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Sarah B. Scruggs, Jei Wang, Peipei Ping, UCLA David Geffen School of Medicine, Departments of Physiology, Medicine/Cardiology, and Computer Science, NIH BD2K Center of Excellence for Biomedical Computing at UCLA (HeartBD2K)
The Cardiovascular BD-HPP Initiative welcomes a new era of precision medicine that capitalizes on future-forward proteome technologies to identify cardiovascular disease mechanisms and therapeutic targets. Proteome signatures have gained recognition as the main driving force in cellular phenotypes, and several extraordinary and quantitative innovations can now elucidate these dynamic fingerprints at unprecedented speed, sensitivity, specificity and scale. Significant advancements that are forging new heights in proteomic analysis include new multiplexed mass spec sample preparation workflows and developments in novel, quantitative phospo- and splice variant proteoform resolution using top-down mass spec technologies.
New and exciting data from Ying Ge’s laboratory unveiled the complex proteoform profile of cardiac Troponin T (cTnT), a critical thin filament regulatory molecule of cardiac contraction/relaxation and clinical biomarker of myocardial infarction. Using online liquid chromatography and immunoaffinity purification coupled to high resolution top-down MS and several complementary fragmentation approaches (CAD, ECD, ETD), they were able to unequivocally identify seven unique cTnT splice variants, each in an unphosporylated or mono-phosphorylated form. This study represents the unprecedented power of top-down MS in isolating various intracellular proteoforms that likely elicit distinctive biological functions.
Exciting work by Jennifer van Eyk’s group includes the development of a streamlined, multiplexed workflow that enables the processing of 96 complex biological samples for MS in a matter of 5 hours. This methodology demonstrated remarkable reproducibility for both plasma and serum samples, showcasing CV’s less than 20% and holding up across replicates, days, instruments, and laboratory sites. Overall, this valuable approach will empower the proteomics community with tools to minimize artefactual introductions and enhance the native biological presentation of peptides during MS analysis. Moreover, we are seeing unparalleled developments in multiplexed, aptamer-based technologies which now target nearly 5000 proteins in biofluids. A collaborative study between Robert Gerszten and Lori Jenning’s groups demonstrated the application of this method to identify circulating biomarkers for myocardial infarction in derivation and validation patient cohorts, with orthogonal technical validation using targeted mass spectrometry
Computational innovations incorporating global proteome dynamics into data science platforms have emerged. The integration of transcript abundance, protein abundance, and protein turnover data and the extraction of multi-dimensional molecular signatures affords a 75% gain in discovering disease gene candidates. These recent developments in data science tools will facilitate protein marker discoveries, including clustering as well as signature extraction.
In summary, new technologies are paving the way to resolving comprehensive, spatio-temporal, dynamic maps of cardiovascular and other organelle proteomes that expand and articulate our understanding of how protein networks propel myriad phenotypes. Capitalizing on this knowledge to ultimately reduce the number of cardiovascular disease-related deaths is truly on the horizon.
Gilbert Omenn, University of Michigan, Michigan
The HUPO Human Proteome Project (HPP) annually reports on progress throughout the field in credibly identifying and characterizing the human protein parts list and making proteomics an integral part of multi-omics studies in medicine and the life sciences. neXtProt release 2018-01, the baseline for the 6th annual HPP special issue of the Journal of Proteome Research, contains 17,470 PE1 proteins, 89% of all neXtProt predicted PE1-4 proteins, up from 17,008 in release 2017-01 and 13,975 in release 2012-02. Conversely, the number of neXtProt PE2,3,4 missing proteins has been reduced from 2949 to 2579 to 2186 over the past two years. Of the PE1 proteins, 16,092 are based on mass spectrometry results, and 1378 on several other kinds of protein studies, notably enhanced by protein-protein interaction findings. PeptideAtlas has 15,798 canonical proteins, up 625 over the past year, including 269 from a major study of SUMOylation as a post-translational modification. The major limitations in finding expression of the remaining PE2,3,4 missing proteins continue to be protein sequences that cannot yield two proteotypic uniquely-mapping non-nested tryptic peptides of > 9 aa; lack of expression of the corresponding transcript in tissues studied; and especially concentrations of proteins too low to be detected even with the most modern mass spectrometers. Enrichment of low-abundance proteins, recognition of semi-tryptic peptides, and analysis of unusual tissues, especially with evidence of transcript expression, offer avenues to find more missing proteins.
