Summary flow chart of biochemical, mass spectrometric, and bioinformatics methods used to identify putative T. brucei nucleoporins and transport factors. Strategies 1–5 are indicated by the red, blue, green, purple, and black colored arrows, respectively. The boxes are colored as follows: gold, protein recovery steps; light blue, protein separation steps; and brown, mass spectrometry techniques. Following mass spectrometry, the bioinformatics strategy outlined here identified 30 putative TbNPC-associated proteins from the initial pool of 757 identified proteins in the TbNEP. SDS-PAGE of fractions from a representative hydroxyapatite separation of the nuclear envelope fraction is shown at the top left. FW, flow-through and wash. Concentrations of phosphate in the elution buffer are indicated above the gel lanes, and apparent molecular masses (in kDa) are shown to the left of the gel. SDS-PAGE of T. brucei NE proteins that were subjected to chemical extraction is shown at the top right. The three extractions (base, salt and detergent (Det.), and heparin) are separated by vertical dashed lines. The pellet (P) and supernatant (S) are indicated. The number of Nups versus the total number of proteins identified with each successive strategy is depicted in the scatter plot (bottom right). Although the total number of proteins identified increases dramatically with further experimentation, the number of NPC-associated proteins levels off after four strategies. 1-D, one-dimensional.