography and tandem mass spectrometry; Proteomics

DOI:  https://doi.org/10.1007/s12253-019-00651-y
J Occup Med Toxicol. 2019 ;14 9

Comparative proteomic analysis in serum of former uranium miners with and without radon induced squamous lung cancer.

Simone Helmig, Günter Lochnit, Joachim Schneider.

Summary: Former uranium miners of the Wismut Company, East Germany, have been exposed to ionizing radiation from radon decay products and therefore were at high risk for lung cancer. Since histological types of cancer in the so called Wismut cohort revealed an association of high radon exposure with a higher relative frequency of squamous cell carcinoma (SqCC), we used comparative proteomic analysis to identify differentially expressed proteins in serum exposed uranium miners with SqCC.
Methode: Pooled sera of exposed former uranium miners without lung disease and pooled sera of former uranium miners with SqCC were analysed by 2-D gel electrophoresis. MALDI-TOF-MS was performed from reproducable, significantly, at least 5-fold up-regulated protein spots. Proteins were identified by MASCOT peptide mass fingerprint search. Additionally a receiver operating characteristic curve for CYFRA 21-1 was created.
Results: The protein spots were identified as Keratin 10 (K10), Keratin 1 (K1), complement factor H (CFH) and a haptoglobin (Hpt) fragment. The sensitivity for CYFRA 21-1 reveals 60% at a specifity of 95 and 80% at a specifity of 80%. Plotting the sensitivity against specifity reveals an AUC of 0.88.
Conclusion: In SqCC Keratin 10 and 1 were strongly induced. This was associated with CYFRA 21-1, confirming the cytokeratin fragment as a tumormarker.

DOI: https://doi.org/10.1186/s12995-019-0228-y
Int J Hyperthermia. 2019 Mar 26. 1-11

Proteomic and bioinformatic analysis of condyloma acuminata: mild hyperthermia treatment reveals compromised HPV infectivity of keratinocytes via regulation of metabolism, differentiation and anti-viral responses.

Yu-Zhe Sun, Jia-Feng Li, Zhen-Dong Wei, Hang-Hang Jiang, Yu-Xiao Hong, Song Zheng, Rui-Qun Qi, Xing-Hua Gao.

   BACKGROUND: Hyperthermia has proved successful in treating cutaneous human papillomavirus infectious diseases such as plantar wart and condyloma acuminata (CA). Moreover, this treatment provides improved therapeutic efficacy in these conditions as compared with conventional therapies.
OBJECTIVES: To investigate the global proteome changes in CA in response to hyperthermia and achieve a better understanding of the mechanisms of hyperthermia therapy against HPV-infectious diseases.
METHODS: CA tissue was obtained from patients undergoing pathological examinations. Diagnosis was verified as based on results of both HE staining and HPV-DNA PCR assay. Hyperthermia was achieved with a 44 °C water bath. Differentially expressed proteins (DEPs) were identified by iTRAQ labeling, SCX chromatography and LC-MS/MS assay. Validation of proteomic results was performed using real-time qPCR and western blot, while bioinformatic analysis of DEPs was accomplished by R 3.4.1, STRING and Cytoscape softwares.
RESULTS: In response to hyperthermia, a total of 102 DEPs were identified with 37 being upregulated and 65 downregulated. Among these DEPs, hyperthermia induced proteins involved with anti-viral processes such as OAS1, MX1, BANF1, CANX and AP1S1, whereas it inhibited proteins that participated in cellular metabolism, such as GALT, H6PD, EXOSC4 and EXOSC6; protein translation, such as RPS4Y1; as well as keratinocyte differentiation, such as KRT5, KRT27, KRT75, KRT76 and H2AFY2.
CONCLUSIONS: Hyperthermia inhibited enzymes and molecules responsible for metabolism modulation and keratinocyte differentiation in CA tissue, whereas it promoted factors involved in anti-viral responses. Such effects may, in part, contribute to the efficacy of local hyperthermia therapy against HPV infection.

Keywords:  Hyperthermia; antivirus; condyloma acuminatum; iTRAQ; proteomics

DOI:  https://doi.org/10.1080/02656736.2019.1578420
Biomark Insights. 2019 ;14 1177271919835570

Identification of Urinary CD44 and Prosaposin as Specific Biomarkers of Urinary Tract Infections in Children With Neurogenic Bladders.

Catherine S Forster, Wendy D Haffey, Michael Bennett, Kenneth D Greis, Prasad Devarajan.

