Seong Beom Ahn

Accelerating the search for the missing proteins in the human proteome

The Human Proteome Project (HPP) aims to discover high-stringency data for all proteins encoded by the human genome. Currently, ∼18% of the proteins in the human proteome (the missing proteins) do not have high-stringency evidence (for example, mass spectrometry) confirming their existence, while much additional information is available about many of these missing proteins. Here, we present MissingProteinPedia as a community resource to accelerate the discovery and understanding of these missing proteins.

Epithelial and Stromal Cell Urokinase Plasminogen Activator Receptor Expression Differentially Correlates with Survival in Rectal Cancer Stages B and C Patients

Urokinase plasminogen activator receptor (uPAR) has been proposed as a potential prognostic factor for colorectal cancer (CRC) patient survival. However, CRC uPAR expression remains controversial, especially regarding cell types where uPAR is overexpressed (e.g., epithelium (uPARE) or stroma-associated cells (uPARS)) and associated prognostic relevance. In this study, two epitope-specific anti-uPAR monoclonal antibodies (MAbs) could discriminate expression of uPARE from uPARS and were used to examine this association with survival of stages B and C rectal cancer (RC) patients. Using immunohistochemistry, MAbs #3937 and R4 were used to discriminate uPARE from uPARS respectively in the central and invasive frontal regions of 170 stage B and 179 stage C RC specimens. Kaplan-Meier and Cox regression analyses were used to determine association with survival. uPAR expression occurred in both epithelial and stromal compartments with differential expression observed in many cases, indicating uPARE and uPARS have different cellular roles. In the central and invasive frontal regions, uPARE was adversely associated with overall stage B survival (HR = 1.9; p = 0.014 and HR = 1.5; p = 0.031, respectively) reproducing results from previous studies. uPARS at the invasive front was associated with longer stage C survival (HR = 0.6; p = 0.007), reflecting studies demonstrating that macrophage peritumoural accumulation is associated with longer survival. This study demonstrates that different uPAR epitopes should be considered as being expressed on different cell types during tumour progression and at different stages in RC. Understanding how uPARE and uPARS expression affects survival is anticipated to be a useful clinical prognostic marker of stages B and C RC.

Is isolation of comprehensive human plasma peptidomes an achievable quest

The low molecular weight (LMW; <10kDa)* plasma peptidome has been considered a source of useful diagnostic biomarkers and potentially therapeutic molecules, as it contains many cytokines, peptide hormones, endogenous peptide products and potentially bioactive fragments derived from the parent proteome. The small size of the peptides allows them almost unrestricted vascular and interstitial access, and hence distribution across blood-brain barriers, tumour and other vascular permeability barriers. Therefore, the peptidome may carry specific signatures or fingerprints of an individuals health, wellbeing or disease status. This occurs primarily because of the advantage the peptidome has in being readily accessible in human blood and/or other biofluids. However, the co-expression of highly abundant proteins (>10kDa) and other factors present inherently in human plasma make direct analysis of the blood peptidome one of the most challenging tasks faced in contemporary analytical biochemistry. A comprehensive compendium of extraction and fractionation tools has been collected concerning the isolation and micromanipulation of peptides. However, the search for a reliable, accurate and reproducible single or combinatorial separation process for capturing and analysing the plasma peptidome remains a challenge. This review outlines current techniques used for the separation and detection of plasma peptides and suggests potential avenues for future investigation. This article is part of a Special Issue entitled: HUPO 2014.

Characterization of the interaction between heterodimeric αvβ6 integrin and urokinase plasminogen activator receptor (uPAR) using functional proteomics

Urokinase plasminogen activator receptor (uPAR) and the epithelial integrin αvβ6 are thought to individually play critical roles in cancer metastasis. These observations have been highlighted by the recent discovery (by proteomics) of an interaction between these two molecules, which are also both implicated in the epithelial-mesenchymal transition (EMT) that facilitates escape of cells from tissue barriers and is a common signature of cancer metastases. In this study, orthogonal in cellulo and in vitro functional proteomic approaches were used to better characterize the uPAR·αvβ6 interaction. Proximity ligation assays (PLA) confirmed the uPAR·αvβ6 interaction on OVCA429 (ovarian cancer line) and four different colon cancer cell lines including positive controls in cells with de novo β6 subunit expression. PLA studies were then validated using peptide arrays, which also identified potential physical sites of uPAR interaction with αvβ6, as well as verifying interactions with other known uPAR ligands (e.g., uPA, vitronectin) and individual integrin subunits (i.e., αv, β1, β3, and β6 alone). Our data suggest that interaction with uPAR requires expression of the complete αβ heterodimer (e.g., αvβ6), not individual subunits (i.e., αv, β1, β3, or β6). Finally, using in silico structural analyses in concert with these functional proteomics studies, we propose and demonstrate that the most likely unique sites of interaction between αvβ6 and uPAR are located in uPAR domains II and III.