Meanwhile, the Chromosome-centric HPP has launched an initiative to characterize the 1260 PE1 proteins that presently lack functional annotation. The HPP Mass Spectrometry resource pillar is conducting a multi-lab analysis of a standard sample with 96 phosphopeptides. The Human Protein Atlas has released its Cell Atlas, Pathology Atlas, and updated Tissue Atlas, and is applying recommendations from the International Working Group on Antibody Validation. A new resource pillar for Pathology to link with clinical translation has been launched. Finally, there is progress applying the quantitative multiplex organ-specific popular proteins targeted proteomics approach in various disease categories. For the full report, see Omenn, Lane, et al, JPR (in press) and reports from the B/D-HPP and C-HPP at www.hupo.org/hpp.
Péter Horvatovich, University of Groningen, Netherlands
The 19th C-HPP Symposium/workshop organized by Fernando J. Corrales, Concha Gil and Young-Ki Paik was held in conjunction with EuPA 2018 Congress with a scientific theme, “Translating Genome into Biological functions”. In this meeting, 13 Chromosome teams (Chr 2, 5, 6, 9, 11, 13, 14, 15, 16, 17, 18, X and Mt.) presented their work-in progress on missing protein identification and uPE1 (PE1 proteins of unknown function) characterization. Beside the talks mentioned in July 2018 C-HPP HUPOST, in the session of next-CP50 challenge, Gilberto B Domont presented latest data on brain proteomics of fetus bearing Congenital Zika Syndrome. Results indicate that lower expression of proteins compromises the vascular system and upper expression directs the cell machinery for virion production by enhancing neurogenesis, energy generation and protein synthesis. In the Young investigator session, Aida Pitarch from Complutense University (Madrid, Spain) presented a 2D gel-based proteomics approach to identify the molecular mechanisms of the human/host immune response induced by infection with Candida albicans. Juan Antonio Vizcaino, from the European Bioinformatics Institute (EBI) provided an update on ProteomeXchange and recent works done at EBI to reuse and integrate public proteomics data, focusing on multi-omics and proteogenomics data integration and multi-omics datasets available at the EBI data repositories. The talk presented ‘Novel ProBAM and ProBed formats facilitating proteogenomics data integration and the OmicsDI portal (http://www.omicsdi.org/)’ which collects multi-omics datasets. Peter Horvatovich from University of Groningen presented a study assessing the bias in proteomics sample preparation of head and neck tissues such as nasal polyps, parotid gland, and palatine tonsils using fours widely used sample preparation protocols (in-gel, in-solution, on-filter, and on-pellet digestion). The study was using MRM based targeted and DDA discovery LC-MS/MS proteomics profiling and concluded that there is no universal sample preparation protocol, which is best for all tissue with respect of relative losses and discovery potential. Charlotte Macron from Nestlé Institute of Health Sciences described identification of missing proteins such as protein dispatched homolog 3, Augurin, Erythroferrone, Protocaderins from cerebrospinal fluids of Alzheimer patients using deep proteomics and TMT labeling. Miguel Marcilla from Complutense University presented the identification of missing protein of Hyaluronan synthase 1 (HAS1) in Human Mesenchymal Stem Cells Derived from Adipose Tissue and Umbilical Cord using deep proteomics fractionation with SDS gel electrophoresis and basic reverse phase fractionation. Bioinformatics (e.g. text mining, online resource of orthologous genes) and wet lab workflows (e.g. gene silencing with siRNA and CRISPR-cas9) to find function for uPE1 proteins of chromosome 13 was presented by Seul-Ki Jeong (Yonsei Proteome Research Center). The complete program of the workshop with presented materials which we received consent from the authors is available at C-HPP Wiki.
Michelle Hill, QIMR Berghofer Medical Research Institute, and The University of Queensland, Australia
Do you wonder how proteomics contributes to understanding of biology or management of disease? Are you interested in contributing to a BD-HPP initiative but not sure how to start? With many options to choose, HUPO 2018 is a perfect time to get involved.
Click here to check out the enticing sample menu!
Note - There are subsidized fees of $60 for Sunday and $100 for Thursday to defray the extra costs of these activities on top of the Congress registration.
Etienne Caron, ZTH Zurich, Switzerland
The large collection of peptides associated to human leukocyte antigens (HLA) is referred to as the human immunopeptidome. Deciphering the composition of the human immunopeptidome is of paramount importance to understand the immune system and to guide the development of next-generation vaccines and immunotherapies against autoimmunity, infectious diseases and cancers. Until now, mass spectrometry (MS) is the only available technology to interrogate the immunopeptidome in an accurate, systematic and unbiased manner. Therefore, the development of advanced analytical MS workflows is of great importance to enable the deciphering of the immunopeptidome at increasing depth and robustness.
