• 29 May 2018 3:55 PM | Anonymous member (Administrator)

    Nicolle Packer, Macquarie University, Australia

    Performance of current software for automated intact glycopeptide identification and MS/MS spectral annotation in glycoproteomics

    Aim of study: To assess the performance of current glycoproteomics software for automated intact N- and O-glycopeptide identification from high resolution MS/MS spectral data across laboratories.

    Note - We would need to get your feedback by June 30, 2018 so that we can present the preliminary outcomes of this study at HUPO 2018 in Orlando. You will of course be a co-author on the manuscript that will be a compilation of the results of the study.

    Please let us know the following:

    1. Whether you will be a participant

    2. Whether you are a developer and/or user

    Introduction: Glycoproteomics, the study of intact glycopeptides in complex biological systems, is a growing discipline [1]. Analytical advances have now facilitated LC-MS/MS-based glycoproteomics studies reporting hundreds and even thousands of unique intact glycopeptides from a single experiment. However, significant bottlenecks clearly exist in the accurate annotation of the large volumes of resulting MS/MS spectral data and in the confident identification and reporting of the corresponding intact glycopeptides. The identification of intact glycopeptides has previously relied heavily on manual interpretation and expert curation support. However, as the field of glycoproteomics transitions to rely increasingly on the use of large data sets in our experimental designs, the development of efficient software for accurate automated glycopeptide identification becomes absolutely essential.

    Over the past 5-10 years the field of glycoproteomics has seen the development of multiple exciting tools that show promise for automated or semi-automated annotation and identification of glycopeptides from MS/MS spectral evidence [2].

    This first study of the newly established HUPO Glycoproteomics Initiative (HGI) sets out to study the performance of the current informatics capabilities in this specialised field. Documenting the current status of glycoproteomics is vital to drive further technological developments and promote applications of system-wide glycopeptide analysis.

    Overview of study: This informatics-focused glycoproteomics study consists of two parts:

    Part A) Comparative study in which developers of glycoproteomics software (of academic and industrial origin) identify and report intact glycopeptides from provided LC-MS/MS glycopeptide datasets using exclusively their own developed software. The developers may improve their existing software in this process but need to provide an exact description of how their software was utilised to obtain the reported glycopeptides and provide software access and experimental conditions to the study committee to allow them to reproduce and interrogate the reported findings.

    Part B) Comparative study in which expert users (research teams) in Glycoproteomics (from academic and industrial origin) identify intact glycopeptides from the same (as in A) provided LC-MS/MS glycopeptide datasets using one or more tools, which they routinely use for glycopeptide analysis including manual interpretation as support. Users must report how they obtained their findings.

    Most participants will fit in either Part A or B; however, a research group can contribute to both parts, but must adhere to the provided guidelines and must provide separate reports from these two efforts. Two LC-MS/MS datasets containing mixtures of intact O- and N-glycopeptide data are provided to all involved participants, which should be reported on according to the provided guidelines.

    Study details and the fine-print:

    About the analysed glycoprotein samples: Human serum containing a complex mixture of N- and O-linked glycoproteins was used in this study (Thermo Fisher Scientific #31876). Briefly, the proteins were reduced, carbamidomethylated and digested exhaustively with porcine trypsin. The resulting peptide mixtures were split and analysed in their native form after enrichment using two different LC-MS/MS acquisition styles.

    About the two LC-MS/MS acquisition style and access to the provided data: The glycopeptides were separated using nano-LC (C18) separation and detected in positive polarity using alternating fragmentation modes (HCD, ETciD, EThcD and CID, see “Terminology” below for definition) on a Thermo Orbitrap Lumos LC-MS/MS platform [3]. Complementary fragmentation types were used within the same LC-MS/MS run to satisfy various software packages for glycopeptide identification. Precursor and product ions were recorded in “profile” mode at high resolution in the Orbitrap (OT) and at low resolution in the ion trap (IT). Two unprocessed data (raw) files acquired using two different acquisition styles are provided to participants:

    Data file A: HCD (OT) – ETciD (OT) – CID (OT) (File: “A_glycopepnew_HCDETciDOTCIDpeptide.raw”)

    Data file B: HCD (OT) – EThcD (OT) – CID (IT) (File: “B_glycopepnewHCDEThcDiTCIDpeptide.raw”)

    https://cloudstor.aarnet.edu.au/plus/index.php/s/koiUv4LCOjobrih (Password:HGI2017)

    Since some participants may not have access to Thermo (proprietary) software for data processing, the data files are also provided after .mgf or .mzxml. conversion. The link also provides access to other critical files for the glycopeptide identification and the reporting template.

