Presenter: R. Neil, Dalton, London, UK
Authors: R. Neil Dalton
Key Learning Objectives:
1. An understanding of MRM based mass spectrophotometric methods for both metabolite and protein/peptide analysis
2. The distinction between speculative and knowledge based screening for biomarkers
3. Appreciating the importance of validity testing of platforms with known biomarkers
The development of soft ionisation techniques, particularly electrospray, for both metabolite and protein/peptide analysis has enabled the application of a range of mass spectrometric scans to provide insights into biological systems. In the search for potential new biomarkers it is important to differentiate between speculative and knowledge based screening, but appreciate that mass spectrometry (MS) is applicable to both approaches, usually on the same preparation of biological sample.
For speculative metabolomic investigations, in its basic MS form, the supernatant from a methanol precipitation of whole blood, plasma, or urine can be simply injected into the mass spectrometer, without chromatography, and the sample interrogated in both positive and negative ion modes across a suitable mass/charge ratio (m/z) range, usually 20-600. Interesting MS signals can then be positively identified in MSMS mode by product ion scanning. On the same sample preparation, MSMS scans for particular compound classes, e.g. amino acids, acylcarnitines, bile salts, can provide the first steps in a knowledge based acquisition system. However, it is the capability to specifically and quantitatively scan for a large number of compounds of known biological significance, using rapid chromatography and multiple reaction monitoring (MRM) mode, that is becoming the primary MSMS metabolomic platform. Sample preparation can be modified to accommodate both hydrophilic and lipophilic targets.
The same approaches can be applied to proteomic analysis. Although electrospray MS has proved a valuable tool in the elucidation of isolated protein structure, it is relatively insensitive for native biological samples; the key to viable proteomic analysis is the use of tryptic (other endopeptidases can be used) digests. MS and MSMS peptide analysis can be performed using exactly the same techniques as for metabolites, with similar advantages and constraints. However, it is the capability to specifically and quantitatively scan for a large number of peptides of known biological significance, using rapid chromatography and MRM mode, that is becoming the primary MSMS proteomic platform. The development of peptide MRM databases is revolutionising this approach but it is limited by the analytical sensitivity of even the best MSMS equipment, though it can be coupled with preliminary multiplexed immunoconcentration.
In developing and applying new metabolomic and proteomic systems it is essential, if possible, that they are validated with already recognised biomarkers. In organ transplantation, early graft function is a primary determinant of long term graft survival. Consequently, our focus has been on organ protection during transplantation and maximising early graft function. Plasma asymmetric dimethylarginine, a new biomarker of hepatic blood flow, has been used to validate our metabolomic platform in liver transplantation. In renal transplantation, urine retinol binding protein, a biomarker of proximal tubular anoxia, and plasma creatinine and symmetric dimethylarginine, biomarkers of glomerular function, have been used to validate our proteomic/peptidomic and metabolomic platforms, respectively.
In conclusion, mass spectrometry is a very powerful tool for both metabolomic and proteomic investigation of organ transplantation and should lead to the identification of clinically valuable new biomarkers.
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