Uncovering large peptide biomarkers
Date: 18.8.2006
The search for biomarkers in biological systems continues apace as countless research groups look for that elusive peptide, protein or metabolite that will serve as an indicator of a particular disease or condition. Two of the most prominent techniques in use are two-dimensional electrophoresis and mass spectrometry, both of which can be used to compare the profiles of a normal and diseased state, in order to identify those components that have changed.
Now scientists in the US have extended the use of mass spectrometry by taking advantage of a new type of hybrid mass spectrometer that incorporates a linear ion trap alongside a Fourier transform ion cyclotron resonance cell. Steven Taylor and colleagues from Amylin Pharmaceuticals, Inc., devised a general LC/MS-based method that should be applicable to other labs working in biomarker or drug discovery where their target compounds are large peptide hormones.
As Taylor points out, mass spectrometric methods involving laser desorption/ionisation are often used to study the differential patterns of peptides but, for larger peptides, some pre-separation is required. With LC/MS, some degree of pre-separation is built in, but problems can occur with larger peptides due to the overlapping signals from multiple-charged states. However, their new procedure can analyse large peptides in a top-down approach, identifying them on the chromatographic timescale.
They demonstrated the technique with cell cultures of Rin-m5F insulinoma, a pancreatic tumour that produces excess insulin. Changes in secretion of the peptides were monitored following treatment with an external stimulant. The cells were grown in the absence or presence of forskolin, a diterpenoid that is commonly used to raise levels of cyclic AMP, a signal carrier that is required for the control of hormones. Biological variability was minimised by pooling samples of the stimulated or non-stimulated cultures, which were processed by solid-phase extraction on a peptide microtrap to obtain the peptides.
With no further treatment, the extracted mixture was analysed by LC/MS with electrospray ionisation, via a nanotrap and an analytical column in series. For each eluted peptide, full-scan FT/ICR mass spectra and MS/MS spectra were collected at high resolution (100,000) and full-scan ion trap and MS2 and MS3 spectra were also recorded.
Peptides were identified from the tandem mass spectra using the SEQUEST programs, allowing for particular post-translational modifications such as methionine oxidation (+16 u) and N-terminal acetylation (+42 u). The candidates from this procedure were confirmed from the accurate mass data obtained by FT/ICR. For large peptides that were not identified this way, FT MS/MS data were employed, along with the MS Tag module of the free Protein Prospector programs.
From the same data, FT peptide mass maps were constructed and compared using a commercial software package (DeCyder MS) that represented the LC/MS data signal intensity maps for easy differentiation of peptide yields from the stimulated or non-stimulated cultures. These maps were used to determine the relative quantities of 37 of the 41 identified peptides, displaying them on simple 2D bar charts that facilitated comparison and the selection of potential biomarkers.
Using amylin as an example, FT MS and ion trap MS/MS signals were detected in two of the forskolin-treated samples, indicating that physiological levels (20 pM) of peptide hormones were measurable.
The greatest increases in abundance following treatment with forskolin were seen for peptides derived from chromogranin A and B, compounds which are used as cancer biomarkers, as their circulations are normally increased during certain cancers. Five chromogranin A peptides and 21 chromogranin B peptides were identified, including their C-terminal sequences with different post-translational modifications, such as amidation and phosphorylation. The greatest changes in abundance were found for glycine-containing precursors of the C-terminal peptides, indicating that forskolin induced newly synthesised, minimally processed peptide.
In addition, 4 new peptides of cocaine and amphetamine-regulated transcript (CART) were identified, along with other secreted peptides. Two identified proteins that were unchanged after treatment were ubiquitin and copper-zinc superoxide dismutase (SOD), both detected on the LC time scale.
The combination of the hybrid mass spectrometer and profiling software proved successful in identifying and comparing the abundances of the peptides within the one experiment. Yet, 57 peptides with molecular masses below 15 kDa remained unidentified. Some of these gave good fragmentation spectra and, according to Taylor, "await further advances in top-down proteomics informatics."
Despite its success, the spread of the technique may be limited by the high cost of the instrumentation, much of which originates from the large magnet required for the FT MS. The recent introduction of the hybrid ion trap-orbitrap combination may provide a more affordable alternative.
"Source":[ http://www.separationsnow.com/coi/cda/detail.cda?id=14059&type=Feature&chId=0&page=1]
