Developing mass spectrometry towards applications in clinical proteomics

Proteome versus proteins Each organism contains a single genome in virtually all its cells and thus the difference between the numerous cell types within the same organisms are attributed mainly to genetic imprinting, epigenetics, transcriptional and post-transcriptional processes. Thus studying all the protein molecules present in the living system (termed proteomics) at any given time and physiological condition will logically provide insights into the mechanism of life to an unprecedented depth. Historically when proteins were studied 'one at a time' achieving a systems view of the cell or organelle was a daunting and laborious endeavor to undertake. Further when proteins are studied individually the results could be occasionally bewildering owing to the crosstalk between the signaling pathways and nodes 1, 2. Though there are successful structural biology technologies on a small scale and techniques like yeast two hybrid 3 and phage display system 4 to characterize the protein-protein interaction at a larger scale they do not provide a system wide view apart from establishing the interaction between groups of proteins. Further the false positive and false negative rates are difficult to estimate and the information is less quantitative (if at all) to construct a stoichiometric protein complex 5. Mass spectrometry based proteomics Mass Spectrometry (MS)-one the most sensitive analytical techniques-has played an enormous role in the development of proteomics to its existing capabilities. The advent of ESI and MALDI techniques initially led to mass spectrometry being used for individual protein sequencing and identification of gel bands thus gradually replacing the Edman degradation method. At first, proteomics was associated with 2-dimensional gel electrophoresis techniques (2DE) 6 but 2DE has serious limitations 7. When protein and peptide separation techniques like liquid chromatography were combined with mass spectrometry 8 , complex protein and peptide 6 mixtures were successfully analyzed leading to the emergence of MS-based proteomics 9-13. Complementary to advances in separation strategies, development of new mass spectrometers especially the FT ICR 14 and Orbitrap 8, 15-18 hybrid instruments, have facilitated routine large scale high accuracy and high resolution MS. Mass spectrometry is not quantitative by itself and strategies have been developed to obtain relative quantification between conditions either using non-radioactive isotopes or the spectral information itself 19-21. Proteins or peptides are labeled with light and different heavy non-radioactive isotopes which are identical in terms of biochemical properties including the ionization efficiency and differ only by mass and thus can be distinguished in …