Correlations between Integrin ανβ6 Expression and Clinico-Pathological Features in Stage B and Stage C Rectal Cancer

Integrin ανβ6 is highly expressed in a range of human cancers and frequently correlates with patient survival. This study examines correlations between ανβ6 expression and patient clinico-pathological features in Stage B and Stage C rectal cancer, including overall survival. Expression of ανβ6 was measured in 362 Stage B or C rectal cancer tissue samples at the tumour central region, invasive tumour front and adjacent non-neoplastic mucosa using immunohistochemistry. Distribution of ανβ6 was found to be significantly higher at the invasive front compared to central regions of the tumour (p<0.001) or adjacent non-neoplastic mucosa (p<0.001) suggesting ανβ6 plays a role in tumour cell invasion. However, integrin ανβ6 expression was not associated with clinico-pathological features or overall survival indicating it is not an independent prognostic marker differentiating Stage B or C rectal cancer. Previous ανβ6 studies have suggested the expression of ανβ6 is involved in the earlier stages (i.e. Stages A/B) of tumour progression rather than the later stages (i.e. Stages C/D). However, our study has revealed that in rectal cancer ανβ6 expression does not increase between Stages B and C, but may occur earlier, namely before or during Stage B cancer.

A site for direct integrin αvβ6·uPAR interaction from structural modelling and docking

Integrin αvβ6 is an epithelially-restricted heterodimeric transmembrane glycoprotein, known to interact with the urokinase plasminogen activating receptor (uPAR), playing a critical role in cancer progression. While the X-ray crystallographic structures of segments of other integrin heterodimers are known, there is no structural information for the complete αvβ6 integrin to assess its direct interaction with uPAR. We have performed structural analysis of αvβ6·uPAR interactions using model data with docking simulations to pinpoint their interface, in accord with earlier reports of the β-propeller region of integrin α-chain interacting with uPAR. Interaction of αvβ6·uPAR was demonstrated by our previous study using immunoprecipitation coupled with proteomic analysis by mass spectrometry. Recently this interaction was validated with proximity ligation assays and peptide arrays. The data suggested that two potential peptide regions from domain II and one peptide region from domain III of uPAR, interact with αvβ6 integrin. Only the peptide region from domain III is consistent with the three-dimensional interaction site proposed in this study. The molecular basis of integrin αvβ6·uPAR binding using structural data is discussed for its implications as a potential therapeutic target in cancer management.

Evaluation of two high-throughput proteomic technologies for plasma biomarker discovery in immunotherapy-treated melanoma patients

BackgroundSelective kinase and immune checkpoint inhibitors, and their combinations, have significantly improved the survival of patients with advanced metastatic melanoma. Not all patients will respond to treatment however, and some patients will present with significant toxicities. Hence, the identification of biomarkers is critical for the selection and management of patients receiving treatment. Biomarker discovery often involves proteomic techniques that simultaneously profile multiple proteins but few studies have compared these platforms.MethodsIn this study, we used the multiplex bead-based Eve Technologies Discovery assay and the aptamer-based SomaLogic SOMAscan assay to identify circulating proteins predictive of response to immunotherapy in melanoma patients treated with combination immune checkpoint inhibitors. Expression of four plasma proteins were further validated using the bead-based Millipore Milliplex assay.ResultsBoth the Discovery and the SOMAscan assays detected circulating plasma proteins in immunotherapy-treated melanoma patients. However, these widely used assays showed limited correlation in relative protein quantification, due to differences in specificity and the dynamic range of protein detection. Protein data derived from the Discovery and Milliplex bead-based assays were highly correlated.ConclusionsOur study highlights significant limitations imposed by inconsistent sensitivity and specificity due to differences in the detection antibodies or aptamers of these widespread biomarker discovery approaches. Our findings emphasize the need to improve these technologies for the accurate identification of biomarkers.

