Biomolecular Analysis

Proteomics

The John Innes Centre Proteomics Facility provides proteomic services using mass spectrometry to the institutions across the Norwich Research Park.

Proteomic technologies, and supporting expertise, enable us to identify the entire protein complement (proteome) of a sample, whether from a cell or whole organism.

Comparing a partial amino acid sequence for a protein with an organism’s genome sequence immediately enables recognition of both the corresponding gene and the protein’s complete amino acid sequence.

Proteomics links the static information contained in the organism’s DNA (its genotype) to the dynamic physiology of the whole-cell and/or whole-organism (its phenotype).

By analysing the abundance of proteins under different physiological conditions, or in different mutants, it is possible to identify groups of proteins that are important under those particular physiological conditions or are affected by that specific mutation. The amino acid sequence of the proteins identifies the genes involved.

Ongoing advances in automated sample preparation, mass spectrometry and bio-informatics are making it possible to quickly and accurately relate proteins to genes.

The facility is equipped with four state-of-the-art mass spectrometers covering a wide range of applications and a number of state-of-the-art programmes for data processing, protein identification by database search, and quantification, so that optimal applications are available for different needs of sample analysis.

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

The Proteomics equipment and services at the John Innes Centre include;

  • Orbitrap-Fusion (Thermo) with nanoLC
  • Synapt G2Si (Waters) with UPLC, nanoLC, Maldi, DESI
  • Autoflex Speed (Bruker)
  • Xevo TQS Triple Quad (Waters) with nanoLC
  • Analysis of intact proteins: intact mass by Maldi-Tof or LCMS-ESI , N-terminal sequencing (Maldi ISD)
  • DESI imaging on Synapt G2Si
  • Identification/ characterisation of individual proteins by Maldi PMF or nanoLC Oribtrap
  • Analysis of complex protein samples (total cell extracts, CoIP’s etc) using nanoLC Orbitrap or nanoLC MSE (Synapt G2Si)
  • Quantitative analysis of proteomics samples by label free quantification or using isotope labelling (e.g. iTRAQ, TMT, 15N)
  • Ion mobility spectroscopy on Synapt G2Si
  • Maldi imaging on Synapt G2Si and on Autoflex speed

“The John Innes Centre Proteomics Facility provides services in the area of proteomics using mass spectrometry. The facility is equipped with four 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”

– Dr Gerhard Saalbach, Proteomics Senior Scientist

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 10 µl containing 100 pmoles of protein, can be a mixture of proteins and may contain buffer salts etc.

Protein sequencing

Mostly done on the Maldi-TOF using ISD.

Both termini can be sequenced up to 30 aa, but the terminal 10 aa can only be estimated if sequence is known. Provide 10 µl containing 1 nmole of pure protein in water or weak buffer, success depends on individual protein and purity of sample.

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 the Orbitrap will used.

Provide excised gel bands or protein solution (10 µl containing 50 pmoles, detergent-free). For gel staining use instant blue or similar and do not over stain. Do NOT use silver staining. Destain with 30% ethanol at 65°C. If you want to submit more than 8 gel bands, you should prepare them for the trypsin digestion according to the preparation of gel slices for Trypsin digestion protocol.

Analysis of complex protein samples

For protein identification

Done by nanoLC-MS/MS (Orbitrap Fusion or Synapt G2-Si).

The Orbitrap Fusion is sensitive and fast. The Synapt G2-Si uses MSe fragmentation and ion mobility. Complex samples can be offline fractionated by IEX or RP HPLC Database searches will be performed using “Mascot” and in addition search results will be provided in “Scaffold” files.

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 (peptides). Samples can be run into an SDS gel and lanes cut into slices. For detection of specific low abundant proteins, enrichment is advised before sample submission.

For protein quantitation (protein expression analysis)

MS analysis is the same as for protein identification (see above). Special Software (MaxQuant, Progenesis, Scaffold) will be used for label-free quantitation. For more accurate quantitation labelling with stable isotopes is recommended. For iTRAQ, chemical labelling will be provided. iTRAQ analysis will be performed on the Synapt G2-Si. For SILAC and 15N no labelling service is provided. Depending on type, quantitation will be performed using different software including Mascot, Mascot Distiller, MaxQuant.

Sample preparation as above. Accurate, reproducible sample preparation is required for good quantitation. 15N labelling is recommended for plants (cell cultures) and bacteria. SILAC labelling is recommended for animal/human cell cultures and bacteria. iTRAQ labelling will be applied after protein/peptide preparation if no metabolic labelling is possible.

Special applications

Special applications can be discussed with the Proteomics Facility Manager.

Detection of post-translational modifications

Modifications can be detected after MS/MS in a normal database search taking into account 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.

Ion mobility analysis

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 on Synapt G2-Si

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

Maldi 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.

Equipment

Our proteomics facility is equipped with four state-of-the-art mass spectrometers covering a wide range of applications.

We also have a range of state-of-the-art programmes for data processing, protein identification by database search, and quantification, so that optimal applications are available for different needs of sample analysis.

Thermo Orbitrap Fusion LC-MS

The Orbitrap Fusion mass spectrometer combines quadrupole, Orbitrap and ion trap mass analysis in a Tribrid architecture.

It enables analysis of challenging low-abundance, high-complexity, or difficult samples to identify more compounds more quickly, quantify more accurately, and elucidate structures more thoroughly.

The Orbitrap Fusion is coupled to a Thermo Scientific Dionex UltiMate 3000 UHPLC system for analysis of complex proteomics samples.

The system allows for reporter ion quantification (iTRAQ, TMT) using synchronous precursor selection.

Bruker Autoflex™ Speed Maldi-TOF/TOF

The Bruker Autoflex Speed Maldi-TOF/TOF is a dedicated instrument for fast mass analysis of a broad range of samples including small molecules, peptides and proteins.

It can be used for protein sequencing (ISD) and protein identification using Peptide Mass Fingerprint (PMF).

Analysis of glycopeptides is supported by the Glycoquest software.

Maldi imaging of tissue samples is also possible using the Fleximaging software.

A special function is available for direct analysis of TLC plates.

Waters Synapt G2-Si QTOF LC-MS

The Waters Synapt G2-Si mass spectrometer (QTOF) can be configured with different LC systems and MS sources to suit different applications. These include:

  • Waters Acquity UPLC for protein analysis and metabolomics
  • Waters NanoAcquity UPLC for complex proteomics
  • Options to use MALDI or DESI sources, primarily for imaging

A special feature of the Synapt G2-Si is its ion mobility function allowing high resolution analysis of compounds based on their shape and structure.

Waters Xevo TQS Triple Quad LC-MS

The Waters Xevo TQ-S is a triple quadrupole instrument and is used for targeted quantitation using SRM methodology.

HPLC

  • 3 nanoACQUITY UPLC (Waters, Manchester, UK)
  • 2 ACQUITY UPLC (Waters, Manchester, UK)
  • 1 Ultimate 3000 HPLC (Dionex/Thermo, Leeds, UK)
  • 1 626 HPLC System (Waters, Manchester, UK)

Robots

  • 1 MALDI-AutoPrep (MAP II) Spotting Robot (Bruker, Coventry, UK)
  • 1 LC-MALDI Fraction Collector Proteineer fc (Bruker, Coventry, UK)

Software

The Proteomics Facility is equipped with a number of state-of-the-art programmes for data processing, protein identification by database search, and quantification, so that optimal applications are available for different needs of sample analysis.

  • Mascot Server
  • 2.4 Mascot Distiller
  • Proteome Discoverer
  • Scaffold 3
  • MaxQuant
  • Progenesis LC-MS