   Purpose: Distinguishing urinary tract infection (UTI) from urinary tract colonization (UTC) in children with neurogenic bladders who require clean intermittent catheterization (CIC) is challenging. Our objective was to identify urinary proteins to distinguish UTI from UTC in CIC-dependent children that have potential to serve as objective markers of UTI.
Experimental design: A total of 10 CIC-dependent children were included in the mass spectrometry analysis (UTI = 5, UTC = 5). Quantitative profiling of urine proteins with isobaric protein labeling was performed using tandem mass spectrometry. Candidate markers were normalized using a collective mixture of proteins from all samples. Relative quantitative abundance of proteins across all samples were compared. Proteins with >50% change in the average abundance were identified as proteins of interest, which were then measured using enzyme-linked immunosorbent assay (ELISA) in an additional 40 samples (no growth = 10, UTC = 15, UTI = 15).
Results: Mass spectrometry revealed 8 differentially expressed proteins. Of these, apolipoprotein D, alpha-amylase 2B, non-secretory ribonuclease, CD44 antigen, and prosaposin were measurable by ELISA. Concentrations of both CD44 and prosaposin were significantly higher in UTI, with area under the curves (AUCs) of 0.72 and 0.78, respectively.
Conclusion: Urinary CD44 and prosaposin are candidate markers that may assist with the diagnosis of UTI in CIC-dependent children.

Keywords:  biomarkers; neurogenic bladder; urinary tract infection

DOI:  https://doi.org/10.1177/1177271919835570
Biomed Rep. 2019 Mar;10(3): 165-174

Identification of diagnostic upper gastrointestinal cancer tissue type-specific urinary biomarkers.

Holger Husi, Marco Fernandes, Richard J Skipworth, Janice Miller, Andrew D Cronshaw, Kenneth C H Fearon, James A Ross.

  Several potential urinary biomarkers exhibiting an association with upper gastrointestinal tumour growth have been previously identified, of which S100A6, S100A9, rabenosyn-5 and programmed cell death 6-interacting protein (PDCD6IP) were further validated and found to be upregulated in malignant tumours. The cancer cohort from our previous study was subclassified to assess whether distinct molecular markers can be identified for each individual cancer type using a similar approach. Urine samples from patients with cancers of the stomach, oesophagus, oesophagogastric junction or pancreas were analysed by surface-enhanced laser desorption/ionization-time-of-flight mass spectrometry using both CM10 and IMAC30 (Cu2+-complexed) chip types and LC-MS/MS-based mass spectrometry after chromatographic enrichment. This was followed by protein identification, pattern matching and validation by western blotting. We found 8 m/z peaks with statistical significance for the four cancer types investigated, of which m/z 2447 and 2577 were identified by pattern matching as fragments of cathepsin-B (CTSB) and cystatin-B (CSTB); both molecules are indicative of pancreatic cancer. Additionally, we observed a potential association of upregulated α-1-antichymotrypsin with pancreatic and gastric cancers, of PDCD6IP, vitelline membrane outer layer protein 1 homolog (VMO1) and triosephosphate isomerase (TPI1) with oesophagogastric junctional cancers, and of complement C4-A, prostatic acid phosphatase, azurocidin and histone-H1 with oesophageal cancer. Furthermore, the potential pancreatic cancer biomarkers CSTB and CTSB were validated independently by western blotting. Therefore, the present study identified two new potential urinary biomarkers that appear to be associated with pancreatic cancer. This may provide a simple, non-invasive screening test for use in the clinical setting.

Keywords:  SELDI-TOF; mass spectrometry; pancreatic cancer; upper gastrointestinal cancer; urine biomarker

DOI:  https://doi.org/10.3892/br.2019.1190
Sci Rep. 2019 Mar 26. 9(1): 5154

Improved survival prognostication of node-positive malignant melanoma patients utilizing shotgun proteomics guided by histopathological characterization and genomic data.

Lazaro Hiram Betancourt, Krzysztof Pawłowski, Jonatan Eriksson, A Marcell Szasz, Shamik Mitra, Indira Pla, Charlotte Welinder, Henrik Ekedahl, Per Broberg, Roger Appelqvist, Maria Yakovleva, Yutaka Sugihara, Kenichi Miharada, Christian Ingvar, Lotta Lundgren, Bo Baldetorp, Håkan Olsson, Melinda Rezeli, Elisabet Wieslander, Peter Horvatovich, Johan Malm, Göran Jönsson, György Marko-Varga.

Metastatic melanoma is one of the most common deadly cancers, and robust biomarkers are still needed, e.g. to predict survival and treatment efficiency. Here, protein expression analysis of one hundred eleven melanoma lymph node metastases using high resolution mass spectrometry is coupled with in-depth histopathology analysis, clinical data and genomics profiles. This broad view of protein expression allowed to identify novel candidate protein markers that improved prediction of survival in melanoma patients. Some of the prognostic proteins have not been reported in the context of melanoma before, and few of them exhibit unexpected relationship to survival, which likely reflects the limitations of current knowledge on melanoma and shows the potential of proteomics in clinical cancer research.

DOI: https://doi.org/10.1038/s41598-019-41625-z
Alzheimers Dement (Amst). 2019 Dec;11 270-276

A proteomic signature for dementia with Lewy bodies.

Sid E O'Bryant, Tanis J Ferman, Fan Zhang, James Hall, Otto Pedraza, Zbigniew K Wszolek, Tori Como, David Julovich, Sravan Mattevada, Leigh A Johnson, Melissa Edwards, James Hall, Neill R Graff-Radford.