Foundation of the Human Immuno-Peptidome Project
The Human Immuno-Peptidome Project (HUPO-HIPP) was created to accelerate research toward robust and comprehensive analysis of immunopeptidomes (https://hupo.org/human-immuno-peptidome-project/). On May 4–5th, 2017, 40 leading scientists and industry representatives from 18 universities and 9 companies convened in Zurich, Switzerland, for the first international HUPO-HIPP workshop (see picture). During this event, participants identified specific challenges toward the goal of HUPO-HIPP, and within this framework, described the structure of a multipronged program aimed at addressing these challenges and implementing solutions at a community-wide level. The identified programs are: (1) method and technology development, (2) standardization, (3) effective data sharing, and (4) education (see details in Caron et al. Immunity, 2017)
1st HUPO-HIPP summer school: September 10-13 2018, Madrid, SpainMembers of HUPO-HIPP recognized that implementation of robust analytical workflows is a major hurdle to the expansion of the immunopeptidomics field. To circumvent this limitation and to disseminate the expertise on currently adopted methods by the immunopeptidomics community, the 1st HUPO-HIPP summer school was launched (http://www.hipp-summerschool.com/). Thus, the major goal of the summer school is to provide attendees with critical information about the main challenges of immunopeptidomics approaches with special emphasis on sample preparation, liquid chromatography-MS/MS analysis, bioinformatics and data sharing. Most importantly, the HUPO-HIPP summer school will contribute to boost the accessibility to immunopeptidomic technologies and to open this scientific niche to a bigger audience.
Next stepsHUPO-HIPP will soon publish the Minimal Information about an Immuno-Peptidomics Experiments (MIAIPE) to provide technical guidelines that represent the minimal information required to sufficiently support the evaluation and interpretation of immunopeptidomics experiments. HUPO-HIPP also plans to 1) establish partnerships with MS developers and other initiatives (e.g. Human Vaccines Project) to increase the impact of MS-based immunopeptidomics, 2) establish partnerships with journals’ editors and funding agencies (NIH and EC) to enforce sharing of immunopeptidomic data, 3) further develop the SysteMHC Atlas (https://systemhcatlas.org) for deposition and open sharing of immunopeptidomic datasets, 4) launch an international human immunopeptidome project consortium (HIPPC), and 5) continuously promote the visibility of HUPO-HIPP in publications, conferences, workshops and elsewhere. Progress and future plans will be discuss at HUPO-Orlando 2018.
If you would like to become a member of HUPO-HIPP complete the attached Membership Form and email to the address provided.
June 16-17, 2018
The 19th C-HPP Symposium/workshop organized by Fernando J. Corrales, Concha Gil and Young-Ki Paik was held in conjunction with the EuPA 2018 Congress with a scientific theme, “Translating Genome into Biological functions”. During this meeting, 13 Chromosome teams (Chr 2, 5, 6, 9, 11, 13, 14, 15, 16, 17, 18, X and Mt.) presented their progress on missing protein identification and uPE1 (PE1 proteins of unknown function) characterization. The workshop program started with an update of the Human Proteome Project (HPP Chair and Chr 17 PI, Gilbert Omenn), on neXt-CP50 initiative, (C-HPP Chair and Chr 13 PI, Young-Ki-Paik) and progress reports on each team according to neXt-MP50 teams, Bioinformatics teams, neXt-CP50 teams and Young Investigators. The final session was devoted to discussion on the C-HPP organization long-term plans.
Here are some highlights from the talks: Gilbert Omenn presented a new computational approach I-TASSER and COFACTOR algorithms to predict function of uPE1 proteins based on primary amino acid sequence. The talk presented predicted gene ontology-based functions of biological, molecular processes and cellular component of 66 uPE1 of Chromosome 17. A new challenge termed neXt-CP50, which aims to characterize at least 50 uPE1s in 3 years, and subsequent long-term plans with experimental strategies on uPE1s were presented and discussed by Young-Ki Paik. Jong Shin Yoo from chromosome 11 team presented and discussed a next generation proteomics pipeline to identify missing proteins (MPs) and protein sequence variants such alternative splicing and single amino acid variants in proteomics datasets. His group identified multiple MPs, splice junctions and single amino acid variant peptides in human brain tissue using deep proteomics and LC-MS/MS with ETD & CID fragmentation. Charles Pineau from chromosome 14 gave an update status on identification of missing proteins from human sperm and build of Human sperm proteome database in collaboration with MASSIVE. He also discussed the contribution of Epididymis proteome to the detection of MPs. Lydie Lane from chromosome 2 showed earlier uPE1 works on zebrafish and human cells. In particular she presented the characterization of enzymes such as APIP, involved in the methionine salvage pathway, mitochondrial proteins such as C11orf82, and developmental proteins such as C2orf62. Elena Ponomarenko and Ekaterina Ilgisonis from chromosome 18 team presented bioinformatics strategies including text mining in PubMed and protein-protein interaction (STRING) databases. Several options for reorganization of the C-HPP were presented and extensively discussed that included continuing a selected chromosome approach by only the currently active chromosome teams, plus the addition of new teams dedicated for bioinformatics/annotation teams, targeted MP stalking, identification of whole families of proteins in a chromosome agnostic manner and in protein – protein interaction data from large scale BioID and similar studies in human cells. The materials presented in this meeting will be available at C-HPP Wiki as soon as consent from the authors is received. More highlights from this meeting will also be presented in the C-HPP section of HUPOST in August 2018.
