Clinical Proteomics

Introduction Glycans have unique characteristics that are significantly different from nucleic acids and proteins in terms of biosynthesis, structures, and functions. Moreover, their isomeric nature and the complex linkages between residues have made glycan analysis a challenging task. Disease development and progression are usually associated with alternations in glycosylation on tissue proteins and/or blood proteins. Glycans released from tissue/blood proteins hence provide a valuable source of biomarkers. In this postgenome era, glycomics is an emerging research field. Glycome refers to a repertoire of glycans in a tissue/cell type, while glycomics is the study of glycome. In the past few years, attempts have been made to develop novel methodologies for quantitative glycomic profiling and to identify potential glycobiomarkers. It can be foreseen that glycomics holds the promise for biomarker discovery. This review provides an overview of the unique features of glycans and the historical applications of such features to biomarker discovery. Future Prospective The concept of glycomics and its recent advancement and future prospective in biomarker research are reviewed. Above all, there is no doubt that glycomics is gaining momentum in biomarker research.

During the past several decades, the understanding of cancer at the molecular level has been primarily focused on mechanisms on how signaling molecules transform homeostatically balanced cells into malignant ones within an individual pathway. However, it is becoming more apparent that pathways are dynamic and crosstalk at different control points of the signaling cascades, making the traditional linear signaling models inadequate to interpret complex biological systems. Recent technological advances in high throughput, deep sequencing for the human genomes and proteomic technologies to comprehensively characterize the human proteomes in conjunction with multiplexed targeted proteomic assays to measure panels of proteins involved in biologically relevant pathways have made significant progress in understanding cancer at the molecular level. It is undeniable that proteomic profiling of differentially expressed proteins under many perturbation conditions, or between normal and "diseased" states is important to capture a first glance at the overall proteomic landscape, which has been a main focus of proteomics research during the past 15-20 years. However, the research community is gradually shifting its heavy focus from that initial discovery step to protein target verification using multiplexed quantitative proteomic assays, capable of measuring changes in proteins and their interacting partners, isoforms, and post-translational modifications (PTMs) in response to stimuli in the context of signaling pathways and protein networks. With a critical link to genotypes (i.e., high throughput genomics and transcriptomics data), new and complementary information can be gleaned from multi-dimensional omics data to (1) assess the effect of genomic and transcriptomic aberrations on such complex molecular machinery in the context of cell signaling architectures associated with pathological diseases such as cancer (i.e., from genotype to proteotype to phenotype); and (2) target pathway-and network-driven changes and map the fluctuations of these functional units (proteins) responsible for cellular activities in response to perturbation in a spatiotemporal fashion to better understand cancer biology as a whole system.

Background: Neurotrauma or injuries to the central nervous system (CNS) are a serious public health problem worldwide. Approximately 75% of all traumatic brain injuries (TBIs) are concussions or other mild TBI (mTBI) forms. Evaluation of concussion injury today is limited to an assessment of behavioral symptoms, often with delay and subject to motivation. Hence, there is an urgent need for an accurate chemical measure in biofluids to serve as a diagnostic tool for invisible brain wounds, to monitor severe patient trajectories, and to predict survival chances. Although a number of neurotrauma marker candidates have been reported, the broad spectrum of TBI limits the significance of small cohort studies. Specificity and sensitivity issues compound the development of a conclusive diagnostic assay, especially for concussion patients. Thus, the neurotrauma field currently has no diagnostic biofluid test in clinical use.

A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26-27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

Background: Cryptococcus neoformans, a basidiomycetous fungus of universal occurrence, is a significant opportunistic human pathogen causing meningitis. Owing to an increase in the number of immunosuppressed individuals along with emergence of drug-resistant strains, C. neoformans is gaining importance as a pathogen. Although, whole genome sequencing of three varieties of C. neoformans has been completed recently, no global proteomic studies have yet been reported. Results: We performed a comprehensive proteomic analysis of C. neoformans var. grubii (Serotype A), which is the most virulent variety, in order to provide protein-level evidence for computationally predicted gene models and to refine the existing annotations. We confirmed the protein-coding potential of 3,674 genes from a total of 6,980 predicted protein-coding genes. We also identified 4 novel genes and corrected 104 predicted gene models. In addition, our studies led to the correction of translational start site, splice junctions and reading frame used for translation in a number of proteins. Finally, we validated a subset of our novel findings by RT-PCR and sequencing. Conclusions: Proteogenomic investigation described here facilitated the validation and refinement of computationally derived gene models in the intron-rich genome of C. neoformans, an important fungal pathogen in humans.

