Institute for Research in Biomedicine (IRB Barcelona), Spain

What is the focus of your lab’s research?

Our goal is to provide the research community at IRB Barcelona and their co-workers with state-of-the-art tools and methodologies for the MS analysis of a broad range of biological species, from large proteins and DNA to small molecules. The final purpose is to get insight into these molecules’ identity, structure, interaction with other molecules and biological function in order to help in drug design, protein mechanism elucidation and in the search for biomarkers. We have implemented methods specialized in top-down proteomics and we are pioneers in this MS strategy in Spain.

As a core facility, we are responsible for working with different biologic molecules, and we are required to change methods constantly and efficiently.

How does the TriVersa NanoMate® align with your research goals?

Originally, we purchased the TriVersa NanoMate® for its chip-based direct infusion mode for noncovalent interaction analysis, but we learned quickly that it could be applied to other areas of our research. Prior to using the TriVersa NanoMate®, the steps involved in collecting fractions were painful and time-consuming. With the TriVersa NanoMate®, we can run LC/fraction collection or infusion without changing the setup and wasting time with stabilization. We do not experience the problems typical with traditional nanoelectrospray sources.

One aspect of the TriVersa NanoMate® that impressed us was the ability to analyze complicated top-down samples with the LC compatibility. It is not possible to analyze these samples on an LC time scale, and the fraction collection capability allowed us to analyze in a way that was not possible previously.

To whom would you recommend the TriVersa NanoMate® for their research?

We use the TriVersa NanoMate® for everything; noncovalent interactions, top-down, middle-down, bottom-up, basic infusion, LC coupled to fraction collection. The instrument is useful in all of its different set-ups, especially without having to change sources and waiting for a stable spray.

The reliability of the system is one of the greatest benefits especially for people who have to change frequently between applications. We have found the spray sensing feature to be very valuable because we know our precious samples will not be lost.

Do you have any publications or presentations using the TriVersa NanoMate®?

Publication Highlight

Characterization of Human Sperm Protamine Proteoforms Through a Combination of Top-Down and Bottom-Up Mass Spectrometry Approaches
Soler-Ventura et al. J Proteome Res, 2020, 19(1), 221-237. DOI: 10.1021/acs.jproteome.9b00499
Identified the sperm protamine proteoforms profile, including their post-translational modifications, in normozoospermic individuals using a top-down MS approach and a proteinase-K-digestion-based bottom-up MS approach.

Other Publications
  • Arauz-Garofalo et al. Protamine characterization by top-down proteomics: Boosting proteoform identification with DBSCAN. Proteoms, 2021. DOI: 10.3390/proteomes9020021
  • Yero et al. The Pseudomonas aeruginosa substrate-binding protein Ttg2D functions as a general glycerophospholipid transporter across the periplasm. Comm Bio, 2021. DOI: 10.1038/s42003-021-01968-8
  • Molnar et al. The histone code reader PHD finger protein 7 controls sex-linked disparities in gene expression and malignancy in Drosophila. Sci Adv, 2019. DOI: 10.1126/sciadv.aaw7965
  • Nadal et al. Structure of the homodimeric androgen receptor ligand-binding domain. Nat Commun, 2017. DOI: 10.1038/ncomms14388
  • Testoni et al. Lack of glycogenin causes glycogen accumulation and muscle function impairment. Cell Metabolism, 2017. DOI: 10.1016/j.cmet.2017.06.008
  • Izquierdo-Serra et al. Optical control of endogenous receptors and cellular excitability using targeted covalent photoswitches. Nat Commun, 2016. DOI: 10.1038/ncomms12221
  • Pujol-Pina et al. SDS-PAGE analysis of Aβ oligomers is disserving research into Alzheimer’s disease: Appealing for ESI-IM-MS. Sci Rep, 2015. DOI: 10.1038/srep14809
  • Saez et al. Influence of PPh3 moiety in the anticancer activity of new organometallic ruthenium complexes. J Inorg Biochem, 2014. DOI: j.jinorgbio.2014.03.002
  • Borg et al. Spectral counting assessment of protein dynamic range in cerebrospinal fluid following depletion with plasma-designed immunoaffinity columns. Clin Proteomics, 2011. DOI: 10.1186/1559-0275-8-6

Max Planck Institute of Molecular Cell Biology and Genetics, Germany

Q: WHAT IS THE FOCUS OF YOUR LABS RESEARCH?

A: The laboratory is split 50/50 between proteomics and lipidomics research. While working on protein analysis, such as identifying protein interaction networks or characterizing the proteomes of organisms that are related very distantly to organisms with sequenced genomes, we also attempt to better quantify the lipidome of various organelles, cells and tissues.

Q: WHAT ARE YOUR PRIMARY GOALS?

