Technology and Research Platforms

Proteomics

Our Proteomics Facility uses mass spectrometry and is equipped with six state-of-the-art mass spectrometers covering a wide range of applications.

This allows for a broad range of methods including analysis of individual proteins (e.g. intact mass, sequencing, identification), the qualitative and quantitative analysis of complex protein samples (e.g. pull-downs, expression studies, modifications, isotope labelling), as well as mass spectrometry imaging and ion mobility.

The facility is equipped with a broad range of mass spectrometers including some of the most advanced instruments for proteomics, including the latest model of the Orbitrap series with advanced capabilities for proteomics using nano LCMS.

Service requests are welcome from across the Norwich Research Park as well as from external institutions.

Proteomics services

  • Protein and peptide off-line fractionation (HPLC)
  • Determination of protein intact mass
  • Bottom up protein identification (after trypsin digestion); Gel bands and Complex mixtures: CoIP, over-expression, pull downs, total cell lysates
  • Top down protein identification
  • Protein mass fingerprinting (PMF) using MALDI
  • Phosphoproteomics; Phosphopeptide enrichment (including non-canonical) and Phosphopeptide identification and quantification
  • Study of post translational modifications (PTM)
  • Quantification – Cellular protein expression analysis; Label free, Targeted quantification (SRM), Isobaric labelling: iTRAQ or TMT, Stable Isotope Labeling: SILAC
  • Mass spectrometry imaging
  • Ion mobility for small molecule analysis
  • Data analysis

Equipment

Mass Spectrometers

Two Thermo Scientific™ Orbitrap Tribrid™ mass spectrometers

  1. Thermo Scientific™ Orbitrap Eclipse™ Tribrid™ mass spectrometer LCMS with Thermo UltiMate® 3000 RSLCnano LC system
  2. Thermo Scientific™ Orbitrap Fusion™ Tribrid™ mass spectrometer LCMS with Thermo UltiMate® 3000 RSLCnano LC system

Plus

Software

The following programmes for proteomics data processing, protein identification by database search, and quantification, are available team to cover the different needs of sample analysis:

  • Mascot Server 2.7
  • Mascot Distiller 2.7.1
  • Proteome Discoverer 2.4 (Thermo)
  • PEAKS 8.5
  • Scaffold 4.10.0
  • MaxQuant
  • Progenesis LC-MS 1.1
  • R
  • Masslynx (Waters)
  • HDI 1.4 (Waters Imaging)
  • FlexAnalysis (Bruker)

Applications and Sample Submission

Analysis of individual proteins

The proteomics team can run a range of analyses of individual proteins, including;

Intact mass analysis

Can be done on the Maldi-TOF or by LCMS on the QTOF (Synapt G2-Si).

For Maldi provide 10 µl containing 200 pmoles of protein in water or weak buffer/salt solution (no glycerol). For QTOF provide at least 10 µl at 50 µM protein, can be a mixture of proteins and may contain buffer, salts, but no detergent.

Protein sequencing

Can be done on the Maldi-TOF using ISD.

Both termini can be sequenced up to 20-30 aa, but the terminal 10 aa can only be estimated if sequence is known and success depends on individual protein and purity of sample.

Provide 10 µl containing at 100 µM of pure protein in water or weak buffer.

Identification of individual proteins

Mostly done by peptide mass fingerprinting (PMF) after trypsin digestion on the Maldi-TOF; for weak samples and mixtures of protein LC-MS/MS on an Orbitrap Tribrid will used.

Provide excised gel bands (processed according to the Trypsin digestion protocol) or protein solution (10 µl containing some 50 pmoles, detergent-free). For gel staining use instant blue or similar and do not over stain. Do NOT use silver staining.

Analysis of complex protein samples

For protein identification

Done by nanoLC-MS/MS  on Orbitrap instruments or on the timstof Pro (Bruker)

The available instruments provide high sensitive and speed for analysis of complex samples.

Samples can be of different origin (cell lysates, tissue extracts, fractions, CoIP’s) and can be in solution or in gel. Solutions should not contain detergents and excess of other compounds.