Following the introduction of routine Prostate Specific Antigen (PSA) screening in the early 1990′s, Prostate Cancer (PCa) is often detected at an early stage. There are also a growing number of treatment options available and so the associated mortality rate is generally low. However, PCa is an extremely complex and heterogenous disease and many patients suffer disease recurrence following initial therapy. Disease recurrence commonly results in metastasis and metastatic PCa has an average survival rate of just 3–5 years. A significant problem in the clinical management of PCa is being able to differentiate between patients who will respond to standard therapies and those who may benefit from more aggressive intervention at an earlier stage. It is also acknowledged that for many men the disease is not life threatenting. Hence, there is a growing desire to identify patients who can be spared the significant side effects associated with PCa treatment until such time (if ever) their di...

Purpose: Human urine is an ideal candidate for use in clinical diagnostics. It is easily available, as untrained personnel can collect it. It correlates well with the pathophysiology of a number of diseases, making it a useful source for clinical proteomics. Experimental design: In this article, we give an update of the human urinary peptide database derived from over 13 000 data sets of CE-MS by now. Results: Urine samples from both patients and healthy subjects were analyzed by CE-MS; these included 47 different pathophysiological conditions. Besides defining biomarkers by their experimental parameters, information on their sequences provides fundamental data into the pathological pathways of diseases. Therefore, we have sequenced 953 urinary peptides by using state-of-the-art top-down MS/MS. Identified biomarkers of all clinical proteomic CE-MS studies including their regulation are also listed in this work. Conclusions and clinical relevance: Biomarker discovery can be used in the management of a wide range of diseases, by combining these data sets of the database. Taking this approach, we can reveal details, at a molecular level, on the pathogenesis of a number of diseases, in particular those associated with urine production and excretion.

Background: The urinary proteome of patients undergoing cardiopulmonary bypass (CPB) may provide important insights into systemic and renal changes associated with the procedure. Such information may ultimately provide a basis to differentiate changes or properties associated with the development of acute kidney injury. While mass spectrometry (MS) analysis offers the potential for in-depth compositional analysis it is often limited in coverage and relative quantitation capacity. The aim of this study was to develop a process flow for the preparation and comparison of the intraoperative urinary proteome. Methods: Urines were collected from patients at the start of CPB and 1-h into CPB. Pooled samples (n = 5) from each time point were processed using a modified Filter Assisted Sample Preparation protocol. The resulting peptides were analyzed by 2D-LC-MS/MS and by 1D-LC-MS/MS SWATH (Sequential Window acquisition of All Theoretical fragment ion spectra). Results: The 2D-LC-MS/MS analysis identified 1324 proteins in the two pools, of which 744 were quantifiable. The SWATH approach provided quantitation for 730 proteins, 552 of which overlapped with the common population from the 2D-IDA results. Intensity correlation filtering between the two methods gave 475 proteins for biological interpretation. Proteins displaying greater than threefold changes (>log 2 1.59) at 1-hour CPB relative to the initiation of CPB (26 down-regulated and 22 up-regulated) were selected for further analysis. Up-regulated proteins were enriched in GO terms related to humoral immune response, predominantly innate immunity (C4b, lactotransferrin, protein S100-A8, cathelicidin, myeloperoxidase) and extracellular matrix reorganization (e.g. MMP-9). Conclusions: This study describes a scheme for processing urine from patients undergoing CPB for mass spectrometry-based analysis. The introduction of SWATH into the workflow offers a sample and instrument sparing approach to obtaining consistent in-depth sample analysis. The design of the methodology is such that it can be readily applied to large numbers of clinical samples with the potential for automation. The results also suggest that activation of the innate immune responses occur during cardiac bypass surgery.

Background: Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis. Methods: Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine. Results: Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state. Conclusions: This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions.

Background: Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. Methods: In total, 16 urine samples having more than 10 5 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. Results: Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. Conclusions: Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.