A: In lipidomics, we forge the alliance with developmental biology. The primary goal of the group is to combine lipidomics with developmental biology. As organisms grow and develop from a single cell, newly differentiated tissues require their own unique membrane lipid composition. We hope to characterize these tailored changes to better understand how inherited defects in lipid metabolism cause disease. We are equally interested in lipidomes of membrane microdomains and the biological significance of its remarkable complexity.

Q: WHY DID YOU INCORPORATE THE TRIVERSA NANOMATE® INTO YOUR LABORATORY?

A: We had a need for automated nanoflow direct-infusion capabilities. Shotgun lipidomics relies on low and stable flow rates, and the TriVersa NanoMate® has this demonstrated ability. We have purchased three additional instruments because they allow us to rapidly switch between lipids and proteomic analysis.

Q: DO YOU HAVE ANY PUBLICATIONS OF PRESENTATIONS USING THE TRIVERSA NANOMATE®?
Publication Highlight
2021: Hormone-Sensitive Lipase Couples Intergenerational Sterol Metabolism to Reproductive Success
Christoph Heier Is a corresponding author , Oskar Knittelfelder, Harald F Hofbauer, Wolfgang Mende, Ingrid Pörnbacher, Laura Schiller, Gabriele Schoiswohl, Hao Xie, Sebastian Grönke, Andrej Shevchenko, Ronald P Kühnlein
Hormone-sensitive lipase (Hsl) was identified as an ancestral regulator of SE degradation, which improves intergenerational sterol transfer and reproductive success in flies.

Other Publications:

  • Knittelfelder, O., Prince, E., Sales, S., Fritzsche, E., Woehner, T., Brankatschk, M., Shevchenko, A. (2020) Sterols as dietary markers for Drosophila melanogaster. BBA – Molecular and Cell Biology of Lipids, 1865 (7), 1388-1981. DOI: 10.1016/j.bbalip.2020.158683
  • Trautenberg, L.C., Knittelfelder, O., Hofmann, C., Shevchenko, A., Brankatschk, M., Prince, E. (2020) How to use the development of individual Drosophila larvae as a metabolic sensor. Journal of Insect Biology, 126, 0022-1910. DOI: 10.1016/j.jinsphys.2020.104095
  • Wang, Y., Hinz, S., Uckermann, O., Hoenscheid, P., von Schoenfels, W., Burmesiter, G., Hendricks, A., Ackerman, J.M., Baretton, G.B., Hampe, J., Brosch, M., Schafmayer, C., Shevchenko, A., Seissig, S. (2020) Shotgun lipidomics-based characterization of the landscape of lipid metabolism in colorectal cancer. BBA – Molecular and Cell Biology of Lipids, 1865 (3), 1388-1981. DOI: 10.1016/j.bbalip.2019.158579
  • Finkelstein, S. Gospe III, S.M., Schuhmann, K., Shevkenko, A., Arshavsky, V.M., Lobanova, E.S. (2020) Phophoinositide profile of the mouse retina. Cells 9(6), 1417. DOI: 10.3390/cells9061417
  • Brankatschk, M., Gutmann, T., Knittelfelder, O., Palladini, A., Prince, E., Grzybek, M., Brankatschk, B., Shevchenko, A., Coskun, U., Eaton, S. (2018) A temperature-dependent switch in feeding preference improves drosphila development and survival in the cold. Developmental Cell, 46, 6, 781-793.e4. DOI: 10.1016/j.devcel.2018.05.028
  • Fernandez, C., Sandin, M., Sampaio, J.L., Almgren, P., Narkiewicz, K., Hoffmann, M., Hedner, T., Wahlstrand, B., Simons, K., Shevchenko, A., James, P., Melander, O. (2013) Plasma lipid composition and risk of developing cardiovascular disease. PLOS One. DOI: 10.1371/journal.pone.0071846
  • Ghosh, A., Kling, T., Snaidero, N., Sampaio, J.L., Shevchenko, A., Gras, H., Geurten, B., Goepfert, M.C., Schulz, J.B., Voigt, A., Simons, M. (2013) A global in vivo drosophila RNAi screen identifies a key role of ceramide phosphothanolamine for glial ensheathment of axons. PLOS Genetics. DOI: 10.1371/journal.pgen.1003980
  • Schuhmann, K., Almeida, R., Baumert, M., Herzog, R., Bornstein, S.R. and Shevchenko, A. (2012) Shotgun lipidomics on a LTQ Orbitrap mass spectrometer by successive switching between acquisition polarity modes. J. Mass. Spectrom., 47: 96-104. DOI: 10.1002/jms.2031
  • Carvalho, M., Sampaio, J.L., Palm, W., Brankatschk, M., Eaton, S., Shevchenko, A. (2012) Effects of diet and development on the Drosophila lipidome. Mol Syst Biol, 8:600. DOI: 10.1038/msb.2012.29
  • Luerschner, L. Richter, D., Hannibal-Bach, H.K., Gaebler, A., Shevchenko, A., Ejsing, C.S., Thiele, C. (2012) Exogenous ether lipids predominantly target mitochondria. PLOS ONE. DOI: 10.1371/journal.pone.0031342
  • Sampaio, J.L., Gerl, M.J., Klose, C., Ejsing, C.S., Beug, H., Simons, K., Shevchenko, A. (2011) Membrane lipidome of an epithelial cell line. PNAS, 108 (5), 1903-1907. DOI: 10.1073/pnas.1019267108
  • Ejsing, C.S., Sampaio, J.L., Surendranath, V., Duchoslav, E., Ekroos, K., Klemm, R.W., Simons, K., Shevchenko, A. (2009) Global analysis of the yeast lipidome by quantitative shotgun mass spectrometry. PNAS, 106 (7), 2136-2141. DOI: 10.1073/pnas.0811700106