A Systematic Bioinformatics Approach to Identify High Quality Mass Spectrometry Data and Functionally Annotate Proteins and Proteomes

In the past decade, proteomics and mass spectrometry have taken tremendous strides forward, particularly in the life sciences, spurred on by rapid advances in technology resulting in generation and conglomeration of vast amounts of data. Though this has led to tremendous advancements in biology, the interpretation of the data poses serious challenges for many practitioners due to the immense size and complexity of the data. Furthermore, the lack of annotation means that a potential gold mine of relevant biological information may be hiding within this data. We present here a simple and intuitive workflow for the research community to investigate and mine this data, not only to extract relevant data but also to segregate usable, quality data to develop hypotheses for investigation and validation. We apply an MS evidence workflow for verifying peptides of proteins from ones own data as well as publicly available databases. We then integrate a suite of freely available bioinformatics analysis and annotation software tools to identify homologues and map putative functional signatures, gene ontology and biochemical pathways. We also provide an example of the functional annotation of missing proteins in human chromosome 7 data from the NeXtProt database, where no evidence is available at the proteomic, antibody, or structural levels. We give examples of protocols, tools and detailed flowcharts that can be extended or tailored to interpret and annotate the proteome of any novel organism.

Addition of protease inhibitors to EDTA containing blood collection tubes does not deliver significant advantage for preservation of plasma for down-stream analysis