Background: Calcific aortic stenosis (CAS) is the most common heart valve disease in the elderly, representing an important economic and social burden in developed countries. Currently, there is no way to predict either the onset or progression of CAS, emphasizing the need to identify useful biomarkers for this condition. Methods: We performed a multi-proteomic analysis on different kinds of samples from CAS patients and healthy donors: tissue, secretome and plasma. The results were validated in an independent cohort of subjects by immunohistochemistry, western blotting and selected reaction monitoring. Results: Alpha 1 antichymotrypsin (AACT) abundance was altered in the CAS samples, as confirmed in the validation phase. The significant changes observed in the amounts of this protein strongly suggest that it could be involved in the molecular mechanisms underlying CAS. In addition, our results suggest there is enhanced release of AACT into the extracellular fluids when the disease commences. Conclusions: The significant increase of AACT in CAS patients suggests it fulfils an important role in the physiopathology of this disease. These results permit us to propose that AACT may serve as a potential marker for the diagnosis of CAS, with considerable clinical value.

More than 200 proteins associate with human spliceosomes, but little is known about their relative abundances in a given spliceosomal complex. Here we describe a novel two-dimensional (2D) electrophoresis method that allows separation of high-molecular-mass proteins without in-gel precipitation and thus without loss of protein. Using this system coupled with mass spectrometry, we identified 171 proteins altogether on 2D maps of stage-specific spliceosomal complexes. By staining with a fluorescent dye with a wide linear intensity range, we could quantitate and categorize proteins as present in high, moderate, or low abundance. Affinitypurified human B, B act , and C complexes contained 69, 63, and 72 highly/moderately abundant proteins, respectively. The recruitment and release of spliceosomal proteins were followed based on their abundances in A, B, B act , and C spliceosomal complexes. Staining with a phospho-specific dye revealed that approximately one-third of the proteins detected in human spliceosomal complexes by 2D gel analyses are phosphorylated. The 2D gel electrophoresis system described here allows for the first time an objective view of the relative abundances of proteins present in a particular spliceosomal complex and also sheds additional light on the spliceosome's compositional dynamics and the phosphorylation status of spliceosomal proteins at specific stages of splicing.

Meningiomas are one of the most prevalent primary brain tumors. Our study aims to obtain mechanistic insights of meningioma pathobiology using mass spectrometry-based label-free quantitative proteome analysis to identifying druggable targets and perturbed pathways for therapeutic intervention. Label-free based proteomics study was done from peptide samples of 21 patients and 8 non-tumor controls which were followed up with Phosphoproteomics to identify the kinases and phosphorylated components of the perturbed pathways. In silico approaches revealed perturbations in extracellular matrix remodeling and associated cascades. To assess the extent of influence of Integrin and PI3K-Akt pathways, we used an Integrin Linked Kinase inhibitor on patient-derived meningioma cell line and performed a transcriptomic analysis of the components. Furthermore, we designed a Targeted proteomics assay which to the best of our knowledge for very first-time enables identification of peptides from 54 meningioma patients via SRM assay to validate the key proteins emerging from our study. This resulted in the identification of peptides from CLIC1, ES8L2, and AHNK many of which are receptors and kinases and are difficult to be characterized using conventional approaches. Furthermore, we were also able to monitor transitions for proteins like NEK9 and CKAP4 which have been reported to be associated with meningioma pathobiology. We believe, this study can aid in designing peptide-based validation assays for meningioma patients as well as IHC studies for clinical applications.

The present review reports the principles, fundamentals and some applications of two-dimensional difference gel electrophoresis for analytical proteomics based on plant proteome analysis, also emphasizing some advantages of 2-D DIGE over 2-D PAGE techniques. Some fluorescent protein labeling reagents, methods of protein labeling, models of 2-D DIGE experiments, and some limitations of this technique are presented and discussed in terms of 2-D DIGE plant proteomes. Finally, some practical applications of this technique are pointed out, emphasizing its potentialities in plant proteomics.

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at ,1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample.

