“Scientists can apply different MS approaches to answer discreet scientific questions relevant to specific steps of the biomarker discovery and validation pathway,” agreed Carsten Baessmann, Ph.D., head of applications development LC/MS, Bruker Daltonik.
“Bruker’s new maXis impact™ ultra high resolution TOF mass spectrometer enables us to use all common approaches for discovery and validation on a single instrument. Moreover, maXis impact delivers the analysis over the four orders of dynamic range with sub-ppm mass accuracy.”
The team explored the performance of the maXis impact using three different options for targeted proteomics. The first approach was to narrowly define the target mass range and to determine the dynamic range and the lower limits of quantitation in a one-dimensional MS separation.
“We were able to define the mass range within millidaltons of the target ion,” said Stephanie Kaspar, proteomics scientist in applications development. “This means that we can successfully distinguish the target from the matrix, even at the concentrations as low as 25–50 attomoles.”
Alternatively the instrument can be switched to the middle-band CID (collision induced dissociation) mode. In this mode, a narrow mass window of 26 daltons is selected to generate the fragments in the second MS dimension. By using overlapping 26 dalton windows, the entire mass range can be scanned.
The resulting ion chromatograms are analyzed to derive either relative sample-to-sample quantitation or absolute quantitation against the reference standard. High-resolution power of maXis impact UHR-TOF enables efficient separation of the adjacent ions, which is critical for identification of unknowns in complex biological samples.
“Using the third approach, the broad-band CID, we quantify target peptides over the entire mass range and confirm the identity via fragment information, without selecting any particular mass window,” continued Dr. Baessmann.
In the first MS dimension, all peptides are quantified. Next, the identity of each peptide is verified by fragmentation and analysis of the resulting ions. For forensic toxicology this approach results in the least number of false positives.
For example, co-eluting dibenzipin and a doxpin metabolite, with a mass difference of 11.2 millidaltons, were correctly identified in authentic urine samples. Further studies are aimed at applying these technologies to identifying and quantifying peptides from biological and clinical samples.
Clinical Diagnosis of Alzheimer Disease
Proteome Sciences has perfected SRM technology for the quantitation of Tau phospho-peptides, one of the main components of Alzheimer disease (AD). Tau is believed to stabilize microtubules in cells and may play a role in regulating synaptic signaling.
Phosphorylation of Tau results in detachment from the cellular scaffold, followed by formation of tangles characteristic for AD pathology. Tau can be phosphorylated at over 30 specific sites. The pattern of Tau phosphorylation seems to correlate with severity of pathological progression of the AD.
“If we detect and quantify Tau phosphorylation at individual amino acids, we will be able to aid in clinical diagnostics and to stratify patients for clinical trials,” commented Ian Pike, Ph.D., COO, Proteome Sciences.
In collaboration with Professor Brian Anderton at King’s College London, Proteome Sciences progressively identified kinases responsible for the phosphorylation process and their unique sites. The team singled out the CK1 delta kinase to be an early player in the sequence of Tau phosphorylation.
While pursuing validation of this kinase as a therapeutic target for its internal drug discovery program, Proteome Sciences secures contract biomarker services with pharmaceutical partners. The company provides SRM-based assays for absolute quantitation of total Tau protein and its phosphorylation at 10 sites. Tau can be tested in brain tissue, cells, and, soon, in cerebrospinal fluid.
“Not all required phosphorylation sites could be measured accurately using antibodies, and some of the phosphorylation sites are only one amino acid apart. So we spent a lot of effort optimizing the transitions for a multiplexed SRM assay,” continued Dr. Pike.
“In some models Tau contains mutations that lose the required trypsin cleavage point, therefore, we used an alternative peptidase to generate target peptides. Furthermore, we optimized the assay to detect Tau in different cellular fractions.”
In early 2012, expert international workgroups convened by the Alzheimer’s Association and the National Institute on Aging, an agency of the U.S. National Institutes of Health, have expanded AD diagnostic guidelines to include Tau. Proteome Sciences works with clients to develop customized assays for other phosphorylation sites to support drug development.
“SRM is the ideal technology for this application, providing highly discriminating measurements and high sensitivity,” concluded Dr. Pike.