Blood collection protocols are a crucial pre-analytical variable that must be carefully defined during the development of any reproducible biomarker assay. It is essential to limit endogenous proteolysis during collection and preservation of the plasma sample in a state compatible with down-stream analysis. This study tested the proteome and peptidome stability of matched plasma samples collected from healthy individuals. Each set of samples was donated simultaneously and stored/handled identically using three different types of blood collection tubes; (i) spray-dried EDTA (ii) EDTA with polymer gel, and (iii) EDTA tubes containing a proprietary cocktail of protease inhibitors (P100TM). All samples were investigated with proteomic and peptidomic approaches using 2D difference gel electrophoresis (2D DIGE) and solid phase extraction (SPE) followed by ESI-MS/MS (Electrospray Ionization) analysis. Results from 2D DIGE showed no significant influence of the different anticoagulant tubes on the level of plasma proteins observed by 2D DIGE, although minor variations in one protein per individual in each individual were observed, which statistically appears to be a random occurrence. In contrast, results obtained from the peptidomic analysis demonstrated a lower number of endogenous peptides suggesting preservation of some proteins in plasma treated with the protease inhibitor cocktail. However every plasma sample analysed showed endogenous peptides obtained from six proteins (namely Fibrinogen alpha chain, Complement C4-A, Complement C4-B, Alpha-2-antiplasmin, Complement C3 and Apolipoprotein E) irrespective of the anticoagulant used for collection. These data should assist researchers in their choice of ideal anticoagulants for plasma proteome and peptidome research. Correspondence to: Mark S. Baker, Australian School of Advanced Medicine, Rm1, Level 1, 75 Talavera Road, Macquarie University, NSW 2109, Australia, Tel: +61 2 9850 8211; Fax: +61 2 9850 7433; E mail: mark.baker@mq.edu.au Received: September 23, 2016; Accepted: October 27, 2016; Published: October 31, 2016 Introduction Clinical proteomics aims to discover, verify and validate diseasespecific protein biomarkers that can be applied for diagnostic, prognostic or therapeutic purposes[1-4]. Blood is the most commonly available biological sample for proteomic analysis leading to the identification of biomarkers [5]. Biomarkers can exist in a variety of different molecular and cellular forms e.g., proteins, DNA, circulating tumor cells. Development of biomarker assays and analytical validation are complex processes, that need to consider a number of potential factors (analytical variables, biological variables and cohort composition) and the nature of the application (e.g., invasiveness of the test, privacy, patient compliance and cost) [6].In order to identify and quantify a biomarker accurately, and to ensure the clinical success of candidate biomarkers, they must be stable during sample collection, preparation and purification and must be present at a detectable concentration. Therefore the preanalytical and storage conditions [7,8] need to be carefully established to prevent any degradation during sample handling, processing, transfer or storage [9].Plasma profiling is an essential part of proteomic research that combines protein separation and characterization technologies [10-12]. Following individual attempts by several industry and clinical proteomics/peptidomics research groups, the HUPO Plasma Proteome Project [13-15]was established in 2002 with the goal of comprehensively characterising the protein constituents of human plasma. The project involved 35 laboratories from around the globe and initially identified 3,020 plasma proteins[16] with high stringency (two or more peptides). Most laboratories focussed exclusively on plasma proteins and contributed to one of the major aims, namely that of creating a plasma proteome inventory. Although there was a growing awareness that reproducibility would be compromised if variability between samples was ignored, the amount of data required to substantiate the effect of such variables on results obtained from proteomics experiments from plasma was not insignificant [17]. In 2005, the HUPO Plasma Proteome Project (PPP) [17] Sample Collection and Handling Committee released a list of variables that need to be considered for the plasma collection and handling process (sample type, collection system, handling issues e.g., stabilization, processing storage and potential effects of additives) [18,19]. A number of plasma proteomic studies have reported that many of the observed peptides were generated through the activity of endogenous proteases [20] (e.g. amino peptidases[21] and carboxypeptidases [22]). Proteolysis commonly occurs as a time dependant degradation process that can significantly influence comparative analyses between blood samples [20,23,24]. Therefore complications and lack of consistency in the sample collection process Mahboob S (2016) Addition of protease inhibitors to EDTA containing blood collection tubes does not deliver significant advantage for preservation of plasma for down-stream analysis Clin Proteom Bioinform, 2016 doi: 10.15761/CPB.1000115 Volume 1(3): 2-9 can hinder the discovery and accurate measurement of protein biomarkers from plasma [25]. It is clear therefore, that a standardized blood sample collection strategy is essential to limit the generation of ex vivo artefacts [26] which not only involves a suitable collection process but also the preservation of the sample in a stable state suitable for downstream analysis [27]. For this reason the idea of blood collection tubes containing protease inhibitors was introduced to control sample degradation by minimizing proteolysis [28]. However, additional aspects of the blood collection protocol (i.e. use of anticoagulant in collection tube, centrifugation speed, centrifugation time) have met the attention of the research community [29-31] and will also play an essential role in obtaining quality data for downstream data processing [32]. Common additives in plasma preparation methodologies include anticoagulants such as ethylenediaminetetraacetic acid (EDTA), sodium citrate, heparin and warfarin [28]. However citrate, heparin and warfarin are not suitable for downstream proteomic analysis for various reasons [33-35]. Citrate, as a liquid reagent contained in the blood-drawing tube, generally dilutes the plasma sample which needs to be taken into account during any further study [36]. In addition, citrate can bind calcium and has resulted in falsely lowered readings of immunoassay measurements for multiple analytes[19]. Heparin is a polymeric glucosaminoglycan, which shows up as a characteristic repeating building-block polymer in MS, masking signals from protein and peptide components and rendering downstream MS analysis challenging [36]. A more commonly used and MS compatible additive is EDTA, a polyprotic acid containing four carboxylic acid groups and two amine groups with lone-pair electrons, that chelates calcium and several other metal ions[37,38]. Although EDTA inhibits the coagulation pathway and prevents clot formation, it does not prevent the activation of the first enzymatic steps for which the possibility of consequent ex-vivo generation of peptides still remains [38]. To avoid protease activity during and after blood collection, and to address the disadvantages mentioned above, tubes containing protease inhibitors (PI) have been designed which are intended to stabilize the blood proteome during the first 15 min of blood extraction. To date, to our knowledge, no comprehensive study has been performed to investigate the efficacy of the different blood collection tubes available in terms of low abundant proteins or at the endogenous/non-tryptic peptide level. Our current study was designed to assist proteomic researchers by establishing a cost effective and standardized collection protocol that will preserve the plasma samples at an optimum condition for downstream analysis. There are currently three major varieties of EDTA containing tubes that are commercially available that use: EDTA only as anticoagulant (‘E’: BD Vacutainers EDTA Tube), a related EDTA containing tube has a dense polymer gel base layer which, upon centrifugation, partitions cellular bodies into the gel (‘G’: BD Vacutainers Plasma Preparation Tube PPTTM) and an EDTA containing tube with a proprietary cocktail of protease inhibitors (PIs) (‘P’: BD P100TM Blood Collection and Preservation System for Plasma Protein Analysis)[39]. The dense gel in ‘G’ tube forms a physical barrier between plasma and blood cells during centrifugation. The ‘P’ tube claims to reduce sample degradation by endogenous proteases. However, it needs to be attached to a specially designed blood collection kit to prevent backflow of the blood and avoid adverse patient reaction from contamination with protease inhibitors. Furthermore, a mechanical separator is required that will provide a solid barrier between plasma and cellular material during centrifugation. The P tubes with the complete blood collection kit costs US

Adenosine monophosphate deaminase 3 activation shortens erythrocyte half-life and provides malaria resistance in mice.

30.00 while the EDTA containing tubes (E tube and G tube) are far cheaper (US