   Introduction: We sought to determine if a proteomic profile approach developed to detect Alzheimer's disease would distinguish patients with Lewy body disease from normal controls, and if it would distinguish dementia with Lewy bodies (DLB) from Parkinson's disease (PD).
Methods: Stored plasma samples were obtained from 145 patients (DLB n = 57, PD without dementia n = 32, normal controls n = 56) enrolled from patients seen in the Behavioral Neurology or Movement Disorders clinics at the Mayo Clinic, Florida. Proteomic assays were conducted and analyzed as per our previously published protocols.
Results: In the first step, the proteomic profile distinguished the DLB-PD group from controls with a diagnostic accuracy of 0.97, sensitivity of 0.91, and specificity of 0.86. In the second step, the proteomic profile distinguished the DLB from PD groups with a diagnostic accuracy of 0.92, sensitivity of 0.94, and specificity of 0.88.
Discussion: These data provide evidence of the potential utility of a multitiered blood-based proteomic screening method for detecting DLB and distinguishing DLB from PD.

Keywords:  Biomarker screening; Blood biomarkers; Dementia with Lewy bodies; Detection; Diagnostic accuracy; Parkinson's disease; Proteomics

DOI:  https://doi.org/10.1016/j.dadm.2019.01.006
Gut. 2019 Mar 26. pii: gutjnl-2018-317571. [Epub ahead of print]

Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis.

Sjoerd van der Post, Karolina S Jabbar, George Birchenough, Liisa Arike, Noreen Akhtar, Henrik Sjovall, Malin E V Johansson, Gunnar C Hansson.

   OBJECTIVE: The colonic inner mucus layer protects us from pathogens and commensal-induced inflammation, and has been shown to be defective in active UC. The aim of this study was to determine the underlying compositional alterations, their molecular background and potential contribution to UC pathogenesis.
DESIGN: In this single-centre case-control study, sigmoid colon biopsies were obtained from patients with UC with ongoing inflammation (n=36) or in remission (n=28), and from 47 patients without colonic disease. Mucus samples were collected from biopsies ex vivo, and their protein composition analysed by nanoliquid chromatography-tandem mass spectrometry. Mucus penetrability and goblet cell responses to microbial stimulus were assessed in a subset of patients.
RESULTS: The core mucus proteome was found to consist of a small set of 29 secreted/transmembrane proteins. In active UC, major structural mucus components including the mucin MUC2 (p<0.0001) were reduced, also in non-inflamed segments. Active UC was associated with decreased numbers of sentinel goblet cells and attenuation of the goblet cell secretory response to microbial challenge. Abnormal penetrability of the inner mucus layer was observed in a subset of patients with UC (12/40; 30%). Proteomic alterations in penetrable mucus samples included a reduction of the SLC26A3 apical membrane anion exchanger, which supplies bicarbonate required for colonic mucin barrier formation.
CONCLUSION: Core mucus structural components were reduced in active UC. These alterations were associated with attenuation of the goblet cell secretory response to microbial challenge, but occurred independent of local inflammation. Thus, mucus abnormalities are likely to contribute to UC pathogenesis.

Keywords:  mucins; mucosal barrier; mucosal protection; mucus; ulcerative colitis

DOI:  https://doi.org/10.1136/gutjnl-2018-317571
Reprod Fertil Dev. 2019 Mar 29.

Quantitative proteomics suggest a potential link between early embryonic death and trisomy 16.

Ting Yao, Haiyan Hou, Guozhong Liu, Jun Wu, Zhe Qin, Yang Sun, Xiaohan Jin, Jun Chen, Yaqiong Chen, Zhongwei Xu.

Activation of extracellular signal-regulated kinase (ERK) signalling, alteration of the uterine microenvironment and a reduction in human chorionic gonadotrophin production have been linked with fetal trisomy 16-induced early embryonic death (EED). However, the detailed biological mechanism of EED remains unclear. Using quantitative proteomics we successfully screened differentially expressed proteins in the villous tissues from patients with EED and fetal trisomy 16 (EEDT16), patients with EED but normal fetal chromosomes (EEDNC) and patients undergoing elective abortion with normal fetal chromosomes (EANC) as the reference group. Compared with the reference group, we identified 337 and 220 differentially expressed proteins in EEDT16 patients and EEDNC patients respectively; these were involved in critical biological processes including immune response, superoxide metabolism, inflammatory responses and so on. We found that differential expression of immunological function-related molecules, such as human leukocyte antigen-g (HLA-G), HLA-C, Fc Fragment Of IgG Receptor III (FcγR III), also named CD16, interleukin 18 (IL-18) and transforming growth factor β1 (TGF-β1), might induce EED in both EEDT16 and EEDNC patients. More severe immunological dysfunction was observed in EEDT16 patients than that in EEDNC patients. Furthermore, differential expression of implantation and invasion-related molecules, such as cytochrome b-245 light chain (CYBA), neutrophil cytosol factor 2 (NCF2), Mitogen-activated protein kinase kinase kinase 4 (MAP3K4), matrix metalloproteinase 2 (MMP2), MMP9 and tumour necrosis factor α (TNF-α) might induce EED in both EEDT16 and EEDNC patients, although more severe dysfunction in the implantation and invasion ability of villous tissues was observed in EEDT16 patients.

