Combine and conquer

Considered by many the promised land of biological research – proteomics is as complex as it is broad. At its heart however has to be good analysis, and for that you need both good data and good bioinformatics. Here we look at how one research facility combines the two

MODERN proteomics research requires advanced technology such as mass spectrometry and bioinformatics. Complex protein mixtures are enzymatically digested and the resulting peptide mixture then analysed using high-performance liquid chromatography (HPLC) followed by mass spectrometry. These kinds of analyses provide information on the peptide masses and their sequences. The data are searched against computer-predicted spectra generated from protein databases derived from genome sequences. The result is peptide (and protein) identification and sequence determination.

The Advanced Mass Spectrometry Facility in the School of Biosciences at the University of Birmingham has extensive experience of this technology for different applications and especially for analysis of complex peptide mixtures separated on-line by fast HPLC. The facility boasts two Quadrupole Time of Flight (Q-TOF) mass spectrometers (Waters Q-TOF II and a Waters Q-TOF Ultima Global) and a hybrid Linear Ion Trap - Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometer (a Thermo Finnigan LTQ FT). The FT-ICR mass spectrometer is equipped with a chip-based nanoelectrospray system (AdvionBiosciences TriVersa NanoMate). Data analysis is performed using Sequest (Bioworks, Thermo Fisher), Mascot (Matrix Science) or OMSSA (NCBI) software.

The Waters Q-TOF Ultima Global mass spectrometer combines electrospray and MALDI sample introduction with high sensitivity MS and MS/MS analysis of protein and peptide mixtures. Combined with the CapLC chromatography system it can be used to analyse complex mixtures. The Q-TOF can be used for LC-coupled MS/MS product and precursor ion experiments, in addition to direct infusion analyses.

For studies requiring extra resolution and mass accuracy the LTQ FT instrument is available. The Thermo Finnigan LTQ-FT is a 7 Tesla FT-ICR mass spectrometer whose tandem mass spectrometry capabilities include electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) in the ICR cell, and collision-induced dissociation (CID) in the ion trap. Although CID fragmentation occurs in the ion trap, the fragments may be detected in the ICR cell for additional mass accuracy. The LTQ FT is coupled with electrospray ionisation.

The ultra high-resolution and mass accuracy inherent to the LTQ-FT enable the following analyses:

• Determination of elemental composition (CcHhNnOoSsPp) based on mass alone (MW up to ~ 450)
• Resolution of ions very closely spaced in mass

The technique is therefore ideally suited to the analysis of complex mixtures, e.g., those found in metabolomic or petroleomic studies. Furthermore, as a result of the ability to detect in the linear ion trap, automatic gain control is employed which greatly reduces space charging effects in the ICR cell, regardless of ion flux.

The unique resolution and mass accuracy also allow the determination of molecular weight of large species (up to ~ 70,000 Da). For example, the FT-ICR instrument is applied to top-down analyses of proteins. Because the FT-ICR is coupled with electrospray ionisation, it is possible to generate intact non-covalent complexes in the gas phase, allowing protein interactions and secondary structure to be investigated.

The ECD capabilities are particularly advantageous for peptide and protein analysis. ECD is a technique that is unique to FT-ICR and offers many advantages for the analysis of peptides and proteins, including more extensive cleavage and therefore sequence information. Unlike traditional MS/MS techniques, ECD fragments retain post-translational modifications (PTM) e.g. phosphorylation and glycosylation, enabling localisation of the PTM. It is therefore possible to perform protein PTM mapping using ECD. Multiple amino acid sites of post-translational modification may be identified unambiguously from a single sample.

For bottom-up proteomics experiments, the high complexity of peptide mixtures resulting from enzymatic protein digestion requires either fast scan cycles, to acquire mass spectral data for co-eluting peptides or long chromatographic gradients, to improve chromatographic resolution. The linear trap of LTQ-FT-ICR is used to perform ion accumulation and activation prior to detection in the ICR cell which increases the scan rate. Increased duty cycle allows for data-dependent mass analysis of co-eluting peptides to be acquired, increasing protein sequence coverage without increasing the gradient length. The linear trap can be used as an ion detection device to perform simultaneous detection of tandem mass spectra with full scan mass spectral detection in the ICR cell, resulting in the fastest scan cycles for performing bottom-up sequencing of protein digests.

The mass spectrometry group at the School of Biosciences is also undertaking several research studies including phosphorylation enrichment and quantitative mass spectrometry analysis.

All these applications can be performed using the Advanced Mass Spectrometry Facility at University of Birmingham, which is part of Functional Genomics and Proteomics Unit. This Unit provides additional services such as DNA sequencing, DNA microarraying, real-time PCR and genotyping. The sequencing facilities are supported by an Applied Biosystems 3700 DNA Analyser, Roboseq 4204 and Qiagen-Biorobots 3000 and 8000 (automated DNA minipreps) which have increased the through-put samples received. The recently acquired MegaBase Sequencing System ensures high through-put sequencing and genotyping.

The Functional Genomics and Proteomics Unit has access to a variety of equipment including IPGphors and Multiphors for running 2-D gels, automated spot cutter, trypsinisation service which uses automated techniques to extract peptides from 2-D gels for use in mass spectrometry. Customers’ gel plugs are received in 96 well plates which are provided by the Proteomics laboratory free of charge. Although the laboratory does contain equipment for running 1D and 2D gels, this service is not offered commercially and customers are asked to provide gel plugs in the 96 well plates which will be dispatched to them. The laboratory does offer training and advice on electrophoresis techniques. Robots perform the necessary extraction procedures to isolate peptides ready for analysis by the QTOF or FTICR mass spectrometers.

The facility and its staff are involved in numerous research efforts in Biosciences, working within such areas as cancer research, cell signalling, microbial gene expression and modulation of plant growth. The facility also services the University’s other departments and external collaborators, both academic and industrial.

By Antony Jones. Antony is Manager of Functional Genomics and Proteomics Services School of Biosciences at the University of Birmingham