"Evaluation of a Novel, Integrated Approach Using Functionalized Magnetic Beads, Batch-Top MALDI-TOF MS with AnchorChip^TM Technology and Pattern Recognition Software for Profiling Potential Biomarkers in Human Plasma"

Xinyi Zhang, Bruker Daltonics, Xinyi.zhang@bdal.com

The advantage of using proteins and peptides as biomarkers is that they can be found readily in blood, urine, and other biological fluids.  Such sample types are readily obtainable and represent a potentially rich palette of biologically informative molecules.  MALDI-TOF mass spectrometry has become a powerful tool for surveying such sample types.  The goal of clinical proteomics is to harness the power of this tool for identifying novel, condition-specific protein fingerprints that may, in turn, lead to the elucidation and use of disease-specific biomarkers.  Here we have evaluated a simple affordable bench-top MALDI-TOF MS to generate protein profiles from human plasma samples of asthma patients and healthy individuals.  We achieve this profiling by using C8-functionalized magnetic beads that enrich a specific subset of plasma proteins based on their absorption to this resin.  This step is followed by elution, transfer onto AnchorChip™ targets and analysis in a bench-top MALDI-TOF MS mass spectrometer (OmniFLEX™, Bruker Daltonics) with AutoXecute™ acquisition control which enables automated operation with reproducible results.   Resulting spectra are compiled and analyzed through the pattern recognition component of ClinProTools™ software. This approach in combination with ClinProTools™ software permits the investigator to rapidly scan for potential biomarker peptides/proteins in human plasma.   The reproducibility of plasma profiles within- and between- days has been evaluated.  The results shows that the novel and facile approach with manual magnetic bead sample preparation and a low-cost bench-top MALDI-TOF MS is suitable to low/medium- throughput plasma protein profiling.

Identification of proteins by mass spectrometry is a well-accepted technique. Once a protein has been identified, however, often many questions arise regarding the biology of that protein. The Celera Discovery System, or CDS can provide answers to many of those questions. The Celera Discovery System is a subscription-based integrated research platform that provides on-line access to comprehensive curated biological data. By searching specially prepared FASTA files incorporating information from CDS, protein molecular functions and biological processes are imported into all protein search results. Direct links to CDS from protein ID software enable further analysis of ontology information as well as comprehensive genomic and biological information. This poster will focus on the different types of information available from CDS and how this information can be used to provide biological insight from various protein ID and quantitation applications.

The discovery stage of proteome analysis typically involves the comparison of different states of a cell or tissue such as diseased vs. normal, mature vs. immature or drug-treated vs. non-treated. This profiling traditionally has been done by two-dimensional gel electrophoresis. While this technique has successfully resolved proteomes, it is labor intensive, time consuming and, at best, semi-quantitative. This paper presents a two- dimensional chromatographic alternative for proteome profiling and fractionation. A complete system, the ProteomeLab PF 2D, has been developed with integrated hardware, software and chemistry. The first dimension separation is done by chromatofocusing over a pH range from 8.5 to 4.0 where proteins are separated by their isoelectric points. In this dimension, fractions are collected based on pH intervals as detected by a pH monitor. The proteins are detected by UV absorbance at 280 nm. The first dimension fractions are then resolved in the second dimension by high resolution reversed phase chromatography with a gradient of trifluoroacetic acid (TFA) in acetonitrile and TFA in water. The proteins are detected by a second UV detector at 214 nm. The two dimensions are linked by a combination fraction collector/auto-injector. This allows the second dimension to commence automatically when the first dimension is complete. The proteome profiles of human plasma from a fasting and non-fasting state were compared with this new system to determine if there were any qualitative and/or quantitative differences. Data from this comparative study with this new proteome profiling and fractionation system will be presented.