Editorial and Review: 29th ASMS Sanibel Conference on Mass Spectrometry—Peptidomics: Bridging the Gap between Proteomics and Metabolomics by MS
The 29th ASMS Sanibel Conference on Mass Spectrometry, organized by Per E. Andrén (Uppsala University), Lingjun Li (University of Wisconsin), and Jonathan Sweedler (University of Illinois), was held on January 19–22, 2017 at the Hilton Clearwater Hotel in Clearwater Beach, FL. The topic of the conference was “Peptidomics: Bridging the Gap between Proteomics and Metabolomics by Mass Spectrometry”. Peptidomics is the characterization and study of the dynamic peptide complement of a sample and includes the areas of peptide-based biomarker discovery, serum and biological fluid peptide measurements, and neuropeptide/brain peptide characterization. As one critical example, neuropeptides modulate the functioning of every neuron in the brain, affect our mood, and are the targets for drugs of abuse, but surprisingly, many cell-cell signaling peptides used in our brain have yet to be characterized.
Peptidomics, the study of the peptide complement used in a tissue or organ, is of growing importance, and the rate of peptide discovery is accelerating, with many novel peptides being reported each year. This acceleration is due to striking technological advances in mass spectrometry that enable the peptidome to be characterized from ever smaller samples with ever increasing chemical detail. The conference highlighted both the unique challenges for endogenous peptide detection and the latest technology development and emerging applications in peptidomics, including wide ranging topics from novel sampling strategies, chemical characterization and large-scale peptidome discovery, computational and informatics tools, to therapeutic approaches, spatial mapping, in vivo analysis and the functional study of these intriguing signaling molecules.
The Friday morning program started with a session devoted to discussions and presentations on unique challenges for measurement of endogenous peptides’ spanning sequence analysis, peptide stability and discovery. The session began by a presentation by Robert Kennedy (University of Michigan) on “Observation of Peptide Dynamics In Vivo Evoked by Behavior and Optogenetic Stimulation using LC-MS.” Kennedy discussed the challenges for in vivo neurochemical monitoring and presented strategies and progress on improving peptide recovery rate via microdialysis sampling. Applications in neurochemistry of motivation and feeding behavior using rodent model systems were highlighted. Following this talk was a presentation by Kurt Boonen (KU Leuven) on “Peptidomics in Neuroendocrine Research” where invertebrate model organisms including tsetse fly and Caenorhabditis elegans were utilized for peptidomic analysis. Boonen presented an overview of a peptidomics approach where LC-MS is combined with in silico mining of neuropeptide precursor sequences. Using this approach, the group was able to identify an unprecedented number of 203 mature neuropeptides from C. elegans whole-body extracts, thus creating a useful resource for the peptidomics community. The session ended with a presentation by Lingjun Li (University of Wisconsin) on the “Multi-faceted Approach for Functional Discovery of Neuropeptides” where she discussed the challenges for neuropeptide discovery from model organisms that lack of a fully sequenced genome and strategies to improve de novo sequencing of neuropeptides. Li presented their recent progress on the development of mass spectral imaging, affinity-enhanced microdialysis, and isotopic labeling strategies for comparative peptidomic analysis in feeding. Several on-going studies with rodent model system and multiplex quantitation enabled by high-resolution MS and dimethylated leucine (DiLeu) tags were also highlighted.
The second session in the morning focused on informatics for peptidome discovery. Nuno Banderia (UC San Diego) delivered a presentation entitled “Spectral Networks for De Novo Sequencing.” In contrast to traditional spectrum identification algorithms that analyze each tandem mass spectrum in isolation, spectral networks define a new computational approach that finds and simultaneously interprets sets of spectra from overlapping peptides. Banderia presented the evolutionary development of multiple versions of de novo sequencing programs based on this new approach. A key aspect of this approach is to recognize variants in similar peptide sequences, which facilitates the characterization of modified peptides and peptide and protein isoforms. Following this, Andrew Christie (University of Hawaii) spoke on “In Silico Prediction of Crustacean Peptidome” where he presented strategies for combining MS accurate mass matching and de novo sequencing with in silico genome and transcriptome mining for accelerated neuropeptide discovery in crustacean model organisms. Tissue-specific de novo assembled transcriptomes can be mined to allow in silico prediction of neuropeptides followed by MS confirmation. The final presentation in this session was given by Marshall Bern (Protein Metrics) on “Bacterial Peptidoglycan Analysis using a Proteomics Search Engine”. He presented the use of bottom-up proteomics methods by high-resolution MS and automated analysis of HCD and ETD fragmentation spectra with Byonic software for a systematic and detailed structural analysis of peptidoglycans in bacteria.