Membrane proteins play important roles in various plasma membrane (PM) activities such as signal transduction and cell recognition. However, a comprehensive proteomic study of membrane proteins remains difficult. Different strategies have been employed to study PM proteome, but little effort has been made to systematically evaluate them. In the present work, liver PM was prepared by subcellular fractionation and an aliquot was washed by sodium carbonate. After evaluation of the PM fraction by electron microscopy and Western blotting, proteins in both original and carbonate washed PM were identified by either 2-DE coupled MALDI-TOF-MS or shotgun strategies. Then protein characteristics such as molecular weight, pI, grand average hydrophobicity, subcellular location, and transmembrane domains were systematically compared. The comparative analysis showed that shotgun strategies were more suitable to identify membrane proteins, while 2-DE-based strategies may serve as a complement. Furthermore, carbonate washing obviously enriched the integral membrane proteins. All the results suggested that the strategy combining carbonate washing and shotgun identification was the optimum strategy to study human liver PM proteome. Using this strategy, 260 high-confidence proteins were identified, wherein 139 were integral membrane proteins which had 1–17 transmembrane domains.

Data mining application to proteomic data from mass spectrometry has gained much interest in recent years. Advances made in proteomics and mass spectrometry have resulted in considerable amount of data that cannot be easily visualized or interpreted. Mass spectral proteomic datasets are typically high dimensional but with small sample size. Consequently, advanced artificial intelligence and machine learning algorithms are increasingly being used for knowledge discovery from such datasets. Their overall goal is to extract useful information that leads to the identification of protein biomarker candidates. Such biomarkers could potentially have diagnostic value as tools for early detection, diagnosis, and prognosis of many diseases. The purpose of this review is to focus on the current trends in mining mass spectral proteomic data. Special emphasis is placed on the critical steps involved in the analysis of surface-enhanced laser desorption/ionization mass spectrometry proteomic data. Examples are drawn from previously published studies and relevant data mining terminology and techniques are explained.

Over the last few decades of biomedical research, animal models of neuromuscular diseases have been widely used for determining pathological mechanisms and for testing new therapeutic strategies. With the emergence of high-throughput proteomics technology, the identification of novel protein factors involved in disease processes has been decisively improved. This review outlines the usefulness of the proteomic profiling of animal disease models for the discovery of new reliable biomarkers, for the optimization of diagnostic procedures and the development of new treatment options for skeletal muscle disorders. Since inbred animal strains show genetically much less interindividual differences as compared to human patients, considerably lower experimental repeats are capable of producing meaningful proteomic data. Thus, animal model proteomics can be conveniently employed for both studying basic mechanisms of molecular pathogenesis and the effects of drugs, genetic modifications or cell-based therapies on disease progression. Based on the results from comparative animal proteomics, a more informed decision on the design of clinical proteomics studies could be reached. Since no one animal model represents a perfect pathobiochemical replica of all of the symptoms seen in complex human disorders, the proteomic screening of novel animal models can also be employed for swift and enhanced protein biochemical phenotyping.

The cerebrospinal fluid (CSF) circulates within the CNS where it plays an essential physiological role in homeostasis of neuronal cells. This biological fluid has an important protein diversity that results from both filtration of serum through the blood-brain barrier and production/secretion of neuronal peptides and proteins. Changes in CSF composition depend on blood proteome, CSF circulation alterations, as well as physiological or pathological brain status. Hence, CSF proteomic analysis gives a unique opportunity to detect and describe biomarkers in neurological affections. Although lumbar puncture is considered as invasive, post lumbar puncture events remain rare and minor. Nevertheless, CSF biological analysis is currently limited to a small number of parameters and clinical situations suggesting mainly meningitis, malignancies or dementia. Few CSF proteomic studies have been performed in comparison to those on blood. In this review, we will provide a proteomics description of the CSF, summarize the current clinical use of this fluid and describe its clinical proteomics examination.