Very complex samples can be fractionated by IEX or RP HPLC.

For protein quantitation (protein expression analysis)

LCMS analysis is the same as for protein identification (see above).

Special Software (MaxQuant, PEAKS, Scaffold) will be used for label-free quantitation.

For more accurate quantitation of complex samples, labelling with stable isotopes is recommended. TMT up 16plex is the method of choice for complex expression analysis.

The mass spectrometer with the most advanced methods for TMT based quantification is the new Orbitrap Eclipse (Thermo) which is available in our facility. It features advanced hard- and software for improved TMT analysis including multiple precursor MS3 for the TMT ions as well as real-time search for specific and reliable peptide quantification.

For TMT and iTRAQ, chemical labelling will be provided, whereas other labelling methods (like SILAC, 15N) cannot be provided by our facility. Mass spectrometric analysis can be performed for all labelled samples.

Special applications

Special applications can be discussed with the Proteomics Team

Detection of post-translational modifications

Modifications can be detected after MS/MS in a database search considering the possible mass differences. Enrichment of modified peptides is necessary for low abundance modifications.

Phosphopeptides can be enriched by chromatographic and affinity-based methods, e.g. using metal-affinity methods.

Phosphopeptide enrichment is provided in our facility. It includes Titanium-based enrichment of standard phosphopeptides (modification of Ser, Thr, Tyr residues) as well as SAX-based enrichment of non-canonical phosphopeptides.

Ion mobility analysis

Ion mobility is available on the Synapt G2-Si (Waters) and the timstof Pro (Bruker).

Isobaric isomers can be distinguished by their different ion mobility drift time. Collisional cross sections can be determined if suitable calibrants are available.

Analysis of small molecules

Small molecules can be analysed by direct infusion or LC-MS/MS on the Synapt G2-Si with high mass accuracy (1 ppm).

Maldi- and DESI-Imaging

Maldi imaging of tissues and tissue sections can be performed with the Bruker Autoflex Speed Maldi-TOF/TOF and with the Waters Synapt G2-Si equipped with a Maldi source.

A DESI source is also available for imaging using the Waters Synapt G2-Si.

Successful applications with the available instruments include localisation of small molecules in leaves and leaf sections, coffee beans, trichomes, as well as peptide localisation in human skin sections.