After a few hours’ break and relaxing time to explore the beautiful Clearwater Beach in the afternoon, the evening session featured the second keynote lecture by Dominic Desiderio (University of Tennessee) on “Peptidomics Research Before It Was Called Peptidomics.” Widely regarded as one of the pioneers who worked on neuropeptide analysis by mass spectrometry, Desiderio gave a historical overview of the field of neuropeptide discovery, characterization, and evolutionary role of MS to accelerate these discoveries. Both qualitative and quantitative analytical strategies were discussed with special emphasis on opioid neuropeptides in human pituitary tissues. Comparative peptidomics and proteomics enabled better elucidation of molecular mechanisms involved in tumor formation as defects in neuropeptidergic system processing is a contributing factor to human anterior pituitary tumor formation. The keynote lecture was followed by six 3-min highlighted poster presentations given by Bruce Southey (University of Illinois; “A bioinformatics pipeline for genome sequence-based characterization of neuropeptidomes”), Matt Baird (Wichita State University; “Isomeric characterization of D-amino acid containing peptides and N-linked glycopeptides by differential ion mobility spectrometry”), Amanda Buchberger (UW-Madison; “Qualitative and quantitative analysis of crustacean neuropeptides after hypoxia exposure”), Christopher Lietz (UC San Diego; “Lithium modulation of regulated neuropeptide secretion in adrenal medullary chromaffin cells of the sympathetic nervous system”), Qing Yu (UW-Madison; “Discovery and identification of O-glycosylated signaling peptides from mouse pancreatic islets”), and Sven Van Bael (KU Leuven; “Characterization of neuropeptide-amidating enzymes in C. elegans by mass spectrometry”). The evening concluded with a full poster session where 24 conference attendees presented their posters and extensive discussions and refreshments were enjoyed by all (Fig. 3).
Saturday morning started with a session focused on “Peptide Imaging Using Mass Spectrometry.” Per Andrén (Uppsala University) presented a talk on “Sample Handling, Quantification and Imaging of Brain Signaling Molecules.” Andrén discussed the utility of focused heat stabilization for enhanced detection of neuropeptides, and presented MALDI MS imaging of neuropeptides and small molecule neurotransmitters where novel derivatization agents and reactive matrices were developed to enable simultaneous imaging of multiple neurotransmitters. Isotope-incorporated matrix was used to obtain relative quantitation information. The application to Parkinson’s disease model system was presented. Peter Verhaert (Delft University of Technology) delivered a presentation on “Tissue Imaging of Neuropeptides Using MALDI Orbitrap MS” where he discussed unique features of high resolution accurate mass measurements for more confident peptide identification. He compared MALDI images obtained from insect tissue samples prepared by flash frozen and paraffin-embedded sections for their neuropeptide content and image qualities. The final talk of this session was presented by Ron Heeren (Maastricht University) on “High Resolution MS Imaging: Translational Research in Action”. Heeren discussed a wide range of high-resolution imaging MS tools and their utilities for diagnostics and prognosis in the context of clinical setting. He updated the audience with some new developments in time-of-flight secondary ion mass spectrometry (TOF-SIMS), including parallel imaging and matrix-enhanced SIMS to enable peptide sequencing directly from tissue with high-resolution SIMS. Complementary to this, Heeren also presented results from high-resolution liquid extraction surface analysis micro-LC-MS and new development of funnel-based MALDI FTMS.