A proof-of-concept demonstration of the use of label-free quantitative glycoproteomics for biomarker discovery workflow is presented here, using a mouse model for skin cancer as an example. Blood plasma was collected from 10 control mice, and 10 mice having a mutation in the p19 ARF gene, conferring them high propensity to develop skin cancer after carcinogen exposure. We enriched for N-glycosylated plasma proteins, ultimately generating deglycosylated forms of the modified tryptic peptides for liquid chromatography mass spectrometry (LC-MS) analyses. LC-MS runs for each sample were then performed with a view to identifying proteins that were differentially abundant between the two mouse populations. We then used a recently developed computational framework, Corra, to perform peak picking and alignment, and to compute the statistical significance of any observed changes in individual peptide abundances. Once determined, the most discriminating peptide features were then fragmented and identified by tandem mass spectrometry with the use of inclusion lists. We next assessed the identified proteins to see if there were sets of proteins indicative of specific biological processes that correlate with the presence of disease, and specifically cancer, according to their functional annotations. As expected for such sick animals, many of the proteins identified were related to host immune response. However, a significant number of proteins also directly associated with processes linked to cancer development, including proteins related to the cell cycle, localisation, trasport, and cell death. Additional analysis of the same samples in profiling mode, and in triplicate, confirmed that replicate MS analysis of the same plasma sample generated less variation than that observed between plasma samples from different individuals, demonstrating that the reproducibility of the LC-MS platform was sufficient for this application. These results thus show that an LC-MS-based workflow can be a useful tool for the generation of candidate proteins of interest as part of a disease biomarker discovery effort.

Bifunctional dCTP deaminase – Deoxyuridine triphosphatase (dUTPase) from Mycobacterium tuberculosis was selected as drug target from database DDTRP (Database of Drug Targets for Resistant Pathogens) that provides a list of potential drug target for different drug resistant infectious diseases including Tuberculosis (TB).The Deoxycytidine triphosphate deaminase ( dCTP) was docked with 131 molecules screened from four different databases, Pubchem, Drugbank, Zinc, and Chembank. Ninety three molecules were successfully docked with dCTP. Eight molecules, two from each database screened were suggested as better molecules over others to bind with dCTP. These eight molecules taken further studies to check their efficiency in vitro.

Background Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease. Results Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia. Conc...

Background: ALK tyrosine kinase inhibition has become a mainstay in the clinical management of ALK fusion positive NSCLC patients. Although ALK mutations can reliably predict the likelihood of response to ALK tyrosine kinase inhibitors (TKIs) such as crizotinib, they cannot reliably predict response duration or intrinsic/extrinsic therapeutic resistance. To further refine the application of personalized medicine in this indication, this study aimed to identify prognostic proteomic biomarkers in ALK fusion positive NSCLC patients to crizotinib. Methods: Twenty-four patients with advanced NSCLC harboring ALK fusion were administered crizotinib in a phase IV trial which included blood sampling prior to treatment. Targeted proteomics of 327 proteins using MRM-MS was used to measure plasma levels at baseline (including pre-treatment and early treatment blood samples) and assess potential clinical association. Results: Patients were categorized by duration of response: long-term responders [PFS ≥ 24 months (n = 7)], normal responders [3 < PFS < 24 months (n = 10)] and poor responders [PFS ≤ 3 months (n = 5)]. Several proteins were identified as differentially expressed between long-term responders and poor responders, including DPP4, KIT and LUM. Next, using machine learning algorithms, we evaluated the classification potential of 40 proteins. Finally, by integrating the different analytic methods, we selected 22 proteins as potential candidates for a blood-based prognostic signature of response to crizotinib in NSCLC patients harboring ALK fusion. Conclusion: In conjunction with ALK mutation, the expression of this proteomic signature may represent a liquid biopsy-based marker of long-term response to crizotinib in NSCLC. Expanding the utility of prognostic biomarkers of response duration could influence choice of therapy, therapeutic sequencing, and potentially the need for alternative or combination therapy.

The “omics revolution: beyond genomics” satellite meeting, run under the auspices of the Australian Peptide Association, The Human Proteome Organisation (HUPO) and the HUPO Australia/New Zealand Chromosome 7 initiative, was held at the Oaks Resort, Port Douglas, Queensland, Australia, on 8th September 2019, immediately prior to the 13th Australian Peptide Conference. The meeting, which had over 100 participants representing Australasia, Europe and America, focused on recent advances in omics-related technologies, including mass spectrometry, biosensors and CryoEM, which will assist in the clinical translation of proteomics towards precision/personalized medicine. An overview of the conference and a summary of the oral presentations are presented.

Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders. In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach. To ...