    About the proposed identification and reporting of intact glycopeptides: Participants are requested to report on identified intact glycopeptides in a tabulated form using a provided Excel template. The template also hosts detailed guidelines for the identification of intact glycopeptides (e.g. Protein and Glycan search space) and the requirements for the data reporting. The developers (Part A) are also expected to provide annotated MS/MS spectra of the identified (reported) glycopeptides (PDF/PPT preferred) if their software allows for this since annotated spectral evidence is a requirement of many journals in the reporting of glycopeptide data sets. Users are encouraged to do the same.

    About the data comparison, disclosure of participant reports and dissemination of outcome: The reported data of the participants will be compiled by the HGI study committee. The identity of the participating developers and their software as well as the relative performance of their software (compared to other software and the user groups) will be disclosed. Should participating developers decide to not return their findings for various reasons, their identity and participation will not be disclosed in any dissemination of the study outcome. User groups will remain anonymous. Results will be compiled, compared, published and presentation (see below). All participants (developers and users alike) that return glycopeptide reports adhering to the study guidelines will be acknowledged for their efforts by being offered a co-authorship in the publication(s) arising from this study and mentioned by name in oral/poster presentations.

    Time-line for 1st HGI study:

    Sep 2016: HGI formed. Head and committee selected.

    July 2017: LC-MS/MS glycoproteomics data generated by Thermo and quality validated by committee.

    2017: Calls for participation in HGI study.

    Sep 2017: HGI study introduced at World HUPO 2017.

    March 2018: Data files and reporting template made available to registered participants.

    30 June 2018: Deadline participant reporting of data.

    July-September 2018: Compilation and comparison of data.

    Sep 2018: Preliminary data presented World HUPO 2018.

    Early – Mid 2019: Outcome(s) published.

    Sep 2019: Outcomes presented at World HUPO 2019.

    HGI study committee:

    ·HGI chair: Prof. Nicolle H. Packer, Macquarie University, Sydney, and Glycomics Institute, Griffith University, Gold Coast, Australia (nicki.packer@mq.edu.au)

    ·HGI deputy chair: Dr. Morten Thaysen-Andersen, Macquarie University, Sydney, Australia (morten.andersen@mq.edu.au)

    Additional HGI study committee members:

    ·A/Prof. Daniel Kolarich, Griffith University, Gold Coast, Australia (d.kolarich@griffith.edu.au)

    ·Prof. Kai-Hooi Khoo, Academia Sinica, Taiwan (kkhoo@gate.sinica.edu.tw)

    ·Prof. Katalin Medzihradszky, UCSF, CA (folkl@cgl.ucsf.edu)

    ·Prof. Joe Zaia, Boston University, MA (jzaia@bu.edu)

    ·Prof. Goran Larson, Gothenburg, Sweden (goran.larson@clinchem.gu.se)

    ·Dr. Stuart Haslam, Imperial College, UK (s.haslam@imperial.ac.uk)

    ·Prof. Giuseppe Palmisano, University of Sao Paulo, Brazil (palmisano.gp@usp.br)

    ·Prof. Jong Shin Yoo, Korea Basic Science Institute, Korea (jongshin@kbsi.re.kr)

    Acknowledgement: Dr Rosa Viner (Thermo) is thanked for providing valuable samples and high quality LC-MS/MS data to this study.


  • 04 May 2018 11:01 AM | Anonymous member (Administrator)

    The 7th newsletter of the Chromosome-centric Human Proteome Project is now available online, read it here.

    In this issue:

    • The New Release of neXtProt
    • Highlights of C-HPP Consortium News
    • Summary of Discussion on the C_HPP Progress
    • 2018 JPR PSecial Issue Call for Papers
    • 19th C-HPP Workshop in Santiago, Spain
    • Future C-HPP Scientific Workshops
    • C-HPP Leadership
    • C-HPP PIC Members
  • 29 Mar 2018 9:40 AM | Anonymous member (Administrator)

    Michelle Hill, QIMR Berghofer Medical Research Institute, and The University of Queensland, Australia

    The last decades saw a steady increase in the incidence of rheumatic and autoimmune diseases such as osteoarthritis (OA) rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel diseases (IBD) and systemic lupus erythematosus (SLE). Rheumatic and autoimmune diseases (RAD) are characterised by inflammation of joints, muscles, and connective tissues around joints and bones. Apart from the debilitating mobility and pain, RAD patients are also at increased risk of cardiovascular and neurodegenerative diseases. Given the huge socioeconomic impact of RAD, broad research efforts have been dedicated these diseases worldwide.

    The RAD B/D-HPP initiative was launched in 2017 by Francisco J. Blanco (Rheumatology Department, INIBIC-Complejo Hospitalario Universitario A Coruña in Spain) with co-chair Paul J. Utz from Stanford Department of Medicine, USA). Currently, 6 research groups from 5 countries participate in RAD-HPP, but the collaborative network has already extended to Cardiovascular-HPP and Chromosome 16-HPP .