DOI: https://doi.org/10.1071/RD17319
J Alzheimers Dis. 2019 Mar 18.

Combined Transcriptomics and Proteomics in Frontal Cortex Area 8 in Frontotemporal Lobar Degeneration Linked to C9ORF72 Expansion.

Pol Andrés-Benito, Ellen Gelpi, Mónica Povedano, Karina Ausín, Joaquín Fernández-Irigoyen, Enrique Santamaría, Isidro Ferrer.

   BACKGROUND: Frontotemporal lobar degeneration with TDP-43 immunoreactive inclusions (FTLD-TDP) may appear as sporadic (sFTLD-TDP) or linked to mutations in various genes including expansions of the non-coding region of C9ORF72 (c9FTLD).
OBJECTIVE: Analysis of differential mRNA and protein expression in the frontal cortex in c9FLTD and evaluation with previous observations in frontal cortex in sFTLD-TDP and amyotrophic lateral sclerosis with TDP-43 inclusions.
METHODS: Microarray hybridization and mass spectrometry-based quantitative proteomics followed by RT-qPCR, gel electrophoresis, and western blotting in frontal cortex area 8 in 19 c9FTLD cases and 14 age- and gender-matched controls.
RESULTS: Microarray hybridization distinguish altered gene transcription related to DNA recombination, RNA splicing regulation, RNA polymerase transcription, myelin synthesis, calcium regulation, and ubiquitin-proteasome system in c9FTLD; proteomics performed in the same tissue samples pinpoints abnormal protein expression involving apoptosis, inflammation, metabolism of amino acids, metabolism of carbohydrates, metabolism of membrane lipid derivatives, microtubule dynamics, morphology of mitochondria, neuritogenesis, neurotransmission, phagocytosis, receptor-mediated endocytosis, synthesis of reactive oxygen species, and calcium signaling in c9FTLD.
CONCLUSION: Transcriptomics and proteomics, as well as bioinformatics processing of derived data, reveal similarly altered pathways in the frontal cortex in c9FTLD, but different RNAs and proteins are identified by these methods. Combined non-targeted '-omics' is a valuable approach to deciphering altered molecular pathways in FTLD provided that observations are approached with caution when assessing human postmortem brain samples.

Keywords:  C9ORF72; FTLD-TDP; frontotemporal lobar degeneration; gene expression; proteomics

DOI:  https://doi.org/10.3233/JAD-181123
Front Immunol. 2019 ;10 423

Caught in a Trap? Proteomic Analysis of Neutrophil Extracellular Traps in Rheumatoid Arthritis and Systemic Lupus Erythematosus.

Elinor A Chapman, Max Lyon, Deborah Simpson, David Mason, Robert J Beynon, Robert J Moots, Helen L Wright.

  Neutrophil Extracellular Traps (NETs) are implicated in the development of auto-immunity in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalization of intracellular neoepitopes e.g., dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy controls, and from patients with RA and SLE to determine if NETs can be differentially-generated to expose different sets of neoepitopes. Ultra-pure neutrophils (>99%) from healthy individuals (n = 3) and patients with RA or SLE (n = 6 each) were incubated ± PMA (50 nM, PKC super-activator) or A23187 (3.8 μM, calcium ionophore) for 4 h. NETs were liberated by nuclease digestion and concentrated onto Strataclean beads prior to on-bead digestion with trypsin. Data-dependent LC-MS/MS analyses were conducted on a QExactive HF quadrupole-Orbitrap mass spectrometer, and label-free protein quantification was carried out using Progenesis QI. PMA-induced NETs were decorated with annexins, azurocidin and histone H3, whereas A23187-induced NETs were decorated with granule proteins including CAMP/LL37, CRISP3, lipocalin and MMP8, histones H1.0, H1.4, and H1.5, interleukin-8, protein-arginine deiminase-4 (PADI4), and α-enolase. Four proteins were significantly different between PMA-NETs from RA and SLE neutrophils (p < 0.05): RNASE2 was higher in RA, whereas MPO, leukocyte elastase inhibitor and thymidine phosphorylase were higher in SLE. For A23187-NETs, six NET proteins were higher in RA (p < 0.05), including CAMP/LL37, CRISP3, interleukin-8, MMP8; Thirteen proteins were higher in SLE, including histones H1.0, H2B, and H4. This work provides the first, direct comparison of NOX2-dependent (PMA) and NOX2-independent (A23187) NETs using quantitative proteomics, and the first direct comparison of RA and SLE NETs using quantitative proteomics. We show that it is the nature of the stimulant rather than neutrophil physiology that determines NET protein profiles in disease, since stimulation of NETosis in either a NOX2-dependent or a NOX2-independent manner generates broadly similar NET proteins irrespective of the disease background. We also use our proteomics pipeline to identify an extensive range of post-translationally modified proteins in RA and SLE, including histones and granule proteins, many of which are known targets of auto-antibodies in each disease.