The second morning session on Saturday featured presentations on “Emerging Tools and Applications.” Carlito Lebrilla (UC Davis) presented a talk on “Peptidomic and Glycopeptidomic Analysis of Bioactive Compounds in Human Milk.” The Lebrilla group has performed extensive MS analysis of the human milk peptidome to reveal thousands of endogenous peptides produced by specific enzymatic cleavages. In-house computational tools were developed to visualize and interpret the data through estimation of the differential enzyme participation. Lebrilla also highlighted recent applications of ion mobility spectrometry (IMS) to screen for different activities as reflected by multiple conformations and PTMs on these milk peptides. Jay Forsythe (College of Charleston) delivered a presentation entitled “Proto-Peptidomics: The First Peptides in Darwin’s Warm Little Pond,” where he presented a fascinating story of using LC-MS tools to unravel high sequence diversity of proto-peptides. Using a dry-wet environmental cycling protocol, hundreds of proto-peptide sequences containing broader combinations of prebiotically plausible monomers were formed over a few day’s reaction. The MS-based sequencing and database searching approach presented by Forsythe could be a powerful tool as the origins-of-life research moves toward systems-level investigations of prebiotic chemistry. David Clemmer (Indiana University, “Developing IMS-MS Techniques for Following Structural Transitions in Peptides in Solution and in Gas Phase”) showed the use of advanced IMS instrumentation to capture intermediate states of peptide and protein ions in the gas phase as they switch between different conformations. Detailed intermediate steps and molecular insights can be gained from measuring collision cross-section distributions of various intermediate structures and performing molecular dynamic simulations. Four model systems including polyproline-13, two neuropeptides, bradykinin and substance P, and a small protein ubiquitin were discussed to demonstrate the utility of this approach. The morning session ended with a short talk by Ryan Julian (UC Riverside) who presented on “Identifying Isomerization and Epimerization in Peptides from Crystallins with Radical Directed Dissociation.” Julian introduced the use of radical directed dissociation (RDD), a gas phase radical-based dissociation method via site-specific photodissociation, to distinguish peptide epimers based on fragmentation patterns and identify four Asp isomers generated via deamidation using LC-MS/MS. Using this method, Julian and his team identified numerous peptide isomerization and epimerization in long-lived crystalline proteins, including some unexpected isomerization sites that might reveal novel molecular insights into aging.
The Saturday afternoon session focused on Peptide Therapeutics. Wendy Zhong (Merck Co.) discussed “Challenges in Structure Determination of Low Level Impurity and Degradation Products of Therapeutic Peptides in Pharmaceutical Industry.” She presented several case studies on the use of electron activated dissociation (ExD) to differentiate isomeric amino acid residues and the combined use of ExD and CAD to determine unknown impurities in a large peptide, which is relevant to the comprehensive characterization of impurities generated from therapeutic peptide synthesis. Albert Lebedev (Moscow State University) presented on “Discovery of New Antibiotics from Frogs – De Novo Sequencing of Long Natural Non-Tryptic Peptides” where he discussed strategies of employing hybrid electron-transfer high-energy collision induced dissociation (EThcD) to discriminate isomeric leucine/isoleucine residues in peptides. The utility of this approach was demonstrated by de novo sequencing of large intact frog skin peptides with intramolecular disulfide loops in a complex mixture of natural non-tryptic peptides. To follow the theme of peptide therapeutics of the session, Michal Bassani-Sternberg (Ludwig Cancer Research Center) presented a fascinating lecture on “Direct Identification of Neoantigens Using In-Depth Immunopeptidomics of Melanoma Tissues for the Development of Anti-Tumor Immunotherapy.” Recent research suggested that targeting neoantigens would enable immune cells to distinguish between normal and cancerous cells, avoiding the risk of autoimmunity. However, discovery of such tumor neoantigens could be very challenging and time consuming. Bassani-Sternberg reported effective protocols for in-depth and accurate MS-based immunopeptidomics, enabling comprehensive interrogation of the naturally presented repertoire of human leukocyte antigen (HLA) binding peptides, including those tissue-derived neoantigens extracted from human tumors and post-translationally modified peptides. This session ended with two short talks by Leslie Hicks (University of North Carolina at Chapel Hill) on “The PepSAVI-MS Pipeline for Natural Product Bioactive Peptide Discovery” and Asoka Ranasinghe (Bristol-Myers Squibb) on “Automated Structural Characterization of Therapeutic Cyclic Peptides and Their Metabolites in Drug Discovery”. Hicks introduced the development of the PepSAVI-MS pipeline that employed MS and statistics to identify bioactive peptide targets from complex biological samples and validated the platform through successful identifications of bioactive peptides from plants and fungal secretomes. Ranasinghe described challenges and development of software tools to characterize cyclic peptides containing disulfide linkages, with two case studies focusing on insulin and atrial natriuretic peptide.
