Figure 5

Comparison of dynamic range of proteomic analyses. Dynamic ranges of identified proteins achieved by the present study are shown in comparison with those of two proteomic analyses of human (Human glomerulus (LMD)) and mouse glomerulus. The former is the result of analysis of 50 human glomerular sections prepared by laser-microdissection from frozen sections of each of 3 different biopsy specimens using conventional HPLC in combination with an LTQ-Orbitrap mass spectrometer [9]. Four replicate runs were conducted for each of the 3 samples and all the identified proteins were integrated to create a high-confidence, non-redundant dataset of identified proteins (604) under the criteria adopted in this study. The high-confidence, non-redundant dataset of mouse glomerulus proteome was taken from Waanders et al. [10] who analyzed the proteins of 50 glomerular sections laser-microdissected from mouse kidneys using a newly developed nanoflow HPLC. The nanoflow HPLC’s long, smaller internal diameter column coupled with the LC-MS interface “Replay” allowed the reanalysis of the injected sample with high sensitivity, and with an LTQ-Orbitrap mass spectrometer, provided 2,670 proteins. Normalized spectral abundance factor (NSAF), a relative protein abundance index, was calculated according to Paoletti et al. [12]. NSAF is based on the number of peptide matches (spectral counts) divided by protein mass or protein length which roughly correlates with protein concentration in a protein mixture (spectral abundance factor, SAF). To accurately account for run to run variation, individual SAF was normalized by dividing the SAF of a respective protein by the sum of SAFs for all identified proteins to give an NSAF representing relative protein abundance. NSAFs for the four key proteins comprising the slit diaphragm of glomerulus (nephrin, podocin, Neph1 and Fyn) are mapped on each of the abundance curves.