Keywords:  NET; citrullinated; histones; neutrophil; neutrophil extracellular trap; rheumatoid arthritis; systemic lupus erythematosus

DOI:  https://doi.org/10.3389/fimmu.2019.00423
mSphere. 2019 Mar 27. pii: e00061-19. [Epub ahead of print]4(2):

Distinct Antibody Signatures Associated with Different Malaria Transmission Intensities in Zambia and Zimbabwe.

Tamaki Kobayashi, Aarti Jain, Li Liang, Joshua M Obiero, Harry Hamapumbu, Jennifer C Stevenson, Philip E Thuma, James Lupiya, Mike Chaponda, Modest Mulenga, Edmore Mamini, Sungano Mharakurwa, Lovemore Gwanzura, Shungu Munyati, Susan Mutambu, Philip Felgner, D Huw Davies, William J Moss.

  Antibodies to Plasmodium falciparum are specific biomarkers that can be used to monitor parasite exposure over broader time frames than microscopy, rapid diagnostic tests, or molecular assays. Consequently, seroprevalence surveys can assist with monitoring the impact of malaria control interventions, particularly in the final stages of elimination, when parasite incidence is low. The protein array format to measure antibodies to diverse P. falciparum antigens requires only small sample volumes and is high throughput, permitting the monitoring of malaria transmission on large spatial and temporal scales. We expanded the use of a protein microarray to assess malaria transmission in settings beyond those with a low malaria incidence. Antibody responses in children and adults were profiled, using a P. falciparum protein microarray, through community-based surveys in three areas in Zambia and Zimbabwe at different stages of malaria control and elimination. These three epidemiological settings had distinct serological profiles reflective of their malaria transmission histories. While there was little correlation between transmission intensity and antibody signals (magnitude or breadth) in adults, there was a clear correlation in children younger than 5 years of age. Antibodies in adults appeared to be durable even in the absence of significant recent transmission, whereas antibodies in children provided a more accurate picture of recent levels of transmission intensity. Seroprevalence studies in children could provide a valuable marker of progress toward malaria elimination.IMPORTANCE As malaria approaches elimination in many areas of the world, monitoring the effect of control measures becomes more important but challenging. Low-level infections may go undetected by conventional tests that depend on parasitemia, particularly in immune individuals, who typically show no symptoms of malaria. In contrast, antibodies persist after parasitemia and may provide a more accurate picture of recent exposure. Only a few parasite antigens-mainly vaccine candidates-have been evaluated in seroepidemiological studies. We examined antibody responses to 500 different malaria proteins in blood samples collected through community-based surveillance from areas with low, medium, and high malaria transmission intensities. The breadth of the antibody responses in adults was broad in all three settings and was a poor correlate of recent exposure. In contrast, children represented a better sentinel population for monitoring recent malaria transmission. These data will help inform the use of multiplex serology for malaria surveillance.

Keywords:  Plasmodium falciparum; malaria; proteomics; serology; surveillance studies

DOI:  https://doi.org/10.1128/mSphereDirect.00061-19
JCI Insight. 2019 Mar 26. pii: 126337. [Epub ahead of print]5

Pro-inflammatory, IL-17 pathways dominate the architecture of the immunome in pediatric refractory epilepsy.

Pavanish Kumar, Derrick Wei Shih Chan, Amanda Lim, Bhairav Paleja, Simon Ling, Lai Li Yun, Su Li Poh, Adeline Ngoh, Thaschawee Arkachaisri, Joo Guan Yeo, Salvatore Albani.

  Drug refractory epilepsy (RE) is a chronic neurological disease with varied etiology that represents a group of patients whose seizures do not respond to anti-epileptic drugs. The immune system may have a role in seizure and epilepsy development, but the specific mechanisms of inflammation that lead to epileptogenesis and contribute to RE are unknown. Here, we used mass cytometry to comprehensively study the immune system of pediatric patients with RE and compared their immune profile and function with patients with age-matched autoimmune encephalitis (AIE) and healthy controls. Patients with RE and AIE displayed similar immune profiles overall, with changes in CD4+ and CD8+ T-cell subsets and an unbalance toward pro-inflammatory IL-17 production. In addition, patients with RE uniquely showed an altered balance in natural killer cell subsets. A systems level intercellular network analysis identified rewiring of the immune system leading to loss of inhibitory/regulatory intercellular connections and emergence of pro-inflammatory pathogenic functions in neuro-inflammatory immune-cell networks in patients with AIE and RE. These data underscore the contribution of systemic inflammation to the pathogenesis of seizures and epileptogenesis and have direct translational implications in advancing diagnostics and therapeutics design.