The Human Proteome Project (HPP) MS-Pillar now extends is Phosphopeptide challenge resource to include affinity capture. Participants are invited to contribute to both phases of phosphopeptide identification methods development by analyzing a set of phosphopeptides by their favorite method and now follow up with an affinity purification step to see improvements in their methods. Together with our launch partner, SynPeptide Co. Ltd in Shanghai (www.synpeptide.com), the MS Resource Pillar has partnered with Resyn Biosciences Pty Ltd in South Africa (www.resynbio.com) to provide a comprehensive phosphopeptide purification kit to use on the SynPeptide-HUPO phosphopeptide mixtures. The Resyn MagReSyn® kit including the magnet separator valued at over US$1000 each are provided free to HUPO members.
The MS-Pillar Phosphopeptide challenge samples are a complex set of human phosphopeptides (Ser, Thr or Tyr) singly and multiply phosphorylated) and their unphosphorylated counterparts that can be used for method development and verification for phosphopeptide enrichment, sequence analysis by mass spectrometry and bioinformatic evaluation.
The first stage of this initiative is for interested members of the HUPO community to obtain a set of the peptides and apply their own methods and bioinformatic analysis to fully characterize the peptides as a neat mixture and in a tryptic digest background. Samples are still available by contacting the email below.
The second stage is now to compare your methods used with the Resyn purification kit for phospho-enrichment and peptide clean-up. The kit contains 2mL of each MagReSyn® Ti-IMAC, Zr-IMAC, and HILIC, and 4-place magnetic separator. As a result of this collaborative endeavor, multiple purification schemes, analytical protocols and data processing strategies will be evaluated, making it possible to determine the approach(es) that provide the highest coverage of phosphopeptides in the mixture.
By partnering with Resyn and SynPeptide the peptide sets and purification kit are being provided free-of-charge to all HUPO members, with the understanding that the analytical and data processing methods and results will be returned to the HPP MS Resource Pillar committee so they can be collated and reported at the HUPO 2018 meeting.
Free aliquots of the peptide mixtures and MagReSyn k it can be obtained by sending an email to HUPO@resynbio.com.
On May 1, 2018, the C-HPP Consortium released the 7th C-HPP Newsletter summarizing 2017 C-HPP activities. Selected highlights from this C-HPP Newsletter are:
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 . 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 .
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 . 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”)
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 (firstname.lastname@example.org)
·HGI deputy chair: Dr. Morten Thaysen-Andersen, Macquarie University, Sydney, Australia (email@example.com)
Additional HGI study committee members:
·A/Prof. Daniel Kolarich, Griffith University, Gold Coast, Australia (firstname.lastname@example.org)
·Prof. Kai-Hooi Khoo, Academia Sinica, Taiwan (email@example.com)
·Prof. Katalin Medzihradszky, UCSF, CA (firstname.lastname@example.org)
·Prof. Joe Zaia, Boston University, MA (email@example.com)
·Prof. Goran Larson, Gothenburg, Sweden (firstname.lastname@example.org)
·Dr. Stuart Haslam, Imperial College, UK (email@example.com)
·Prof. Giuseppe Palmisano, University of Sao Paulo, Brazil (firstname.lastname@example.org)
·Prof. Jong Shin Yoo, Korea Basic Science Institute, Korea (email@example.com)
Acknowledgement: Dr Rosa Viner (Thermo) is thanked for providing valuable samples and high quality LC-MS/MS data to this study.
The 7th newsletter of the Chromosome-centric Human Proteome Project is now available online, read it here.
In this issue:
The Most Trusted Journal in the International Field of Proteomics
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