Currently, microarrays reveal global transcription patterns; but to understand active biological pathways fully, global protein contents and their modification status in cells and tissues must be determined. To accomplish this on a genome-wide scale, we are evaluating adaptation of two-dimensional micro- high pressure liquid chromatography (2D micro-HPLC) followed by MS/MS (mass spectrometric) identification of peptides. The peptides are derived from 20-30 micrograms of total protein by digesting with trypsin; separated based on charge through a strong cation exchange column; collected, concentrated and further fractionated based on hydrophobicity on a reverse phase column; and their sequence are then inferred by MS/MS. In preliminary studies using kidneys from newborn mice we assessed protein extraction procedures and optimization of peptide fractionation steps. From two independent experiments, we were able to identify 33,451 peptides at 95% Confidence Interval and assign them to 11,235 unique proteins. Compared with the estimated number of 14,000-15,000 protein species in a complex tissue, it is conceivable that up to 80% of the proteins were identified in these experiments. Proteins from multiple cellular compartments, with a wide range of net charge, as well as 50% of total ribosomal proteins and transcription factors were included. The differential phosphorylation status of many peptides was also revealed, and the technique has potential to identify other modifications as well. Furthermore, peptides were recovered from 500 proteins that had only existed as constructs based on bioinformatics predictive programs, with no RNA or protein evidence previously adduced. Thus, these assays provide experimental support for the existence of the predicted species. The method overall is robust and can reveal the composition of a complex mixture of proteins and is being extended to other tissues and cell types, and to more resolving chromatographic conditions.