Max Planck Institute for Chemical Ecology, Germany

Q: What is the focus of your lab’s research?

A: One of the topics of our lab is the interaction of insect pheromones and their analogues with pheromone-binding proteins (PBPs) which are part of an extremely sensitive multi-component pheromone detection system.

We utilize binding assays to determine the function of the PBPs through affinity measurements of the protein-ligand receptors, calculate binding constants, the spatial arrangement of the complex and do modeling. In addition, point-mutated PBPs are used for a better understanding of the contribution of individual amino acids to the binding event.

Q: How does the TriVersa NanoMate® (TVNM) align with your research goals?

A: The TVNM enabled us to develop a high-throughput method to study protein-ligand-interactions for large series of different pheromones and their analogues.

As the binding energies involved are very low and we need to preserve the native structure of the molecules, the soft-ionization conditions of the TVNM are perfect for us. Further, these studies are difficult with classic electrospray, due to the stickiness of the samples. They create problems from short cleaning cycles and produce contaminations.

In contrast, the established method with the TVNM is reliable and stable, and it eliminates the sticky sample issues. In addition, multiple experiments with very low quantities of protein (1 nmol) at different cone-voltage conditions are possible.

We recently added the LESA™ (Liquid Extraction Surface Analysis) capability to the TVNM. This enables us to detect putative signal molecules on leaf surfaces and to track down their production and storage sites by comparing the data with extracts from samples derived from the inner compartments of the leaves.

Q: To whom would you recommend the TriVersa NanoMate for their research?

A: I would recommend the TriVersa NanoMate to everybody because it is a universal source. With direct infusion, coupling for fraction collection and surface analysis, it may replace all ionization sources.

LESA – Liquid Extraction Surface Analysis

More analytes and higher sensitivity

Developed in collaboration with Oak Ridge National Laboratory*, the liquid extraction surface analysis (LESA) capability of the TriVersa Nanomate® enables simple, direct ESI mass spectrometric analysis from a variety of surfaces.

Using the TriVersa NanoMate® for Rapid Analyte Identification Based on Static nESI-MS/MS (Infusion MS/MS)

In many cases of routine protein identification, processing times become the major bottleneck at the core facility. Although a nano-LC/MS/MS approach is recognized as a general strategy for protein identification, the typical 30-120 min run-time prohibits fast turnaround times. The TriVersa NanoMate offers a more rapid approach for the infusion of the protein digest, with only a 2-5 min run-time per sample.

Integrated Top-down/Bottom-up Proteomics for the Characterization of Protein Isoforms

Presented by: Ljiljana Paša-Tolić, Ph.D., Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory

Description: Obtaining extended sequence coverage of intact proteins and characterizing multiple post-translational modification (PTM) sites can pose key technical challenges. In this webinar, we describe how we have implemented Advion’s TriVersa NanoMate® and RePlay® technologies to address these issues using an integrated top-down/bottom-up approach.

We demonstrate how the TriVersa NanoMate’s capabilities of chip-based electrospray ionization (ESI) and fraction collection are used to profile isoforms, further investigate targeted proteins at the intact level using ECD or CID, perform bottom-up proteomics on a collected fraction to get confident protein identifications, perform targeted MS/MS on particular fractions of interest, and better characterize where PTMs may have occurred.

We also discuss how RePlay simplifies the data analysis process by allowing you to see the correlation between the intact protein and the observed peptides, even when you are sample limited. Using RePlay, we incorporated an on-line digestion strategy with the integrated top-down/bottom-up approach. This technique allows you to implement LC-MS/MS during both the intact run and a secondary RePlay run with digestion, so you can see the correlation in time between the observed parent ions and the digested ions as well as the MS/MS of the fragment ions.