    After establishing the networks and promoting visibility of the new initiative, the immediate scientific goals of RAD-HPP are to define the proteome of human joint tissues (cartilage, synovial membrane, subchondral bone), and to assemble prioritized lists of proteins clinically relevant in RAD using the ‘popular proteins’ strategy with text mining tools. Collaborations within the RAD-HPP have already resulted in several publications in 2017, including circulating biomarkers for knee radiographic osteoarthritis , use of protein array to discover and validate biomarkers for Osteoarticular Pathologies , and anti-citrullinated protein antibodies in rheumatoid arthritis.

    To spread the word outside of proteomics research circles, RAD-HPP members presented their proteomics work at key RAD clinical meetings around the globe in 2017. These included Osteoarthritis Research Society International (OARSI) World Congress in Las Vegas (USA), the Annual European Congress of Rheumatology-EULAR in Madrid (Spain), Annual Meeting of the American College of Rheumatology in San Diego (USA), and XLIII Congress of the Spanish Rheumatology Society in Bilbao (Spain).

    To boost the development and utility of quantitative proteomics assays, RAD-HPP members conducted focused proteomics training sessions in 2017, with highlights being sessions at “Cutting Edge Osteoarthritis” Symposium at Pembroke College, Oxford, and “Proteomics in rheumatic diseases” lecture at a Summer School on Platform Technologies and Big Data applications in Precision Medicine, in Santander, Spain.

    With the synergistic international effort and strong clinical translational focus, the RAD-HPP initiative is poised to realise the utility of proteomics in RAD diagnosis and management.

  • 06 Dec 2017 1:14 PM | Anonymous member (Administrator)


    Submit your manuscript to Journal of Proteome Research for the 2018 HUPO Human Proteome Project Special Issue. Submission deadline May 31, 2018 

    The JPR Special Issue on the HPP will now consider short definitive reports, submitted in the Letters format, on the discovery of a Missing Protein(s). 

    To be considered for publication, the missing protein(s) must meet the Guidelines v 2.1 and be cast in the context of both the HPP and biological setting in which they were discovered. We anticipate this format will encourage many teams, particularly of the B/D-HPP, to highlight such protein discoveries in a disease and biological context.

    Click here for more info.


  • 20 Nov 2017 5:00 PM | Anonymous member (Administrator)

    AMSTERDAM, NOVEMBER 20 2017 – While colorectal cancer screening already saves many thousands of lives, there is still room for improvement. By looking at a combination of specific proteins rather than at a single blood protein alone, substantially more patients with colorectal cancer and advanced adenomas could be detected. The study describing these findings, conducted by researchers from the Netherlands Cancer Institute and VU University Medical Center, is published in the Annals of Internal Medicine.

    Click here to read the full article. 

  • 20 Nov 2017 4:15 PM | Anonymous member (Administrator)

    The Human Proteome Project continues to make major progress on identifying and characterizing the predicted protein products from all the protein-coding human genes. Using the stringent HPP Guidelines for Interpretation of Mass Spectrometry Data, we have reported that the community has now credibly detected 17,008 of the 19,587 neXtProt PE1,2,3,4 predicted proteins (87%). This curate result in neXtProt version 2017-01 is a substantial increase from one year earlier. That leaves 2579 “missing proteins”. The new Uniqueness Checker from neXtProt facilitates testing novel peptide sequences to see if they each uniquely map to a single predicted protein and qualify as “proteotypic”.

    Additional progress was reported in the Dublin HUPO Congress as early results from the Next50 Missing Proteins Challenge for all of the Chromosome-based HPP teams. Particularly notable were the following strategies: (1) focus on testis and sperm, the richest source of tissue-specific transcripts; (2) examine relatively understudied tissues and organs, like bladder and kidney; (3) achieve relative enrichment and equalization of abundance for low-abundance proteins by use of ProteoMiner hexapeptide-coated beads; (4) search GPMdb and PeptideAtlas for “stranded peptides” and then match their spectra to spectra of corresponding peptide sequences in the SRM Atlas (Kusebauch et al, Cell 2016); and (5) perform extensive biological, immunochemical, and siRNA studies on targeted proteins, like the differentiation of TBL1Y from TBL1X in gender-associated differentiation of cardiac myocytes. At least 73 additional missing proteins were brought forward from these studies.

    The Chromosome-centric HPP has launched a companion initiative to seek functional annotation of the 1232 PE1 proteins which currently have no such annotation. Meanwhile, the Biology and Disease-driven HPP teams are applying the popular proteins/targeted analysis strategy to liver, heart, and other organ-specific proteomes. And the MS Resource Pillar has developed a standard sample with 96 phospho-peptides made available to labs everywhere to facilitate progress on post-translational modifications, starting with phosphorylated sites.