Keywords:  Cellular immune response; Epilepsy; Immunology; Neuroscience; T cells

DOI:  https://doi.org/10.1172/jci.insight.126337
Mass Spectrom Rev. 2019 Mar 28.

MS1 ion current-based quantitative proteomics: A promising solution for reliable analysis of large biological cohorts.

Xue Wang, Shichen Shen, Sailee Suryakant Rasam, Jun Qu.

  The rapidly-advancing field of pharmaceutical and clinical research calls for systematic, molecular-level characterization of complex biological systems. To this end, quantitative proteomics represents a powerful tool but an optimal solution for reliable large-cohort proteomics analysis, as frequently involved in pharmaceutical/clinical investigations, is urgently needed. Large-cohort analysis remains challenging owing to the deteriorating quantitative quality and snowballing missing data and false-positive discovery of altered proteins when sample size increases. MS1 ion current-based methods, which have become an important class of label-free quantification techniques during the past decade, show considerable potential to achieve reproducible protein measurements in large cohorts with high quantitative accuracy/precision. Nonetheless, in order to fully unleash this potential, several critical prerequisites should be met. Here we provide an overview of the rationale of MS1-based strategies and then important considerations for experimental and data processing techniques, with the emphasis on (i) efficient and reproducible sample preparation and LC separation; (ii) sensitive, selective and high-resolution MS detection; iii)accurate chromatographic alignment; (iv) sensitive and selective generation of quantitative features; and (v) optimal post-feature-generation data quality control. Prominent technical developments in these aspects are discussed. Finally, we reviewed applications of MS1-based strategy in disease mechanism studies, biomarker discovery, and pharmaceutical investigations.

Keywords:  LC-MS; MS1 quantification; ion current-based proteomics; large cohorts; reproducible protein measurement

DOI:  https://doi.org/10.1002/mas.21595
Toxicol Appl Pharmacol. 2019 Mar 26. pii: S0041-008X(19)30114-0. [Epub ahead of print]

Validation of a multiplexed LC-MS/MS clinical assay to quantify insulin growth factor binding proteins in human serum and its application in a clinical study.

Nanjun Liu, Elizabeth Tengstrand, Olaf Boernsen, Stephan Bek, Frank Hsieh.

  Circulating insulin-like growth factor-binding proteins (IGFBPs) continue to gain attention as biomarkers of drug activities on insulin like growth factor (IGF)/IGF receptor signaling pathways. A multiplexed LC-MS/MS method was validated for the absolute quantitation of IGFBPs in human serum. The method was used to measure screening concentrations of IGFBPs in spinal and bulbar muscular atrophy (SBMA) patients in a phase 2 clinical trial. Concentrations of IGFBP 1, 2, 3, and 5 were simultaneously determined based on representative signature peptides derived from an optimized trypsin digestion procedure. Signature peptide levels were absolutely quantitated using a sensitive/specific targeted LC-MS/MS method. Corresponding mass-shifted, stable isotope-labeled peptides were employed as internal standards. A true blank matrix for the quantitation of IGFBPs was not available since they are endogenous proteins in human serum. In this method, calibration standards/curves were prepared using authentic synthetic peptides spiked into a surrogate matrix. The surrogate matrix was generated from human serum treated in the same way as the study samples, but using iodoacetic acid instead of iodoacetamide as the alkylation reagent. This surrogate matrix approach allowed for the direct and sensitive/specific quantification of IGFBP 1, 2, 3, and 5 due to the lack of any endogenous background. Equivalent matrix effect and recovery of analytes was achieved for the authentic and surrogate matrices. The fully validated LC-MS/MS assay will allow further evaluation of the utility of IGFBP biomarkers in clinical trials.

Keywords:  Alkylation reagent; Insulin-like growth factor-binding proteins; LC–MS/MS; Matrix effect; Protein quantitation; Spinal and bulbar muscular atrophy

DOI:  https://doi.org/10.1016/j.taap.2019.03.024
J Vis Exp. 2019 Mar 07.

Visualization of Amyloid β Deposits in the Human Brain with Matrix-assisted Laser Desorption/Ionization Imaging Mass Spectrometry.

Masaya Ikegawa, Takashi Nirasawa, Nobuto Kakuda, Tomohiro Miyasaka, Yuki Kuzuhara, Shigeo Murayama, Yasuo Ihara.