The Saturday evening session featured the third keynote lecture presented by Richard Caprioli (Vanderbilt University) on “Next Generation Mass Spectrometry Imaging: Molecular Measurements in Time and Space.” Since his seminal publication in 1997, showing the power of MALDI imaging mass spectrometry for tissue analysis, Caprioli has continuously advanced the technology through innovation and improvements in sample preparation, instrumentation, and informatics approaches. In his talk, Caprioli reviewed the current state-of-the-art development of imaging MS and presented a wide range of clinical applications, ending with discussions about new technology initiatives including higher spatial resolution that enables single-cell analysis, multi-modal image fusion, and 3D MS imaging. Following the keynote lecture was six selected 3-min highlighted poster presentations by Kellen DeLaney (UW-Madison; “Mass Spectrometric Quantitation and Imaging of Neuropeptide Expression Levels in Crustacean Tissue and Circulating Fluid Resulting from Food Intake”), Elena Romanova (University of Illinois; “Strategies for Peptidome Characterization in Non-Model Species”), Elva Fridjonsdottir (Uppsala University; “Structure-specific Peptide Transmission and Processing in a Model of Parkinson’s Disease and L-DOPA-induced Dyskinesia”), Ayaka Maeda-Murayama (Kyushu University; “Development of an Integrated Proteomic and Metabolomics Imaging Technique Using MALDI-MS”), Andre Venter (Western Michigan University; “Improving DESI-MS Analysis of Proteins and Peptides”), and Barbara Larsen (DuPont Co.; “Study of Protein Oxidation in Freeze-dried Cells during Long-Term Storage”). These short introductory talks invited audience to attend the second full poster session where another 24 conference attendees presented their posters and exchanged ideas while enjoying light refreshments and conversations (Fig. 3).
The final half day of the conference on Sunday morning started with a session on “Peptidomic Approaches to Biomarker Discovery and New MS Strategies for Peptide Analysis”. Johan Gobom (University of Gothenburg) delivered a presentation on “Peptide Biomarkers in Neurodegenerative Diseases.” He presented a quantification-driven workflow that relied on spectral clustering to match MS/MS spectra. Multiplex isobaric labeling was employed to analyze endopeptidomes of cerebrospinal fluid from patients at different disease stages (and controls) to allow biomarker discovery. Complementary to the use of biofluid for biomarker discovery, Richard Drake (Medical University of South Carolina) focused his biomarker discovery from clinical tissue samples. His talk on “Prostate Cancer-associated Peptide and Glycan Co-localization in FFPE Tissues by MALDI-FTICR Imaging Mass Spectrometry” highlighted strategies to map the spatial distribution and co-localization patterns of N-linked glycan distributions and tryptic peptides in fresh frozen tissues and formalin-fixed paraffin-embedded tissues where tumor-specific glycans can be grouped relative to nontumor regions.
The last talk of this session was given by Francisco Fernández-Lima (Florida International University) where he discussed “Strategies for Discovery and Targeted Analysis using nESI-TIMS-TOF MS and nESI-TIMS-FTICR MS.” Fernández-Lima in collaboration with the Pacific Northwest National Laboratory (Richland, WA) team evaluated the potential of trapped ion mobility spectrometry (TIMS) coupled to TOF MS for discovery and targeted monitoring of peptide biomarkers from human-in-mouse xenograft tumor tissue. Equipped with high mobility resolution (R = 150–250), the offline nanoESI-TIMS-CID-TOF MS was shown to be complementary to the traditional LC-ESI-MS/MS proteomics workflow.
The second session of the Sunday morning featured presentations on “Single-cell Neuropeptidomics.” Susanne Neupert (University of Cologne) presented work on “Single Cell Peptidomics in Insects.” She showed impressive results for mapping neuropeptide expression from single neurons in a variety of insect species, including cockroaches and fruitflies. N-terminal derivatization using 4-sulfophenyl isothiocyanate and subsequent MS analysis was demonstrated for de novo sequencing of neuropeptides from single cells. Complementary to fruitfly single-cell peptidomic analysis, Tony Stretton (UW-Madison) presented his work on MS mapping of neuropeptides in the nematode Ascaris sum. Stretton reported on the use of three independent techniques, single-cell MS, in conjunction with immunocytochemistry, and in situ hybridization, to localize neuropeptides and their transcripts in the nervous system of A. suum. Using this combined approach, they were able to discover novel neuropeptides in four GABAergic neurons involved in a three-dimensional head movement of the worm. The final presentation of this session was delivered by Peter Nemes (George Washington University) on the topic of “Trace-level Metabolite-Peptide-Protein Detection in Single Cells using High-Resolution MS.” Using the frog Xenopus laevis as a model system, Nemes demonstrated enhanced peptide detection toward single-neuron proteomics by supplementing ultrasensitive capillary electrophoresis (CE)-nanoESI-high-resolution MS with reversed-phase fractionation.