    A total of 26 papers have appeared on line from the Journal of Proteome Research and will appear in the December 2017 hard-copy issue of the journal as the 5th annual special issue of the HUPO Human Proteome Project.

    Gilbert S. Omenn, MD, PhD, HPP Chair


  • 20 Nov 2017 4:09 PM | Anonymous member (Administrator)

    The Chromosome-Centric Human Proteome Project (C-HPP) was launched with the goal to catalogue the entirety of the parts list of the human proteome, specifically to find evidence at protein level of all human protein coding genes. C-HPP contributed with collaborating partners in developing guidelines for mass spectrometry data interpretation, with EBI to organize central data repository ProteomeXchange for proteomics experiences, to deliver stringent peptide and protein identification lists of human proteome from large-scale community deposited LC-MS/MS data in PeptideAtlas and with neXtProt to define 5 categories of protein evidence (PE1-5). In 2012, at the start of C-HPP, neXtProt included 20,059 protein entries, from which 13,664 had evidence at protein level (PE1), including 12,509 with mass spectrometry data. In January 2017, neXtProt accounted for 20,159 entries, from which 572 might correspond to non coding elements (PE5). C-HPP members, collaborating partners and the proteomics community found evidence at protein level (PE1) for 17,008 out of the 19,587 protein coding genes, from which 15,173 have mass-spectrometry evidence in PeptideAtlas, leaving to 2,579 missing proteins (MP) i.e. human protein coding genes with evidences at PE2-4 levels.

    Advancing the HPP

    At the Dublin HUPO congress and HPP Workshop, C-HPP PIs have made two important decisions for future directions.

    First, they resolved to extend the term period of C-HPP from 2022 to 2027 in an attempt to reflect the current progress as we face a slowing in discovery reflecting an increasing proportion of the MPs that are extremely limited in spatial and temporal expression and still evade detection. 2022-2027 will provide extra time to plan and execute the whole C-HPP plans as bench marked from the lessons of the Human Genome Project (HGP). In fact, Dr. Leroy Hood mentioned that HGP group had a similar experience and that they reviewed the progress every 5 year and redirected the HGP based on the progress made during the past years.

    Second, they launched neXt-CP50 challenge that is led by Young-Ki Paik, complementary to neXt-MP50 (see Figure 1), to characterize 1232 known PE1 which have no functional annotation as of 8-8-2017 (neXtProt). Details on the strategy and timeline will be available shortly for those 25 PIs who are involved in this campaign.

    Over the past 5 years, protein coding genes with protein evidence at PE1 level increased from 68.1% to 86.8% and finding evidences for the remaining MPs set a challenge for the proteomics community. To promote these efforts in early 2017, C-HPP launched a new campaign the neXt-MP50 challenge led by Chris Overall, which aims to find evidences for the remaining missing proteins. This campaign has the ultimate goal to find missing proteins by identifying types of human samples not analyzed yet by the proteomics community considering sample location, stimulus, diseases/health, age. By using novel sample preparation techniques such as proteominer, mass spectrometry proteomics profiling or bioinformatics technologies, the neXt-MP50 aims to uncover the remaining dark matter of the human proteome. Proteogenomics methods integrating genomics and proteomics data tightly is important to identify sequence variants of human proteins. Proteogenomics data integration is gaining momentum amongst the proteomics and genomics communities and has a central role in C-HPP. In the next phase, C-HPP is promoting the use of proteogenomics data integration to reveal the amino acid sequence space of the human proteins as well as to promote the identification of peptides with post-translation modifications regarded as an important source of structure variability of human proteins. Proteogenomics data integration, peptides with post-translation modification and next-MP50 challenge to find the evidences for the remaining missing proteins are the goals of the second phase of C-HPP, which is on the agenda for the next five years.


  • 08 Nov 2017 4:06 PM | Anonymous member (Administrator)

    Click here to read the full summary of the Human Proteome Project Post Congress Workshop in Dublin, Ireland.

  • 17 May 2017 11:36 AM | Anonymous member (Administrator)

    The 6th newsletter of the Chromosome-centric Human Proteome Project is now available online, read it here

    In this issue:

    • Editorial
    • C-HPP Leadership Update
    • C-HPP Principal PIC
    • A Brief Introduction Of New PIs and Plans
    • JPR SI Call For Papers
    • Working Group Formation of C-HPP
    • Briefings in the 2016 C-HPP Workshops 
    • Future C-HPP Workshops
  • 25 Jan 2017 1:08 PM | Deleted user

    The first issue of the 2017 B/D-HPP newsletter is now available online. Read it here.

    In This Issue:

    • HUPO 2016 in review
    • Growing HUPO through ECRs
    • Success Story - Cancer Moonshot at HUPO
    • Initiative Spotlight
    • Liver Human Proteome Project
    • Mitochondria Human Proteome Project




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