The neuropathology of Alzheimer's disease (AD) is characterized by the accumulation and aggregation of amyloid β (Aβ) peptides into extracellular plaques of the brain. The Aβ peptides, composed of 40 amino acids, are generated from amyloid precursor proteins (APP) by β- and γ-secretases. Aβ is deposited not only in cerebral parenchyma but also in leptomeningeal and cerebral vessel walls, known as cerebral amyloid angiopathy (CAA). While a variety of Aβ peptides were identified, the detailed production and distribution of individual Aβ peptides in pathological tissues of AD and CAA have not been fully addressed. Here, we develop a protocol of matrix-assisted laser desorption/ionization-based imaging mass spectrometry (MALDI-IMS) on human autopsy brain tissues to obtain comprehensive protein mapping. For this purpose, human cortical specimens were obtained from the Brain Bank at the Tokyo Metropolitan Institute of Gerontology. Frozen cryosections are cut and transferred to indium-tin-oxide (ITO)-coated glass slides. Spectra are acquired using the MALDI system with a spatial resolution up to 20 µm. Sinapinic acid (SA) is uniformly deposited on the slide using either an automatic or a manual sprayer. With the current technical advantages of MALDI-IMS, a typical data set of various Aβ species within the same sections of human autopsied brains can be obtained without specific probes. Furthermore, high-resolution (20 µm) imaging of an AD brain and severe CAA sample clearly shows that Aβ1-36 to Aβ1-41 were deposited into leptomeningeal vessels, while Aβ1-42 and Aβ1-43 were deposited in cerebral parenchyma as senile plaque (SP). It is feasible to adopt MALDI-IMS as a standard approach in combination with clinical, genetic, and pathological observations in understanding the pathology of AD, CAA, and other neurological diseases based on the current strategy.

DOI: https://doi.org/10.3791/57645
J Cell Physiol. 2019 Mar 25.

Gene expression, protein profiling, and chemotactic activity of infrapatellar fat pad mesenchymal stem cells in pathologies of the knee joint.

Beatriz Bravo, Maria Concepcion Guisasola, Javier Vaquero, Irene Tirado, Arancha R Gortazar, Francisco Forriol.

  The infrapatellar fat pad (IPFP) is a periarticular adipose knee tissue. This tissue contains a large number of mesenchymal stem cells (MSCs). In the present work, we wanted to study the IPFP MSCs and their relationship and differences in two groups, anterior cruciate ligament (ACL) ruptures knees and ostheoarthrosis (OA). The IPFP of 42 patients with OA or ACL rupture were analyzed. Isolation, primary culture, and a genetic and proteomic study of MSCs from IPFP were performed. Gene expression of IL-6, tumor necrosis factor (TNF), IL-8, HSPA1A (Hsp70), CXCL10, RANTES, MMP1, MMP3, TIMP1, and BMP7 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). We analyzed MSCs from from 12 diferents patients in two cellular pools (6 from AO disease and 6 from ALC rupture to form two cell pool), for the iTRAQ Proteomic Assay. The conditional media were used in quantitative analysis of MSC soluble factors by Luminex and for de migration assay. A higher gene expression of IL-6, TNF, CXCL10, RANTES, and MMP1 and OPG in MSCs from OA versus ACL (p < 0.05) was observed. Conversely HSPA1A, TIMP1, and RANKL showed a significant lower expression in OA-MSCs (p < 0.05). In the secretome analysis, adipsin and visfantin levels in the supernatants from OA-MSCs were lower (p < 0.05) respect to ACL-MSCs. Also, the monocytic cells migrated two-folds in the presence of conditioned media from OA-MSCs patients versus patients with ACL-MSC. The infrapatellar pad should be considered as an adipose tissue capable of producing and excreting inflammatory mediators directly in the knee joint, influencing the development and progression of knee joint pathologies.

Keywords:  ACL; OA; infrapatellar fat pad; mesenchymal stem cells

DOI:  https://doi.org/10.1002/jcp.28532
J Immunother Cancer. 2019 Mar 29. 7(1): 91

Proteomic test for anti-PD-1 checkpoint blockade treatment of metastatic melanoma with and without BRAF mutations.

Paolo A Ascierto, Mariaelena Capone, Antonio M Grimaldi, Domenico Mallardo, Ester Simeone, Gabriele Madonna, Heinrich Roder, Krista Meyer, Senait Asmellash, Carlos Oliveira, Joanna Roder, Julia Grigorieva.