Background: Rheumatoid arthritis and osteoarthritis are two common musculoskeletal disorders that affect the joints. Despite high prevalence rates, etiological factors involved in these disorders remain largely unknown. Dissecting the molecular aspects of these disorders will significantly contribute to improving their diagnosis and clinical management. In order to identify proteins that are differentially expressed between these two conditions, a quantitative proteomic profiling of synovial fluid obtained from rheumatoid arthritis and osteoarthritis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. Results: We have identified 575 proteins out of which 135 proteins were found to be differentially expressed by ≥3-fold in the synovial fluid of rheumatoid arthritis and osteoarthritis patients. Proteins not previously reported to be associated with rheumatoid arthritis including, coronin-1A (CORO1A), fibrinogen like-2 (FGL2), and macrophage capping protein (CAPG) were found to be upregulated in rheumatoid arthritis. Proteins such as CD5 molecule-like protein (CD5L), soluble scavenger receptor cysteine-rich domain-containing protein (SSC5D), and TTK protein kinase (TTK) were found to be upregulated in the synovial fluid of osteoarthritis patients. We confirmed the upregulation of CAPG in rheumatoid arthritis synovial fluid by multiple reaction monitoring assay as well as by Western blot. Pathway analysis of differentially expressed proteins revealed a significant enrichment of genes involved in glycolytic pathway in rheumatoid arthritis. Conclusions: We report here the largest identification of proteins from the synovial fluid of rheumatoid arthritis and osteoarthritis patients using a quantitative proteomics approach. The novel proteins identified from our study needs to be explored further for their role in the disease pathogenesis of rheumatoid arthritis and osteoarthritis. Sartaj Ahmad and Raja Sekhar Nirujogi contributed equally to this article.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is responsible for 10% of cases of the end stage renal disease. Early diagnosis, especially of potential fast progressors would be of benefit for efficient planning of therapy. Urine excreted proteome has become a promising field of the search for marker patterns of renal diseases including ADPKD. Up to now however, only the low molecular weight fraction of ADPKD proteomic fingerprint was studied. The aim of our study was to characterize the higher molecular weight fraction of urinary proteome of ADPKD population in comparison to healthy controls as a part of a general effort aiming at exhaustive characterization of human urine proteome in health and disease, preceding establishment of clinically useful disease marker panel.

Introduction Saliva is a body fluid that holds promise for use as a diagnostic fluid for detecting diseases. Salivary proteins are known to be heavily glycosylated and are known to play functional roles in the oral cavity. We identified N-linked glycoproteins in human whole saliva, as well as the N-glycoproteins in parotid, submandibular, and sublingual glandular fluids.

Improved methods for the early diagnosis of colorectal cancer by way of sensitive and specific tumour markers are highly desirable. Therefore, efficient strategies for biomarker discovery are urgently needed. Here we present an approach that is based on the direct experimental access to proteins released by SW620 human colorectal cancer cells in vitro. A 2-D map and a catalogue of this subproteome -here termed the secretome -were established comprising more than 320 identified proteins which translate into approximately 220 distinct genes. As the majority of the secretome constituents were nominally cellular proteins, we directly compared the secretome and the total proteome by 2-D-DIGE analysis. We provide evidence that unspecific release through cell death, classical secretion, ectodomain shedding, and exosomal release contribute to the secretome in vitro, presumably reflecting the mechanisms in vivo which lead to the occurrence of tumour-specific proteins in the circulation. These data together with the fact that the SW620 secretome catalogue, as presented here, does comprise a large number of known and novel biomarker candidates, validates our approach to isolate and characterize the tumour cell secretome in vitro as a rich source for tumour biomarkers.

In this review we would like to highlight the importance of acute-phase proteins as sensor of diseases. Both acute-phase protein levels and glycosylation have been reported to be altered in inflammation and other diseases including cancer. Factors that promote acute-phase protein synthesis and enhance the expression of specific glycosyltransferases, such as sialyltransferases and fucosyltransferases, may be up-regulated in some tumours and would explain the changes in acute-phase protein levels and the specific N-glycosylation modifications of some acute-phase proteins in cancer. However, further studies are required to define the potential clinical application of these acute-phase protein cancer-specific modifications as possible cancer diagnostic or monitoring tools.