The conference ended with the final keynote lecture that was presented by Jonathan Sweedler (University of Illinois) on “Measuring Signaling Peptides One Cell at a Time.” Sweedler has focused on the development of new metabolomics and peptidomics technologies for assaying small volume samples, and application of these methods to study novel neurochemistry. He highlighted several neuropeptidomic studies from a range of model organisms spanning from mollusks, insects to mammals, using a suite of ultrasensitive mass spectrometry measurement tools. Recent examples of high throughput mapping of single cell heterogeneity of islets via microscopy-guided single-cell MALDI MS were demonstrated.
Account and Perspective—“Diversity of Neuropeptide Cell-Cell Signaling Molecules Generated by Proteolytic Processing Revealed by Neuropeptidomics Mass Spectrometry” by Vivian Hook, Christopher Lietz, Sonia Podvin, Tomas Cajka, Oliver Fiehn
“Towards Discovery and Targeted Peptide Biomarker Detection Using nanoESI-TIMS-TOF MS” by Alyssa Garabedian, Paolo Benigni, Cesar E. Ramirez, Erin S. Baker, Tao Liu, Richard D. Smith, and Francisco Fernández-Lima
“Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry” by Kelly L. Wormwood, Armand Gatien Ngounou Wetie, Marcus Vinicius Gomez, Yue Ju, Paul Kowalski, Marius Mihasan, Costel C. Darie
“EThcD Discrimination of Isomeric Leucine/Isoleucine Residues in Sequencing of the Intact Skin Frog Peptides with Intramolecular Disulfide Bond” by Tatiana Yu Samgina, Sergey V. Kovalev, Miriam D. Tolpina, Polonca Trebse, Gregor Torkar, Albert T. Lebedev
“Rapid Characterization of Insulin Modifications and Sequence Variations by Proteinase K Digestion and UHPLC-ESI-MS” by Rong-Sheng Yang, Weijuan Tang, Huaming Sheng, and Fanyu Meng
“Fungal Secretome Analysis via PepSAVI-MS: Identification of the Bioactive Peptide KP4 from Ustilago maydis” by Christine L. Kirkpatrick, Nicole C. Parsley, Tessa E. Bartges, Madeline E. Cooke, Wilaysha S. Evans, Lilian R. Heil, Thomas J. Smith, and Leslie M. Hicks
“Quantitative Peptidomics with Five-plex Reductive Methylation Labels” by Alexandre K. Tashima and Lloyd D. Fricker
“A Caenorhabditis elegans Mass Spectrometric Resource for Neuropeptidomics” by Sven Van Bael, Sven Zels, Kurt Boonen, Isabel Beets, Liliane Schoofs, Liesbet Temmerman
“Neuropeptide Mapping of Dimmed Cells of Adult Drosophila Brain” by Max Diesner, Reinhard Predel, Susanne Neupert
“Characterization of Disulfide-linked Peptides Using Tandem Mass Spectrometry Coupled with Automated Data Analysis Software” by Zhidan Liang, Kenneth N. McGuinness, Alejandro Crespo, and Wendy Zhong
“Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry” by Sam B. Choi, Camille Lombard-Banek, Pablo Muñoz-LLancao, M. Chiara Manzini, Peter Nemes
“Exploring the Sea Urchin Neuropeptide Landscape by Mass Spectrometry” by Eric B. Monroe, Suresh P. Annangudi, Andinet A. Wadhams, Timothy A. Richmond, Ning Yang, Bruce R. Southey, Elena V. Romanova, Liliane Schoofs, Geert Baggerman, Jonathan V. Sweedler
“Phosphopeptidomics Reveals Differential Phosphorylation States and Novel SxE Phosphosite Motifs of Neuropeptides in Dense Core Secretory Vesicles” by Christopher B. Lietz, Thomas Toneff, Charles Mosier, Sonia Podvin, Anthony J. O’Donoghue, Vivian Hook
“A Multifaceted Mass Spectrometric Method to Probe Feeding Related Neuropeptide Changes in Callinectes sapidus and Carcinus maenas” by Yuzhuo Zhang, Kellen DeLaney, Limei Hui, Junhua Wang, Robert M. Sturm, Lingjun Li
Overall, the conference organizers thank the ASMS Board of Directors for supporting this meeting on such a timely and important topic, and would like to acknowledge the help and hard work of Brent Watson during the entire planning process, and making the conference proceed smoothly. We thank all conference attendees for their participation and for making the meeting a great success. For those who did not attend, reading the papers in this Focus Issue should help fill in some of the gaps.