A novel approach using MALDI-TOF/TOF mass spectrometry and prestructured sample supports (AnchorChip Technology) for proteomic profiling and protein identification.

Mass spectrometry (MS)-based proteomic profiling and protein identification has become a powerful tool for the discovery of new disease biomarkers. Among the MS platforms, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) MS offers high sample throughput and the flexibility to couple with different off-line sample fractionation techniques. Here, we present a strategy using MALDI-TOF/TOF MS to analyze fractionated human serum samples for proteomic profiling and then identify serum peptides from these proteomic profiles. We achieve the profiling analyses by using different functionalized magnetic beads to enrich specific subsets of serum proteins/peptides based on their absorption to these beads. This step is followed by elution, transfer onto prestructured sample supports (AnchorChip targets), and analysis in a MALDI-TOF/TOF mass spectrometer. Selected serum peptides are then analyzed in the tandem MS (TOF/TOF) mode to generate fragment ions for determination of their amino acid sequences. We have demonstrated that using this approach, proteomic profiling and protein identification can be done in a single MS instrument.

Evaluation of a novel, integrated approach using functionalized magnetic beads, bench-top MALDI-TOF-MS with prestructured sample supports, and pattern recognition software for profiling potential biomarkers in human plasma.

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 easily obtained and represent a potentially rich palette of biologically informative molecules. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) represents a key tool for rapidly interrogating 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 diseasespecific biomarkers that may be used to diagnose disease as well as to evaluate disease severity, disease progression, and intervention efficacy. Here we have evaluated a simple, affordable bench-top MALDI-TOF mass spectrometer 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 by this resin. This step is followed by elution, transfer onto a prestructured sample support (AnchorChip technology), and analysis in a bench-top MALDI-TOF mass spectrometer (OmniFLEX) 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 show that the novel and facile approach with manual magnetic-bead sample preparation and a low-cost bench-top MALDI-TOF mass spectrometer is suitable for preliminary biomarker discovery studies.

Mass spectrometry-based clinical proteomics.

In recent years, mass spectrometry (MS) has been recognized as a 'Gold Standard' tool for the identification and analysis of individual proteins in expression proteomics studies. Moreover, MS has proven useful for the analysis of nucleic acids for single nucleotide polymorphism (SNP) genotyping purposes. With the increased usage of MS as a standard tool for life science applications and the advancement of MS instrumentation, sample preparation and bioinformatics, MS technology has entered novel screening and discovery application areas that are beyond the traditional protein identification and characterization applications. The areas of clinical diagnostics and predictive medicine are just two prime examples of these fields. Predictive markers or biomarkers for early diagnosis of diseases are of growing importance for the human healthcare community. The goal of using MS in clinical proteomics is to generate protein profiles (mass to charge [m/z] ratio versus signal intensity) from readily available body fluids like serum, saliva and urine to detect changes in protein levels that reflect changes in the disease states. Whereas the results originating from individual protein markers may be intriguing, data resulting from the analysis of complex, multiple biomarker patterns may be unequivocal. These biomarker patterns are hidden in complex mass spectra and are not always obvious to the human eye. Sophisticated bioinformatics algorithms have to be applied to determine these unique biomarker patterns. Here, we review the latest developments concerning the use of MS for the discovery of biomarker patterns and for the identification of individual biomarkers in the field of clinical proteomics applications.

Solid-phase profiling of proteins.

Protein array technologies refer to any fabrication and use of any arrays containing multiple proteins captured on solid surfaces. This valuable tool is used for high-throughput protein-protein interaction studies, protein marker discovery and other applications. This unit provides basic protocols for biomarker discovery and protein-protein interactions. Different kinds of recently emerged protein array technologies and related detection methods are reviewed.