  The therapeutic landscape in metastatic melanoma has changed dramatically in the last decade, with the success of immune checkpoint inhibitors resulting in durable responses for a large number of patients. For patients with BRAF mutations, combinations of BRAF and MEK inhibitors demonstrated response rates and benefit comparable to those from immune checkpoint inhibitors, providing the rationale for sequential treatment with targeted and immunotherapies and raising the question of optimal treatment sequencing.Biomarkers for the selection of anti-PD-1 therapy in BRAF wild type (BRAF WT) and in BRAF mutated (BRAF MUT) patients help development of alternative treatments for patients unlikely to benefit, and might lead to better understanding of the interaction of checkpoint inhibition and targeted therapy. In this paper we evaluate the performance of a previously developed serum proteomic test, BDX008, in metastatic melanoma patients treated with anti-PD-1 agents and investigate the role of BRAF mutation status. BDX008, a pre-treatment proteomic test associated with acute phase reactants, wound healing and complement activation, stratifies patients into two groups, BDX008+ and BDX008-, with better and worse outcomes on immunotherapy.Serum samples were available from 71 patients treated with anti-PD1 inhibitors; 25 patients had BRAF mutations, 39 were wild type. Overall, BDX008+ patients had significantly better overall survival (OS) (HR = 0.50, P = 0.016) and a trend for better progression-free survival (PFS) (HR = 0.61, P = 0.060) than BDX008- patients. BDX008 classification was statistically significant in the analyses adjusted for mutation status, LDH, and line of treatment (P = 0.009 for OS and 0.031 for PFS). BRAF WT BDX008+ patients had markedly long median OS of 32.5 months and 53% landmark 2 years survival, with statistically significantly superior OS as compared to BDX008- patients (HR = 0.41, P = 0.032). The difference between BDX008+ and BDX008- in PFS in BRAF WT patients and in OS and PFS in BRAF MUT patients did not reach statistical significance, though numerically was consistent with overall results. The test demonstrated significant interaction with neutrophil-to-lymphocyte ratio (NLR) (PFS P = 0.041, OS P = 0.004). BDX008 as a biomarker selecting for benefit from immune checkpoint blockade, especially in patients with wild type BRAF and in subgroups with low NLR, warrants further evaluation.

Keywords:  Anti-PD-1; BRAF mutations; Immunotherapy; Melanoma; Nivolumab; Pembrolizumab; Proteomic test

DOI:  https://doi.org/10.1186/s40425-019-0569-1
Expert Rev Mol Diagn. 2019 Mar 25. 1-12

Advances in prognostic markers for colorectal cancer.

Lili Duan, Wanli Yang, Xiaoqian Wang, Wei Zhou, Yujie Zhang, Jinqiang Liu, Hongwei Zhang, Qingchuan Zhao, Liu Hong, Daiming Fan.

   INTRODUCTION: Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and contributes significantly to cancer-related deaths. Since CRC is largely asymptomatic until alarm features develop to advanced stages, the incidence and mortality rate of CRC is relatively high and increases year by year. Areas covered: This review mainly summarizes the recent advances of genetic, epigenetic, and proteomic molecular markers that may be used to predict the prognosis of CRC. Moreover, we made an introduction to immune biomarkers and emerging microbial biomarkers. We also discussed the recent movement of molecular pathological epidemiology (MPE) in the context of prognostic biomarkers. Finally, we discussed the pros and cons of various biomarkers for improving prognostic judgment. Expert opinion: A great number of molecular targets have been identified as prognostic markers for CRC. However, because of some limitations, the efficiency of these biomarkers is restricted in clinical application. Combining a panel of several approaches may present a promising alternative tool for clinical practice. Further large-scale clinical trials are required to validate the prognostic value of these biomarkers in clinical use.

Keywords:  Molecular markers; colorectal cancer; epigenetic; genetic; prognosis; proteomic

DOI:  https://doi.org/10.1080/14737159.2019.1592679
J Allergy Clin Immunol. 2019 Mar 26. pii: S0091-6749(19)30410-5. [Epub ahead of print]

Lentiviral gene therapy corrects platelet phenotype and function in Wiskott-Aldrich patients.

Lucia Sereni, Maria Carmina Castiello, Dario Di Silvestre, Patrizia Della Valle, Chiara Brombin, Francesca Ferrua, Maria Pia Cicalese, Loris Pozzi, Maddalena Migliavacca, Maria Ester Bernardo, Claudio Pignata, Roula Farah, Lucia Dora Notarangelo, Nufar Marcus, Lorella Cattaneo, Marco Spinelli, Stefania Giannelli, Marita Bosticardo, Koen van Rossem, Armando D'Angelo, Alessandro Aiuti, Pierluigi Mauri, Anna Villa.

   BACKGROUND: Thrombocytopenia is a serious issue for all classical Wiskott-Aldrich Syndrome (WAS) and X-linked thrombocytopenia (XLT) patients as it causes severe and life-threatening bleedings. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet (PLT) counts remain low in GT -treated patients.
OBJECTIVE: We carefully investigated PLT defects in terms of phenotype and function in untreated WAS patients and assessed the effect of GT treatment on PLT dysfunction.
METHODS: We analyzed a cohort of 20 WAS/XLT patients, fifteen of them receiving GT. PLT phenotype and function were analyzed by electron microscopy, flow cytometry and aggregation assay. PLT protein composition was assessed before and after GT by means of proteomic profile analysis.
RESULTS: We show that PLTs from untreated WAS patients have reduced size, abnormal ultrastructure and hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores PLT size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein (WASp) expression and length of follow-up.
CONCLUSIONS: Our study highlights the coexistence of morphologic and multiple functional defects in PLTs lacking WASp and demonstrates that GT normalizes PLT proteomic profile with consequent restoration of PLT ultrastructure and phenotype, which may explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in XLT only presenting micro-thrombocytopenia.

Keywords:  Gene Therapy; Platelets; Wiskott-Aldrich Syndrome; XLT

DOI:  https://doi.org/10.1016/j.jaci.2019.03.012