Glycosylation studies of plasma proteins can reveal information about the onset and progression of diseases, where in the glycan biosynthetic pathways are disturbed as in rheumatoid arthritis (RA). The present study was focused on analysis of Olinked glycoproteins of plasma in RA patients. Two dimensional gel electrophoresis of jacalin bound plasma of RA patients revealed a number of differentially expressed protein spots as compared to healthy controls. Eighteen protein spots were found to have statistically significant (p,0.05) difference in their expression level from four sets of gels and were identified by MALDI-TOF MS. Most of the identified proteins were predicted to be O-glycosylated proteins by Net-O-Gly 3.1 algorithm. Among these the alpha 2HS glycoprotein (A2HSG) was found to be down regulated whereas inter alpha trypsin inhibitor H4 (ITIH4) was up regulated and this was validated by Western blotting. The glycosylation studies showed the reduced N-linked sialylation of A2HSG in RA patients. Altered glycoprotein expression and functional as well as structural studies of glycans might help in the diagnosis of RA and understanding the disease pathogenesis.

INTRODUCTION: Early detection, assessment of disease progression, and application of an appropriate therapeutic intervention are all important for the care of patients with type 2 diabetes. Currently, however, there is no simple test for early detection of type 2 diabetes. Established diagnostic tests for the disease including oral glucose tolerance, fasting blood glucose, and hemoglobin A1c are relatively late markers where the disease has already progressed. Since blood is in direct contact with many tissues, we hypothesized that pathological tissue changes are likely to be reflected in proteomic profiles of plasma. METHODS: Mice were reared either on regular chow or a high-fat diet at weaning and several physiological responses (i.e., weight, fasting plasma glucose and insulin, and glucose tolerance) were monitored at regular time intervals. Plasma was collected at regular intervals for proteomic analysis by two-dimensional gel electrophoresis and subsequent mass spectrometry. RE...

Introduction: Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite. Methods: In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy. Results and conclusion: We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.

Background: In approximately 80% of patients, ovarian cancer is diagnosed when the patient is already in the advanced stages of the disease. CA125 is currently used as the marker for ovarian cancer; however, it lacks specificity and sensitivity for detecting early stage disease. There is a critical unmet need for sensitive and specific routine screening tests for early diagnosis that can reduce ovarian cancer lethality by reliably detecting the disease at its earliest and treatable stages. Results: In this study, we investigated the N-linked sialylated glycopeptides in serum samples from healthy and ovarian cancer patients using Lectin-directed Tandem Labeling (LTL) and iTRAQ quantitative proteomics methods. We identified 45 N-linked sialylated glycopeptides containing 46 glycosylation sites. Among those, ten sialylated glycopeptides were significantly up-regulated in ovarian cancer patients' serum samples. LC-MS/MS analysis of the non-glycosylated peptides from the same samples, western blot data using lectin enriched glycoproteins of various ovarian cancer type samples, and PNGase F (+/−) treatment confirmed the sialylation changes in the ovarian cancer samples. Conclusion: Herein, we demonstrated that several proteins are aberrantly sialylated in N-linked glycopeptides in ovarian cancer and detection of glycopeptides with abnormal sialylation changes may have the potential to serve as biomarkers for ovarian cancer.

Mitral regurgitation is a frequent valvular heart disease affecting around 2.5 % of the population with prevalence directly related to aging. Degeneration of mitral valve is broadly considered as a passive ongoing pathophysiological process and little is known about its physiological deregulation. The purpose of this study was to highlight new biomarkers of mitral regurgitation in order to decipher the underlying pathological mechanism as well as to allow the diagnosis and the monitoring of the disease. Modulation of various blood proteins expression was examined in patients suffering from different grades of mitral regurgitation (mild, moderate and severe) compared to healthy controls. To this end, several routine clinical assays and the multi analyte profile technology targeting 184 proteins were used. High-density lipoprotein, apolipoprotein-A1, haptoglobin and haptoglobin-α2 chain levels significantly decreased proportionally to the degree of mitral regurgitation when compared t...