Proteomics publications

  • Gomez-Escribano J.P., Castro J.F., Razmilic V., Jarmusch S.A., Saalbach G., Ebel R., Jaspars M., Andrews B., Asenjo J.A., Bibb M.J. (2019) Heterologous Expression of a Cryptic Gene Cluster from Streptomyces leeuwenhoekii C34T Yields a Novel Lasso Peptide, Leepeptin. Appl Environ Microbiol. 85(23), 01752-19
  • Gherghisan-Filip C., Saalbach G., Hatziioanou D., Narbad A., Mayer M. J. (2018) Processing and Structure of the Lantibiotic Peptide Nso From the Human Gut Bacterium Blautia obeum A2-162 analysed by Mass Spectrometry. Scientific reports 8 10077
  • Kuhaudomlarp S., Patron N. J., Henrissat B., Rejzek M., Saalbach G., Field R. A. (2018) Identification of Euglena gracilisß-1,3-glucan phosphorylase and establishment of a new glycoside hydrolase (GH) family GH149. Journal of Biological Chemistry 293 2865-2876
  • Hems E. S., Wagstaff B. A., Saalbach G., Field R. A. (2018) CuAAC click chemistry for the enhanced detection of novel alkyne-based natural product toxins Chemical Communications 54 12234–12237
  • Louveau T., Orme A., Pfalzgraf H., Stephenson M. J., Melton R., Saalbach G., Hemmings A. M., Leveau A., Rejzek M., Vickerstaff R. J., Langdon T., Field R. A., Osbourn A. (2018) Analysis of Two New Arabinosyltransferases Belonging to the Carbohydrate-Active Enzyme (CAZY) Glycosyl Transferase Family1 Provides Insights into Disease Resistance and Sugar Donor Specificity. Plant Cell 30 3038-3057
  • Lichman B. R., Kamileen M. O., Titchiner G. R., Saalbach G., Stevenson C. E. M., Lawson D. M., O’Connor S. E. (2018) Uncoupled activation and cyclization in catmint reductive terpenoid biosynthesis. Nature Chemical Biology 15 71-79
  • Dong H., Dumenil J., Lu F. H., Na L., Vanhaeren H., Naumann C., Klecker M., Prior R., Smith C., McKenzie N., Saalbach G., Chen L., Xia T., Gonzalez N., Seguela M., Inze D., Dissmeyer N., Li Y., Bevan M. W. (2017) Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis. Genes & Development 31 197-208
  • Hatziioanou D., Gherghisan-Filip C., Saalbach G., Horn N., Wegmann U., Duncan S. H., Flint H. J., Mayer M. J., Narbad A. (2017) Discovery of a novel lantibiotic nisin O from Blautia obeum A2-162, isolated from the human gastrointestinal tract Microbiology-SGM
  • Grenga L., Chandra G., Saalbach G., Galmozzi C. V., Kramer G., Malone J. (2017) Analyzing the Complex Regulatory Landscape of Hfq – an Integrative, Multi-Omics Approach. Frontiers in Microbiology 8 1784
  • Ivanova I. M., Nepogodiev S. A., Saalbach G., O’Neill E. C., Urbaniak M. D., Ferguson M. A. J., Gurcha S. S., Besra G. S., Field R. A. (2017) Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in Euglena gracilis membranes. Carbohydrate Research 438 26-38
  • O’Neill E. C., Saalbach G., Field R. A., O’Connor S. E. (2016) Gene Discovery for Synthetic Biology: Exploring the Novel Natural Product Biosynthetic Capacity of Eukaryotic Microalgae. Methods in Enzymology, Vol. 576 Burlington: Academic Press 99-120
  • Little R. H., Grenga L., Saalbach G., Howat A. M., Pfeilmeier S., Trampari E., Malone J. G. (2016) Adaptive remodeling of the bacterial proteome by specific ribosomal modification regulates Pseudomonas infection and niche colonisation PLoS Genetics 12 (2) e1005837
  • Andriotis V. M., Saalbach G., Waugh R., Field R. A., Smith A. M. (2016) The Maltase Involved in Starch Metabolism in Barley Endosperm Is Encoded by a Single Gene. PLoS ONE 11 e0151642
  • Campo V. L., Ivanova I. M., Carvalho I., Lopes C. D., Carneiro Z. A., Saalbach G., Schenkman S., da Silva J. S., Nepogodiev S. A., Field R. A. (2015) Click chemistry oligomerisation of azido-alkyne-functionalised galactose accesses triazole-linked linear oligomers and macrocycles that inhibit Trypanosoma cruzi macrophage invasion Tetrahedron 71 7344-7353
  • Walden M., Edwards J. M., Dziewulska A. M., Bergmann R., Saalbach G., Kan S. Y., Miller O. K., Weckener M., Jackson R. J., Shirran S. L., Botting C. H., Florence G. J., Rohde M., Banfield M. J., Schwarz-Linek U. (2015) An internal thioester in a pathogen surface protein mediates covalent host binding. eLife 4 e06638
  • Derbyshire P., Ménard D., Green P., Saalbach G., Buschmann H., Lloyd C. W., Pesquet E. (2015) Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis. Plant Cell 27 2709-26
  • Jefferson M., Donaszi-Ivanov A., Pollen S., Dalmay T., Saalbach G., Powell P. P. (2014) Host Factors interacting with the Pestivirus N terminal protease, Npro are Components of the Ribonucleoprotein Complex., J Virology 88, 10340-10353
  • Rashid A. M., Saalbach G., Bornemann S. (2014) Discrimination of large maltooligosaccharides from isobaric dextran and pullulan using ion mobility mass spectrometryRapid Comm Mass Spectrometry 28, 191-199