Proteins biomarker molecules

Intact-cell MALDI-TOF analysis offers several attractive features for rapid screening of bacterial collections. Analysis is performed directly on the cells after minimal sample preparation, and data acquisition is complete in only a matter of minutes. Intact biomarkers are introduced into the MALDI-TOF instrument under these conditions. Whether the observed biomarker molecules are desorbed directly from the surface of the cell wall or are extracted from the cells and co-crystallized with the matrix is currently unresolved, but MALDI spectra of intact bacteria generally contain a large number of peaks in the mass range 1-20 kDa [31]. For bacterial cells, proteins are the most often observed biomarkers. While this approach samples only a small percentage of the total proteins produced in the cells, these profiles have been reported by many groups to be suitable for taxonomic identification, down to at least the strain level. The wide availability of the MALDI-TOF instrumentation and its relative ease of use, coupled with relatively simple sample preparation procedures, have been key features in the rapid advancement of this approach. 

Currently, one particularly important field of research is to identify efficient ways of creating biologically meaningful protein arrays. Protein-DNA conjugates have been used as a new approach to form protein arrays which are actually based on the well-established DNA array technology [59]. The capturing proteins were conjugated with DNA to be bound noncovalently and reversibly to surface-bound DNA, and then used to extract biomarker molecules from complex solutions with ultimate detection by MALDl-MS. 

If the scope of mass spectrometry is limitless, why are the applications of clinical MS almost completely small molecules The answer is that most clinical tests analyze small molecules, biomarkers that are either metabolites or steroids and, hence, mass spectrometers would target those first. Perhaps a more complete answer would also include that methods must be very robust, easily reproduced in different labs, reliable, and subjected to an extensive array of validation tests. Although peptide and protein analysis is increasing rapidly in clinical labs, the MS approaches to these assays is lagging behind somewhat. MS techniques targeting these peptides and proteins exist, but they are primarily in the research stage, with few systems and methods subjected to the clinical rigors of validation. Once the necessary validations occur and methods simplified, it will only be a short time before MS is used routinely in clinical proteomics. 

A different study purely focused on isolating carrier molecules and their bound proteins to search for biomarkers of clinical interest. It was not only found that circulating carrier proteins were reservoirs for the accumulation and amplification of putative disease markers but also that the low molecular mass proteins that bound to albumin were distinct from those bound to nonalbumin carriers. Using SELDI-TOF, it was further verified that albumin bound peptides associated with ovarian cancer. This demonstrated that albumin capture was an effective method for harvesting disease-relevant biomarkers [66]. 

Apart from the classic biomarkers, i.e., tau protein(s) and their phosphorylated forms as well as AP peptides, several other candidate biomarkers have been tested, as recently and extensively reviewed by Frank et al. (2003). Neuronal thread proteins (NTPs) are a family of molecules expressed in the CNS. In a post mortem study, brains of AD patients expressed significant increases of NTP immunoreactivity (de la Monte and Wands, 1992). Following this finding, CSF examination for NTP revealed increased concentration of NTP which correlated tvith progression of dementia and neuronal degeneration (de la Monte et al, 1992). Sensitivity and specificity of this protein as a possible marker of AD, however, have not been determined in a large enough number of patients. 

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