Background Intrahepatic cholangiocarcinoma (iCCA) is a malignancy that arises from the intrahepatic biliary tree, showing high mortality rates due to its late clinical presentation and limited treatment options. iCCA is characterized by a dense, reactive desmoplastic stroma marked by a dramatic accumulation of extracellular matrix (ECM). Although recent results strongly suggest a relationship between increasing desmoplastic stroma and the enhanced malignant behaviour of iCCA, the importance of ECM proteins in the pathogenesis of iCCA still have to be addressed. Methods iCCA ECM fibrillar structural organization was characterized by histological analysis. ECM proteome profiles from decellularized iCCA and surrounding noncancerous tissues were analysed by nLC coupled to MALDI-TOF/TOF analysis. Results iCCA tissues displayed high levels of collagen fibers and low abundance of reticular and elastic fibers, suggesting stiffness and loss of polarity. The ECM proteome profiles of iCCA samp...

Background: To develop a new molecular targeted treatment for brain (AVMs), identification of membrane proteins that are localised on the AVM endothelium is crucial. Current treatment methods are surgery and radiosurgery. However, complete occlusion post radiosurgery are achieved within 3 years, while patient remain at risk of haemorrhage. This study aims to identify potential protein targets in AVM endothelial cells that discriminate these vessels from normal vessels; these proteins targets will be investigated for the molecular therapy of brain AVMs to promote rapid thrombosis after radiosurgery. Methods: We employed in vitro biotinylation that we developed, and mass spectrometry to detect cell surfaceexposed proteins in cultures of murine cerebral endothelial cells (bEnd.3). Two forms of mass spectrometry were applied (iTRAQ-MS and MS E) to identify and quantify membrane protein expression at various time-points following irradiation which simulates a radiosurgical treatment approach. Immunocytochemistry was used to confirm the expression of selected membrane proteins. ProteinPilot V4.0 software was used to analyse the iTRAQ-MS data and the MS E data was analysed using ProteinLynx Global Server version 2.5 software. Results: The proteomics data revealed several differentially expressed membrane proteins between irradiated and non-irradiated cells at specific time points, e.g. PECAM-1, cadherin-5, PDI, EPCR and integrins. Immunocytochemistry data confirmed the expression of these proteins. Conclusion: Cell surface protein biotinylation and proteomics analysis successfully identified membrane proteins from murine brain endothelial cells in response to irradiation. This work suggests potential target protein molecules for evaluation in animal models of brain-AVM.

The objective of the present study is to identify proteins that change in the extent of the modification with O-linked N-acetylglucosamine (O-GlcNAcylation) in the kidney from diabetic model Goto-Kakizaki (GK) rats, and to discuss the relation between O-GlcNAcylation and the pathological condition in diabetes. O-GlcNAcylated proteins were identified by two-dimensional gel electrophoresis, immunoblotting and peptide mass fingerprinting. The level of O-GlcNAcylation of these proteins was examined by immunoprecipitation, immunoblotting and in situ Proximity Ligation Assay (PLA). O-GlcNAcylated proteins that changed significantly in the degree of O-GlcNAcylation were identified as cytoskeletal proteins (α-actin, α-tubulin, α-actinin 4, myosin) and mitochondrial proteins (ATP synthase β, pyruvate carboxylase). The extent of O-GlcNAcylation of the above proteins increased in the diabetic kidney. Immunofluorescence and in situ PLA studies revealed that the levels of O-GlcNAcylation of acti...

Background: An early, reliable and noninvasive method of early pregnancy diagnosis is prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also help in to reduce the calving interval and rebreeding time which is beneficial for industries as well as farmers. The aim of this work is to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal we performed DIGE and LFQ for identification of protein which has significant differential expression during pregnancy. Results: DIGE experiment revealed a total of eleven differentially expressed proteins out of which nine were up regulated having fold change ≥1.5 in all time points. The LFQ data analysis revealed 195 differentially expressed proteins (DEPs) out of 28 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Bioinformatics analysis of DEPs showed that a majority of proteins were involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. Conclusion: This is first report on differentially expressed protein during various time points of pregnancy in cow to our best knowledge. In our work, we identified few proteins such MBP, SERPIN, IGF which were differentially expressed and actively involved in various activities related to pregnancy such as embryo implantation, establishment and maintenance of pregnancy. Due to their involvement in these events, these can be considered as biomarker for pregnancy but further validation of is required.