Aberdeen Proteomics incorporates the Aberdeen Proteome Facility, established in 1997, and the COGEME Proteome Service Facility, established in 2000. Headed by Professor Al Brown, the facility is staffed by researchers specialising in the analysis of protein mixtures by high resolution 2-dimensional gel electrophoresis (2DE) together with identification and characterisation of proteins using peptide mass fingerprinting (PMF), automated N-terminal sequencing, and nanoflow LC-tandem mass spectrometry (LC-MSn). A wide variety of samples are analysed by the proteomics facility, including tissue samples, body fluids, plant material, and microbial extracts.

Users of the facility are encouraged to talk to us before preparing samples for submission. If experiments are being planned that involve a substantial proteomics requirement then we can offer advice, costings and letters of support for grant applications. Please contact Al Brown, Phil Cash or Zhikang Yin if you are planning such a project. Expertise in protein extraction and gel electrophoresis is provided by Evelyn Argo, Phil Cash, Laura Selway and Jan Walker. Image analysis of 2-D gels can be provided as a service, or the user can analyse their own experiment, after suitable training, as appropriate – see Phil Cash or ZhikangYin for further information. The main laboratory houses robotics for gel spot picking, digestion and MALDI plate preparation, MALDI-TOF and ion trap mass spectrometers, plus protein sequencing, amino acid analysis, peptide synthesis, and high-performance liquid chromatography (HPLC) systems. Standard protocols and advice about the above techniques are available from Ian Davidson, David Stead and Elizabeth Stewart. All our methods can be tailored to meet individual research requirements.

Robots (from left to right): Genomic Solutions ProPic, ProMS and ProGest; ABiMED digester

High resolution 2D gel electrophoresis remains a key technology in proteomics for the separation of complex protein mixtures. Proteins are separated according to charge in the first dimension and molecular mass in the second dimension. Aberdeen Proteomics uses immobilised pH gradient (IPG) technology for the first dimension separation coupled with SDS-PAGE for the second dimension. The latter separations are carried out in vertical electrophoresis systems. The Facility processes 2D gels in various size formats using either pre-cast or in-house prepared gels. Proteins resolved by 2DE are typically detected with Coomassie blue or silver stains. In many cases these detection methods are compatible with protein identification by mass spectrometry. Proteins can also be transferred to PVDF membranes for Western blotting or N-terminal sequencing. The gels produced by 2DE are imaged using a laser densitometer or high resolution scanner and the images generated can be analysed with Progenesis software (NonLinear Dynamics) with statistical methods for protein expression and cluster analysis.

Dual-screen PC running NonLinear Dynamics Progenesis software

With the continued progress in whole genome sequencing reliable identifications can now be achieved for proteins from many different sources. Peptide mass fingerprinting (PMF) is one of the most widely used approaches for the identification of proteins extracted from 1D or 2D electrophoresis gels. Aberdeen Proteomics is equipped for the semi-automated processing of samples using robots from Genomic Solutions for excising spots from gels, protein digestion, peptide extraction and purification and subsequent target plate spotting for mass spectrometry. This equipment minimises the risks from keratin contamination of the samples. Peptide mass fingerprints from proteins subjected to in-gel trypsin digestion are determined using an Applied Biosystems Voyager DE-STR MALDI-TOF mass spectrometer to achieve mass accuracies of between 10-100 ppm for individual peptides. The current capacity of the facility for PMF is up to 100 samples per day with optional full database search and identification report. Intact protein masses can also be determined with an accuracy of between 500-1000 ppm (0.05 – 0.1%) for proteins up to 300 kDa.

Voyager DE-STR MALDI-TOF mass spectrometer

A Bruker Daltonics HCTultra PTM Discovery System was installed at the beginning of 2006 and is a significant development for the facility. This system comprises a state-of-the-art ion trap mass spectrometer, the HCTultra, coupled to a nanoflow LC system, the Dionex UltiMate 3000. Proteins may be identified by peptide fragment fingerprinting (PFF) down to femtomole levels. The ion trap has the ability to perform multiple stages of mass analysis (up to 10) with fragmentation by collision-induced dissociation (CID) or electron transfer dissociation (ETD), enabling peptide sequencing and characterisation of post-translational modifications such as phosphorylations and glycosylations. Coupling to nano-LC allows complex mixtures to be analysed, enabling experiments such as MudPIT (multi-dimensional protein identification technique) and quantification by ICAT (isotope-coded affinity tags), providing an alternative to gel-based proteomics.

Bruker Daltonics PTM Discovery System: A Dionex UltiMate 3000 nanoflow LC is coupled to
a Bruker HCT ultra ion trap mass spectrometer with ETD capability

Aberdeen Proteomics also has an Applied Biosystems Procise 494cLC sequencing system to determine N-terminal amino acid sequences of proteins and peptides by automated Edman degradation chemistry. The machine handles both liquid samples and those blotted from gels onto PVDF membrane. Internal sequences can be determined by first digesting the sample with an appropriate enzyme or reagent, separating the digested fragments by HPLC and applying the fragments of choice to the sequencer. The capacity is up to 10 samples per week and sequences of between 5-30 amino acids can be achieved from 0.1-20 picomoles of protein or peptide depending on the sample quality. Optional BLAST database searching can be performed from the sequences information obtained for protein identification.

Applied Biosystems Procise 494cLC protein sequencing system

Aberdeen Proteomics run formal and informal hands-on training courses that can be tailored to the requirements of the individual researcher. These courses cover most of the techniques provided as a service from the laboratories (charges may be made for both materials and technical time).